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imad tutorials: fix auth/init, use geemap
Mort, here are some updates.
Auth now adds the auth_mode='notebook' to initiate the typical authentication flow.
Change ee_jupyter to geemap for interactive map display (we are officially supporting geemap).
:)
RE geemap - it is very intuitive - you can use it just like the Code Editor!
m = geemap.Map()
m.centerObject(...)
m.addLayer(...)
m
Under the upperleft menu in the map, you can find the inspector and layer managers. It also includes lots of extra helper functions that I'm less familiar with, but I really like the parity of the map functionality with the Code Editor!
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British North Russia Squadron
The British North Russia Squadron was a squadron of the Royal Navy based at Murmansk from 1917 to 1919.
History
The squadron was formed as part of an initiative by the Entente Powers to keep the Russian Empire in the First World War. One goal was to protect the large stockpiles of Allied material that had begun stockpiling at the ice-free port at Murmansk. Russia's continued involvement in the war was challenged externally by German advances into the East and internally by a strong antipathy to the war amongst the Russian population. This later factor had led to support for the Bolshevik Revolution and had resulted in the Russian Civil War. The squadron would remain in Russia throughout most of the Allied North Russia Intervention, though it served no real role in this.
The squadron was originally placed under the command of Rear Admiral Thomas Kemp with the Canopus-class predreadnought HMS Glory (1899) as his flagship. This battleship had been refitted, with some guns being removed to allow for more accommodation for marines. There was also the cruiser HMS Vindictive (1897), a depot ship, an armed boarding vessel and a variety of trawlers and drifters which had been converted to function as minesweepers.
In October 1918 Rear-Admiral John F.E. Green became senior British naval officer in northern Russia, relieving Admiral Kemp, who returned home. Rear-Admiral Green did not object to the withdrawal of American ships from Murmansk after July 1918, as the U.S. naval ships had been logistically dependent on the British, to whom they had been a burden.
The squadron returned to England in September 1919, shortly after the ratification of peace with Germany, with the signing of the Treaty of Versailles.
Admiral commanding
Post holders included:
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of small light spots down back.
"Underparts, in the male, blue throughout; belly-patches not separated by a lighter or darker mid-ventral line; throat evenly colored, light blue to snout and lips, and lighter in tint than general ventral color; blue of belly not separated from throat patch by a lighter or darker area across gular region (young specimens excepted). Female more richly colored below than in bi-seriatus; lighter than male; belly patches separated by a faintly lighter area; chest lighter than belly; one extensive throat patch as in male; blue not always extending to beneath hind limb."
fourth toe 20 21 22 22 20 21
Remarks. — In size, this subspecies equals the largest speci mens of S. o. biseriatus. The dorsal scales seem more num erous than in that subspecies. Mr. Camp states: "A number of individuals at hand in a large series of S. o. bi-seriatus from the southern Sierras in Kern and Tulare counties and farther north are, of all our specimens, the closest in size and ventral coloration to taylori; they are, however, of greenish and more dusky shades of blue beneath than the new form, and their status must be held questionable pend ing the acquisition of material from the headwaters of the Kings and San Joaquin rivers. A male specimen, one of
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Pliable container for treatment of infectious medical waste
ABSTRACT
A method is provided for treatment of infectious medical waste. The method in a broad sense includes washing the infectious medical waste with a liquid to convert the infectious medical waste into noninfectious waste. In accordance with another aspect, a method is provided which includes a first step of wetting the infectious medical waste with a first liquid to remove loose particles and materials that are soluble in the first liquid from the infectious medical waste, and a second step of wetting the infectious medical waste with a disinfecting liquid to disinfect the infectious medical waste. An apparatus also is provided for treatment of infectious medical waste. The apparatus comprises a substantially liquid-tight volume for containing the infectious medical waste. The volume includes a first input for inputting a first liquid into the volume to wet the infectious medical waste with the first liquid and thereby remove loose particles and materials that are soluble in the first liquid from the infectious medical waste.
RELATED APPLICATION
This application is a divisional of application Ser. No. 08/509,996 filed Aug. 1, 1995, which issued as U.S. Pat. No. 6,113,854.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and apparatus for treatment of medical waste and, more specifically, to methods and apparatus for treatment of medical waste that are not dependent upon incineration.
2. Description of the Related Art
The present invention is useful for the effective treatment of a relatively wide variety of medical wastes, but its applicability and effectiveness are particularly noteworthy as to certain classes of medical wastes. There is a lack of uniformity and convention as to the use of certain terms involved in discussions of medical wastes, and for that reason some discussion of nomenclature would facilitate an understanding of the invention.
The term medical waste is defined by the U.S. Environmental Protection Agency (the “U.S. EPA”) as any solid waste which is generated in the diagnosis, treatment, or immunization of human beings or animals, in research pertaining to these, or in the production or testing of a biological. 40 C.F.R. Ch.1, § 259.10(a); U.S. Environmental Protection Agency, Mar. 24, 1989, pp. 12,373-12,374. Biological as used herein refers to preparations made from living organisms and their products, including vaccines, cultures, etc., intended for use in diagnosing, immunizing or treating humans or animals in research pertaining thereto. Such medical waste may derive from a variety of sources, including hospitals, clinics, health maintenance organizations, and the like. Specific examples of medical wastes are provided below.
The term infectious medical waste is defined as waste containing any organism (such as a virus or bacteria) that is capable of being communicated by invasion of or multiplication in body tissues and is capable of causing disease or adverse health impacts in humans. The closely-related term regulated medical waste, used by some synonymously with the term infectious medical waste, is defined by the U.S. EPA as any solid waste [defined in 40 C.F.R. § 259.10(a)], generated in the diagnosis, treatment, (e.g., provision of medical services), or immunization of human beings or animals, in research pertaining thereto, or in the production or testing of a biological, that is not excluded or exempted under 40 C.F.R. § 259.30(b), and that is listed in the table of regulated medical waste provided at 40 C.F.R. § 259.30(a). 40 C.F.R. § 259.30 (1992).
Technically, medical waste qualifies as infectious or regulated medical waste only if it contains pathogens (microorganisms capable of causing disease) of sufficient quantity and virulence so that human exposure to the medical waste could result in infectious disease. Thus, this class of medical waste is defined in terms of its effect on humans, i.e., the effect of the waste on humans must be known or predicted to classify waste as infectious or regulated medical waste. In view of this definition, the manner in which one determines whether a particular batch of medical waste qualifies as infectious or regulated medical waste has not been reduced to a single, universally-recognized test. Infection Control and Hospital Epidemiology, January 1992, page 40.
One common approach to classification of such wastes involves using a subjective standard in which the waste is deemed to be infectious or regulated medical waste if it is suspected to contain pathogens in sufficient quantity and virulence to cause disease. Infection Control and Hospital Epidemiology, January 1992, page 39.
For purposes of the present invention, the term infectious medical waste as used hereinbelow is interpreted in a broad sense to include the U.S. EPA definition of regulated medical waste at 40 C.F.R. § 259.10(a) as referred to above, including wastes that meet the subjective standard referred to above. Moreover, the term as used in connection with the invention includes any medical waste that is handled or treated as infectious or regulated medical waste as defined by any of the above definitions.
Infectious or regulated medical waste is commonly divided into seven groups or subcategories. These subcategories, which are summarized, e.g., in the table of regulated medical waste at 40 C.F.R. § 259.30(a), include the following:
1. Cultures and Stocks. This group includes cultures from medical and pathological laboratories, cultures and stocks of infectious agents from research and industrial laboratories, wastes from the production of biologicals, discarded live and attenuated vaccines, and culture dishes and devices used to transfer, inoculate and mix cultures.
2. Pathological Wastes. This group includes tissues, organs, and body parts and body fluids that are removed during surgery or autopsy, or other medical procedures, and specimens of body fluids and their containers.
3. Human Blood and Blood Products. This group includes liquid waste human blood, products of blood, items saturated and/or dripping with human blood, or items that were saturated and/or dripping with human blood that are now caked with dried human blood, including serum, plasma, and other blood components, and their containers, which were used or intended for use in either patient care, testing and lavatory analysis, or the development of pharmaceutical. Intravenous bags are also included in this category.
4. Sharps. This group includes sharp items that have been used in animals or human patient care or treatment or in medical research, or industrial laboratories, including hypodermic needles, syringes (with or without the attached needles), pasteur pipettes, scalpel blades, blood vials, needles with attached tubing, and culture dishes (regardless of presence of infectious agents). Also included within this group are other types of broken or unbroken glassware that were in contact with infectious agents, such as used slides and cover slips.
5. Animal Waste. This group includes contaminated animal carcasses, body parts, and bedding of animals that were known to have been exposed to infectious agents during research (including research in veterinary hospitals), production of biologicals, or testing of pharmaceutical.
6. Isolation Wastes. This group includes biological waste and discarded materials contaminated with blood, excretion, exudates or secretions from humans who are isolated to protect others from certain highly-communicable diseases, or isolated animals known to be infected with highly-communicable diseases.
7. Unused Sharps. This category includes unused discarded hypodermic needles, suture needles, syringes, and scalpel blades.
The present invention is well suited for the effective treatment of a wide variety of medical wastes. It is particularly well suited, however, and finds greatest practical advantage, in the treatment of solid or semi-solid infectious medical wastes falling within subcategories 3 and 6 above, i.e., blood and blood products, and isolation wastes. These materials typically comprise synthetic or man-made materials that contain microorganisms, biologicals, or pathogens. Medical waste falling within either of these two subcategories is referred to in this document as treatable medical waste.
Although most infectious medical wastes are generated in hospitals and other health care facilities, relatively little data are available on their specific composition. It is believed that infectious medical wastes typically include a heterogeneous mixture of materials such as plastics, dry cellulosic solids, wet cellulosic solids, and noncombustible materials such as metals. A typical mix of such materials within a batch of medical waste might include, for example, approximately 14% by weight of plastics, about 45% by weight of dry cellulosic material, approximately 18% by weight of wet cellulosic material, and approximately 20% by weight of noncombustibles. Infection Control and Hospital Epidemiology, January 1992, page 40. These wastes often comprise the disposable contents and packaging of single-use products, such as incontinency products, surgeon's instruments, woven towels, non-woven towels, non-woven surgical barrier drapes, non-woven surgical staff apparel, wound dressings, surgical sponges, examination instruments, plastic treatment vessels of many shapes and sizes, patient restraints, etc.
Infectious medical waste contains or is suspected of containing organisms capable of causing disease or adverse health impacts in humans. The term pathogen similarly refers to an agent, organism or microorganism that causes or is capable of causing disease. Examples of pathogens include bacteria, viruses, fungi, mycobacteria and spores. A given item of infectious medical waste typically will have many different types of pathogens on it, and the number of individual pathogenic microorganisms on the item typically will run into the billions. A principal object of the invention is to treat infectious medical waste in a manner that reduces the amount of pathogens so that the waste no longer qualifies as infectious medical waste. Given the extremely large numbers of pathogens encountered, and given the nature of their existence and destruction, measures of the reduction of pathogens in a specific population usually are expressed in statistical terms. In these statistical terms, the amount of pathogens remaining in the population typically are expressed in terms of colony forming units (CFU) per square inch. The State and Territorial Association on Alternate Treatment Technologies recommends requiring an inactivation of vegetable bacteria, fungi, lipophilic/hydrophilic viruses, parasites, and mycobacteria at a 6 log to the 10th power reduction or greater, and inactivation of B. stearothermophilus spores or B. subtilis spores at a 4 log to the 10th power reduction or greater. Technical Assistance Manual: State Reaulatory Oversight Of Medical Waste Treatment Technologies, April, 1994, pages 8-12.
Heretofore, the conventional procedure for handling of medical wastes, and particularly for treatable medical wastes, in health care facilities has been as follows. As the medical wastes are created, e.g., in the health care facility, they are placed in red plastic bags. When the red plastic bags become filled, they are taken to a storage area at the health care facility and placed in large plastic drums, typically 44-gallon polyvinyl chloride drums. Lids are secured over the drums to limit odors and to limit vapor and air escape out of the drums. Periodically, the medical waste in the drums is permanently disposed of.
U.S. EPA regulations grant the individual states fairly broad discretion in regulating the handling and treatment of medical wastes. Generally, disposal of infectious medical wastes by dumping in a sanitary landfill without treatment is not permitted. Placing of infectious medical waste in a landfill can be punishable by severe fines and penalties.
The most widely used permanent disposal technique for medical wastes, including treatable medical wastes, is incineration. The incineration is undertaken at special incineration facilities, usually located at a site away from the generating health care facility. At periodic intervals, the medical wastes are picked up by a transporter at the health care facility and taken to the incineration facility. The contents of the drums then are inputted into an incinerator and incinerated. The medical wastes are thereby converted to solids, such as inert ash, and to stack gases that are released into the atmosphere.
The incineration process has been generally disadvantageous in that it is costly. Moreover, incineration brings to bear a host of environmental concerns, most importantly air quality concerns. The U.S. EPA has recently proposed rules pursuant to the 1990 Clean Air Act amendments that would mandate drastic reductions in medical waste incinerator emissions. 60 C.F.R. § 10654. This is expected to greatly increase the cost of incineration and further reduce its attractiveness as a viable disposal technique.
Steam sterilization of medical wastes has been used as an alternative to incineration. With this approach, the medical wastes are placed in an apparatus similar to an autoclave. In a typical steam sterilization process, the wastes are exposed to steam at approximately 250° F. for about 30 minutes.
Steam sterilization has been disadvantageous for treatment of medical wastes in that it requires relatively large and costly equipment, and in that it is expensive to operate due in large part to the substantial heating requirements. Steam sterilization also is disadvantageous in that it generally does not eliminate highly-objectionable visual evidence of the contamination.
Chemical disinfection, typically accompanied with grinding, is a process in very limited use. This treatment process grinds the wastes in a hammermill in the presence of a chemical disinfectant. This process tends to be expensive.
Thermal inactivation of solid waste, another such process, is accomplished by the application of dry heat in an oven which is usually operated by electricity. This process involves pre-shredding he waste, initially heating it typically with an electric source, and then maintaining a temperature of around 200° F. for approximately two hours in a large enclosed chamber. This process also tends to be expensive.
Irradiation exposes wastes to ultraviolet or ionizing radiation from a source such as cobalt 60 in an enclosed, shielded chamber. Disadvantages are the large initial cost of the equipment, the skilled personnel required for safe operation, and the disposal problem for the decaying radiation source. The method is only effective generally if the ultraviolet radiation reaches the pathogenic waste and there is generally little radiation penetration through the waste which tends not to be ultraviolet transparent.
Objects of the Invention
Accordingly, an object of the present invention is to provide a method and apparatus for treatment of infectious medical waste that does not require incineration.
Another object of the present invention is to provide a method and apparatus for treatment of infectious medical waste that poses less of a threat to environmental quality than incineration.
Still another object of the present invention is to provide an apparatus and method for treatment of medical waste that is less costly relative to incineration.
Additional objects and advantages of the invention are set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing objects, and in accordance with the purposes of the invention as embodied and broadly described in this document, methods and apparatus are provided for treating infectious medical waste to substantially reduce or eliminate pathogen populations so that the treated waste no longer is infectious and may be recycled or disposed of using conventional methods.
The invention utilizes the basic principle that medical waste, and particularly infectious medical waste, may be treated as unregulated waste if such waste has been treated so that it no longer is “infectious.” This departs from conventional approaches such as incineration in which the waste itself is destroyed or physically transformed to eliminate the threat.
The approach used in the method according to the invention in broad terms involves washing the infectious medical waste with a liquid to convert the infectious medical waste into noninfectious waste. This approach is somewhat similar to the manner in which reusable textiles such as clothing are laundered. The standard approach to treating soiled reusable fabrics, of course, is through laundering. The principal objectives in reusable fabric laundering techniques are to remove stains and kill pathogens while at the same time preserving the desirable physical properties of the fabric, such as tensile strength and fiber elasticity, color, size, shape, etc. The present invention takes advantage of the disinfecting features of textile laundering approaches, while adapting such approaches to the particular needs, constraints and nuances of infectious medical waste treatment.
In accordance with the invention, a method for treatment of infectious medical waste is provided which comprises washing the infectious medical waste with a liquid to convert the infectious medical waste into noninfectious waste. The liquid preferably comprises an aqueous system, and more preferably at least one of a detergent solution and a bleaching agent.
A related method according to the invention includes a first step of wetting the infectious medical waste with a first liquid to remove loose particles and materials that are soluble in the first liquid from the infectious medical waste, and a second step of wetting the infectious medical waste with a disinfecting liquid to disinfect the infectious medical waste. The first liquid preferably comprises an aqueous solution. A particularly well suited example of the first liquid would comprise a detergent solution. The effectiveness of the method may be facilitated in some applications by imparting a mechanical action to the infectious medical waste in the first liquid. The disinfecting liquid preferably comprises a bleaching agent, for example, that may include available chlorine.
In accordance with another aspect of the invention, an apparatus is provided for treatment of infectious medical waste. The apparatus includes a substantially liquid-tight volume for containing the infectious medical waste. The compartment includes a first input means for inputting a first liquid into the volume to wet the infectious medical waste with the first liquid and thereby remove loose particles and materials that are soluble in the first liquid from the infectious medical waste. The apparatus may further include a second input means for inputting a disinfecting liquid into the substantially liquid-tight volume to disinfect the infectious medical waste.
According to still another aspect of the invention, a pliable container is provided that has proven useful in containing the medical waste during treatment. The pliable container includes a wall member forming a concavity and having a perimeter, the wall member being permeable to water, a protective surface coupled to the wall member perimeter to enclose the concavity, and an access for selectively opening and closing the container.
The first and second steps of the method may be, and preferably are, carried out sequentially. This is not, however, necessary. The method may be carried out, in some instances advantageously so, by performing the first and second steps simultaneously or with some overlap.
After treatment using the methods and apparatus of the invention, the resultant uncontaminated and unregulated wastes may be discarded in the normal fashion, e.g., by disposal in a sanitary landfill. Selected items from the resultant wastes also may be retrieved for subsequent use or recycling.
BRIEF DESCRIPTION OF THE DRAWING
The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate a presently preferred embodiment and method of the invention and, together with the general description given above and the detailed description of the preferred embodiment and method given below, serve to explain the principles of the invention.
FIG. 1. shows a first preferred embodiment of the invention. This embodiment is designed for batch-type treatment of infectious medical waste.
FIG. 2 shows a mesh bag in accordance with the invention. This mesh bag is useful for containing infectious medical waste during treatment according to the method of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS AND METHODS
Reference will now be made in detail to the presently preferred embodiments and methods of the invention as illustrated in the accompanying drawings, in which like reference characters designate like or corresponding parts throughout the drawings.
The preferred apparatus and methods of the invention are useful for treatment of a variety of infectious medical wastes. They are particularly attractive, however, for treatment of what has been defined herein as treatable medical waste. The apparatus and methods are useful as a general matter for infectious medical waste in which the carrier or vehicle or substrate for the pathogens is a solid or semi-solid material. The medical waste need not be impervious to the liquids or to the washing process. Paper products, which for example typically disintegrate into pulp particles easily viewable with the naked eye when subjected to water-based washing processes, typically are suitable for treatment according to the invention.
In accordance with the invention, an apparatus is provided for treatment of infectious medical waste. The apparatus in broad terms comprises a substantially liquid-tight volume for containing the infectious medical waste. A first input means is provided for inputting a first liquid into the volume to wet the infectious medical waste with the first liquid and thereby remove loose particles and materials that are soluble in the first liquid from the infectious medical waste. Preferably, the apparatus also includes a second input means for inputting a disinfecting liquid into the substantially liquid-tight volume to disinfect the infectious medical waste. The first and second input means in some embodiments may be one and the same, or they may share common or overlapping articles of hardware.
The apparatus of the invention also optionally but preferably includes means for imparting a mechanical action to the infectious medical waste, and evacuation means for evacuating bulk quantities of the first liquid from the substantially liquid-tight volume.
As a specific yet merely illustrative example, a first preferred embodiment 10 of the apparatus according to the invention is shown in FIG. 1. This embodiment comprises a commercial-scale laundering machine 12 such as an Ellis automatic unloading washer (54″×108″), marketed by Ellis Corp. of Itasca, Ill. The internal volume of the machine is approximately 144 cubic feet. Machine 12 includes four volumes in the form of four washer pockets or compartments 14 a-d. Machine 12 has a rotating-drum design in which the treatment volumes or compartments comprise an axially-mounted horizontal cylinder 16 that is rotatable about the longitudinal axis of the compartments. The cylindrical compartments are contained within a rigid and substantially liquid-tight shell 18. Each of the compartments is substantially circular with an internal diameter of about 54 inches, a length of about 27 inches, and an internal volume of about 36 cubic feet. Interior walls 20 a-c separate compartments 14 a-d so that wall 20 a is between compartments 14 aand 14 b, wall 20 b is between compartments 14 b and 14 c, and so on. Each compartment has a rigid exterior wall 22 that is perforated with a relatively large number of perforations or holes of sufficient size (e.g., about 0.25 to 0.5 inches in diameter) that they are readily permeable by the liquids used in the machine in accordance with the invention. Ribs or vanes 24 are fixed on the interior walls to facilitate agitation, as explained more fully below. Each compartment has an opening or aperture 26 for inputting and removing articles such as the infectious medical wastes or containers of such wastes, as further described below. Doors 28 are provided to close shell 18 and provide a substantially liquid-tight seal.
The first input means as embodied in machine 12 comprises a water inlet system for selectively inputting water and other liquids such as detergent solutions, as described more fully below. Water inlet system includes piping 32, a water inlet valve 34, and one or more water inlets 36 into the respective compartments. Water inlet system 30 is coupled to and in fluid communication with a water source (not shown), such as a municipal water supply that provides tap water-quality water. Water inlet system 30 also is coupled to and in fluid communication with a water heating system (not shown), such as a commercially-available hot water heater or industrial heat exchanger, so that the water as it is inputted into the compartment by water inlets 36 can have a temperature, selected by an operator, of from ambient temperature to about 200° F.
Machine 12, and more specifically the first input means, also includes as a preferred option the capability to separately add components of the first liquid. For example, in many applications it is advantageous for the first liquid to include such components as alkaline components (e.g., sodium hydroxide), surfactants (surface active agents), sequestrants (e.g., water softening compounds), etc. Such components may be added directly to the water in water inlet system 30, either individually or as formulated compounds. The component adding means may take a variety of forms, depending upon the form of the additive, the specific application, the design of the machine, the treatment volume, and other factors. For additives that comprise dry supplies such as powdered detergents or for semi-solid additives such as gels, the component adding means may comprise a supply door for manual supply insertion, or a flush system in which, for example, the dry supplies are placed in a hopper and they are automatically flushed into the compartment by a liquid such as water. If the supplies are in liquid form, the component adding means may comprise a supply door through which the supplies may be poured into the compartments. Preferably, however, the component adding means would comprise a liquid feed system or automatic liquid chemical injection system in which the liquid supplies are automatically fed and inputted to the compartments at the appropriate time and in the appropriate amounts.
In the preferred embodiment, the component adding means comprises an automatic liquid chemical injection system 38 as shown in FIG. 1 which includes a plurality of supply containers 40 a-c, flexible tubing 42 connected in fluid communication between supply containers 40 a-c and compartments 14 a-d, and a controller/regulator 44 that times, controls and regulates the flow of supplies from the respective supply containers to the compartments according to preselected settings.
Machine 12 includes means for imparting a mechanical action to the infectious medical waste. The mechanical action means may take virtually any form, provided that the effect is to impart mechanical action to the contents of the treatment compartment within the liquid contained in the compartment so that components of the waste are physically contacted and/or agitated to facilitate physical removal of pathogens from the waste. As noted above, each compartment includes a plurality of ribs or vanes 24. Shell 18 is substantially liquid-tight when doors 28 are closed and latched. A driving means such as a motor, turbine, pump, drive belt or the like (not shown) is motive ly coupled to cylinder 16 to selectively rotate the compartments in a desired rotational direction, at a desired speed and for a desired duration. This rotation causes the contents of the compartments, both liquid and solid, to be subjected to mechanical action. The extent of the mechanical action can be regulated, e.g., by controlling the rotational frequency of the compartments. Mechanical action also can be affected by periodically reversing the rotation of the compartments, and by regulating the frequency and speed at which the compartment rotation is reversed.
It should be noted that other mechanical action means are within the scope of the invention. For example, such means may include protrusions, blades, impellers and the like within the washer compartment, or liquid jets that spray or otherwise direct liquid into the compartments. Mechanical action also may be effected by vibrating or otherwise moving the compartment itself or having a stationary drum with a center agitator such as ones which would be found in a typical home washing machine.
An evacuation means also is provided in machine 12 for evacuating the excess or bulk quantities of liquid from the treatment volume or compartments. The evacuation means preferably comprises a drainage system that includes one or more drains 46, drainage piping 48 and a drainage valve 50. Drains 46 are disposed in shell 18 and are in fluid communication with the washer compartments 14 a-d. Drainage piping 48 is coupled to and in fluid communication with drains 46, and with a discharge such as a municipal sewer system. Drainage valve 50 comprises a pneumatically-actuated valve assembly. It is disposed in the drainage piping to control the flow of liquid through the drainage system. At the appropriate time, the bulk liquid contents of the washer compartments can be drained by actuating drainage valve 50 so that bulk liquids in the compartments flow through drains 46, through drainage piping 48 and drainage valve 50, and ultimately out to the sewer system.
Machine 12 also includes a second input means for inputting a disinfecting liquid such as a bleaching agent solution into compartments 14 a-d to disinfect the infectious medical waste. This second input means preferably is used after the first liquid has been inputted into the compartments. More preferably, it is used to input the disinfecting liquid after the bulk of the first liquid has been drained from the compartments.
The second input means may be separate from the first input means, it may share components with the first input means, or it may be identical to and one and the same with the first input means. Accordingly, the second input means may comprise a water inlet system as described above, a supply door or window, a supply hopper, an injector, etc. In machine 12, the second input means comprises the water inlet system 30 and automatic liquid chemical injection system 38, as described above. This second input means is in fluid communication with compartments 14 a-d so that the disinfecting liquid may be injected directly into the compartments during processing.
The presently preferred method of the invention will now be described as it could be carried out using the apparatus depicted in FIG. 1. It should be noted, however, that the method of the invention is not limited to use with this machine, and may find application in many other contexts and with many other machines.
The method according to the invention may be carried out at a health care facility, at a separate site, or elsewhere. The location is not necessarily limiting. Merely for purposes of illustration, it will be assumed in this document that the method is to be carried out at a separate location, here called a treatment facility, away from the health care facilities that generated the medical waste.
As mentioned above, the medical wastes as they are obtained from the health care facilities typically comprise 44-gallon drums filled with red-bagged waste materials. These drums usually are screened at the health care facility so that only drums that are relatively clean externally and which are properly sealed with lids are transported from the health care facility to the treatment facility. Radiation monitoring also is performed at the health care facility to identify and separate any drums containing radioactive materials prior to transport.
Upon arriving at the treatment facility, the drums are moved into a receiving area, where they are visually inspected for drum integrity, leakage, sealing, visible evidence of stains or contamination, etc. Radiation monitoring may be performed here as well to screen any radioactive materials. Each drum also is weighed. After this initial processing, each drum is opened and the red bags contained in it are unloaded. Each red bag is opened and the contents are removed and inspected. Materials other than treatable infectious medical wastes are removed and placed in separate containers.
It has been learned that the batch treatment of infectious medical waste can be greatly facilitated by containing the medical waste in specially-designed containers having certain desirable properties. Accordingly, as one aspect of the invention, a pliable container or mesh bag 52 is provided which has the following construction, as illustrated in FIG. 2.
Mesh bag 52 has a generally cylindrical shape with a length of about 27 inches and a radius of about 15 inches. This provides sufficient capacity for about 35 pounds of treatable infectious medical waste. It should be noted that the shape and size of the container are not necessarily limiting. The bag shape and size may be selected, for example, to conform to the shape and size of equipment with which the bag is to be used, such as machine 12 and its compartments.
Mesh bag 52 includes a water-permeable wall member 54 forming a concavity 56 and having a perimeter 58. The wall member preferably comprises a mesh material having a mesh size of about {fraction (1/16)} by ⅛ inches. The wall member may comprise, for example, a synthetic fiber mesh material with relatively low water absorptivity and reactivity, such as nylon, polyester, olefin, acrylic, mod acrylic, etc. Different materials and larger or smaller mesh sizes are permissible, however, depending upon such factors as the specific medical waste being treated and the liquids being used in the treatment. The mesh size should be selected so that the wall member is permeable to water and/or other liquids that are useful in the practice of the invention, yet contains particles that might otherwise clog piping or valves, or which might exceed particle size and quantity limitations on effluent water imposed by regulation.
The container further includes a protective surface 60 coupled to wall member perimeter 58 to enclose concavity 56. The protective surface performs the important function of protecting people having access to the container from being injured by sharps or potentially injurious objects in the bag. The protective surface should have sufficient strength and impenetrability so that it will appropriately limit the risk of wounds to workers handling the bag under ordinary processing conditions and circumstances, and while processing the types of medical wastes ordinarily encountered from health care facilities.
Protective surface 60 comprises a polyvinyl chloride sheet. Alternative materials include various flexible plastic materials with high chemical resistant properties. Protective surface 60 is about 25.5 inches long, about 10 inches wide, and about ¼ inch thick. It has an interior surface 62 and an exterior surface 64. Protective surface 60 meets and generally exceeds the criteria for strength and puncture resistance outlined above.
Mesh bag 52 includes an access in the form of a heavy-duty, corrosion-resistant zipper 66 disposed at perimeter 58 of the bag for selectively opening and closing the bag to insert and remove contents. A handle 68 is attached to exterior surface 64 of protective surface 60 to facilitate securing and moving mesh bag 52. Handle 68 comprises a woven nylon ribbon approximately 5 cm wide, approximately 1 cm thick, and running the length of protective surface 60. Handle 68 is secured to protective surface 60 by a plurality of steel rivets or other suitable means.
Returning to the description of the preferred method, after opening and inspecting the contents of the red bags, an operator places the contents of the red bags and the red bags themselves into mesh bag 52. When mesh bag 52 is substantially full, zipper 66 is zipped up to seal the bag and enclose the waste. Three mesh bags filled with infectious medical waste are placed in each of the washer compartments 14 a-d of machine 12. When all washer compartments that are to be filled have been filled, doors 28 and are closed and latched.
In a broad sense, the method of the invention comprises washing the infectious medical waste with a liquid to convert the infectious medical waste into noninfectious waste. A principal objective of the method is to use liquid chemistry techniques and, more particularly, chemical laundering-type techniques, to convert infectious medical waste into benign, non-infectious waste. This requires the reduction of pathogens down to a level sufficiently close to zero in statistical terms as to be acceptable for categorization and handling as non-infectious wastes. For example, the reduction in pathogenic activity would have to be reduced so that the wastes no longer qualify as infectious medical wastes as that term is used in this document, which includes the definitions provided above. The State and Territorial Association on Alternate Treatment Technologies recommends requiring a level of disinfection demonstrated by the ability to inactivate surrogate pathogens such as vegetable bacteria, fungi, lipophilic/hydrophilic viruses, parasites, and mycobacteria at a 6 log to the 10th power reduction or greater; and to inactivate B. stearothermophilus spores or B. subtilis spores at a 4 log to the 10th power reduction or greater. Technical Assistance Manual: State Regulatory Oversight Of Medical Waste Treatment Technologies, April, 1994 pages 8-12. The elimination of pathogens may be through physical removal, through killing or destruction of the pathogens, etc.
In eliminating pathogens using a washing or laundering-type approach, the effectiveness of the process may be regulated or controlled by a number of liquid treatment factors, principal among which include the following: (1) chemical function of the liquid or liquids used (including composition, concentration of the various chemical species present, chemical activity, etc.), (2) temperature, (3) mechanical action, (4) time or duration of processing and of the stages of processing, if the processing is staged (e.g., rinse, detergent cycle, bleaching cycle, etc.), and (5) dilutions (e.g., number of cycles or replacements of liquid baths). Each of these liquid treatment factors includes a number of subsidiary factors, parameters and variables. In addition, there are interactions or trade-offs among the various factors.
As a general matter, aqueous systems (systems based on the use of water) usually are preferred for the treatment of infectious medical wastes in accordance with the invention. Water is a universal solvent that is widely recognized for its unique and attractive properties. In most instances, water is readily available in ample quantities and at low cost.
For most applications, the aqueous system should be mildly alkaline for the predominant portion of the treatment. The pH levels preferably will be in the range of 8 to 10. Water hardness should be avoided to limit the formation of insoluble metal salts, particularly where detergents or other surfactants are employed. The temperature of the aqueous system for most applications should be at least about 140° F., and preferably should be in the range of 180° F. to 205° F. for most applications. In addition, some mechanical action is preferred. In most applications, the mechanical action should be moderate to vigorous.
To illustrate the range of variations in the liquid treatment factors that are permissible within the scope of the invention, the following Examples 1-4 are provided. In each of these examples, the following conditions are assumed. The infectious medical waste to be treated are placed in mesh bags 52. Each bag contains approximately 35 pounds of infectious medical waste. Three of the mesh bags are placed in each of the washer compartments of machine 12, and doors 28 are secured to enclose the compartments in a substantially liquid-tight fashion.
EXAMPLE 1 PREDOMINANT CHEMICAL FUNCTION
Water inlet is actuated to input water into compartments 14 a-d. The water is tap water from a municipal water treatment source. It has an alkalinity of about 50-100 ppm as sodium bicarbonate, and a pH of about 8.0. The incoming water has been heated to a temperature of approximately 160° F. When the water level within compartments 14 a-d reaches approximately 6 inches, valve 34 of water inlet system 30 closes to stop the input of water.
In this example, the liquid consists essentially of a disinfecting liquid. Accordingly, approximately 128 ounces of a 14% aqueous solution of sodium hypochlorite is added to the four compartments 14 a-d through supply system 38. The aqueous system thereby created within compartments 14 a-d constitutes an oxidizing bleach solution of sodium hypochlorite in which the concentration of sodium hypochlorite in which the concentration of available chlorine is about 300 ppm. The compartments of machine 12 are rotated throughout this process at a rotational frequency of about 20-30 rpm so that agitation and mechanical contacting of the contents are continuous.
Upon completion of the cycle (about 15 minutes into the process), drainage valve 50 is actuated to drain the bulk solution from the compartments. Water inlet system 30 is again actuated to input tap water for a brief rinsing cycle, this time at approximately 140° F. to 160° F. but with all other parameters for the water remaining the same. This second water input or phase is continued until the water reaches a level of approximately 6 inches. Again, agitation is continuous. The cycle is continued for approximately 3 minutes, at the completion of which drainage valve 50 is actuated to drain the bulk rinse solution from the compartments.
EXAMPLE 2 PREDOMINANT TEMPERATURE
Water inlet 34 is actuated to input water into compartments 14 a-d. The water is tap water from a municipal water treatment source, as described in Example 1, above. The incoming water in this second example, however, has been heated to a temperature of approximately 200° F. The water level is the same as for Example 1. Approximately 16 ounces of detergent and non-ionic sequestrant (such as JS Concentrate, commercially-available from Industrial Blending of Queen Creek, Ariz.), 10 ounces of alkali (such as caustic soda), and 32 ounces of 14% aqueous sodium hypochlorite are added to compartments 14 a-d through supply system 38. The aqueous system thereby created within compartments 14 a-d constitutes a detergent and oxidizing bleach solution in which the concentration of the sodium hypochlorite is about 300 ppm. Mechanical action is employed as described in Example 1, above, as are the rinse cycle and drain procedures. The process is carried out for about 15 minutes.
EXAMPLE 3 PREDOMINATE TIME FACTOR
This example is carried out as described for Example 1, above, but with the following modifications. The first cycle in which the waste is treated with the detergent solution is carried out for about 20 minutes. Similarly, the second cycle in which the waste is treated with a bleaching solution is carried out for about 30 minutes.
EXAMPLE 4 TIME AND CHEMICAL ACTION COMBINED PREDOMINANT
In this example, a sodium hypochlorite solution of 0.1% is mixed in a large tank of water at ambient temperature. The infectious medical wastes are placed in a basket and submerged completely for a four hour period. The solution is reused until its disinfection properties decline to a point at which testing indicates that the surrogate pathogen test inactivation in the center of the medical waste load no longer meets regulatory requirements. Generally this is a function of volume of sodium hypochlorite solution in proportion to pounds of regulated medical waste processed.
Examples 1-4 illustrate the broad range of parameters that are permissible within the scope of the invention for the effective treatment of infectious medical waste. Each would be expected to yield satisfactory results within the scope of the invention. Examples 1-3 illustrate that the cleaning and disinfecting effectiveness of the processing of the regulated medical wastes is a function of time, temperature, chemical action, mechanical action, and dilution. Example 4 similarly illustrates the effects and interaction of time, temperature, chemical action and dilution. It is possible to achieve a certain level of microorganism inactivation by concurrently varying these five parameters. An increase in the intensity of one action will allow for a decrease in the intensity of one or more of the other actions. If time is increased, then chemical action may be decreased while still maintaining the same level of effectiveness. To illustrate further, Example 4 uses extremes in time and chemical action in order to eliminate the need for elevated temperature, mechanical action, and dilution. The interaction of the parameters is similar but different to their interaction in textile laundering. For example, the importance of mechanical action is significantly less with infectious medical waste processing than with textile laundering. The chemical action in medical waste processing also can be used to provide benefits which linger after the process has ended. The residual coating of sodium hypochlorite left on the medical waste materials after processing is complete continues to have disinfecting benefits. These examples or variants of the key parameters may be ideally suited for a particular infectious medical waste treatment application. In the most typical cases, however, the predominance of one or two factors, as was designed into Examples 1-4, would be non-ideal. For example, the cost of chemicals in Example 1 generally would be unnecessarily high. Similarly, the cost associated with creating the elevated temperature in Example 2 would be unduly high. The cost in terms of operating capacity reductions for Example 4 also would be unnecessarily high for most applications. Therefore, a balance is preferable.
The preferred method of the invention uses a two-stage approach. According to the preferred method, the infectious medical waste is first wetted with a first liquid to remove loose particles and water-soluble materials from the infectious medical waste. This wetting most advantageously involves immersing or saturating the infectious materials with the first liquid. In the preferred method, this is accomplished by washing the waste materials during an initial wash cycle of machine 12.
As described previously, infectious medical waste is placed in each of 12 mesh bags so that each mesh bag contains approximately 35 lbs. of the waste. Three of the mesh bags are placed in each of the four washer compartments 14 a-d in machine 12. Doors 28 are secured to enclose the compartments in liquid-type fashion.
Water inlet valve 34 is actuated to input tap water (as described in Example 1) into the compartments. The water has a temperature of about 140° F. to 170° F., preferably 170° F. with the other parameters for the water being as described in Example 1, above. When the water level reaches about 6 inches, the water input procedure is stopped.
The first liquid of this preferred method comprises an aqueous alkaline detergent solution. Accordingly, at this stage a detergent is added to each of the compartments.
The invention is well suited for use with a variety of alkaline detergent solutions. For example, suitable alkalies include caustic soda, soda ash, sodium orthosilicate, sodium metasilicate, and their equivalent potassium counterparts. Suitable detergent or surfactant components include commercially-available detergents, synthetic detergents, soaps, anionic and nonionic surfactants, the latter having sufficient ethoxylation to provide a cloud point of about 60° C. and 38° C. to 42° C. titre tallow soap, as surfactants; and sodium tripolyphosphate and tetrasodium pyrophosphate, or such chelating agents as disodium dihydride ethylenediamine tetraacetic acid as sequestrants. In most medical waste treatment applications, the alkaline detergent solution should have a detergent concentration sufficient to saponify or emulsify the range of fat-based soils found in infectious medical waste. Preferably, the detergent solution has a detergent concentration of about 500 parts per million (ppm) to 1,000 ppm. A suitable detergent solution may comprise sodium oxide with a concentration of at least about 250 ppm, and a surfactant with a concentration of at least about 250 ppm. The first liquid preferably has a pH of about 10.5 to 12 and an alkalinity of about 250 ppm to 1,000 ppm as sodium oxide.
The detergent composition for use in the preferred method comprises an aqueous solution of.three components. These components and their approximate proportions expressed in mass percent are as follows:
1. tetrasodium ethylenediamine 2.5% tetra acetate 2. tin opal CBS-X 0.4% 3. nonylphenoxy polyethoxy 25% ethanol (9-10 mole ethylene oxide)
The remainder consists essentially of water. The detergent component is made by di solving the tetrasodium ethylenediamine tetra acetate and the tin opal CBS-X in water at about 100° F., then adding the nonylphenoxy polyethoxy ethanol, and finally top off the solution to the desired mass with water. For example, to make a 100 pound batch of the detergent component, 25 pounds of tetrasodium ethylenediamine tetra acetate and 0.4 pounds of tin opal CBS-X are added to water at about 100° F. while mixing. After these ingredients have dissolved, 25 pounds of nonylphenoxy polyethoxy ethanol is added while mixing. When all components have dissolved, water is added to the solution until the total weight reaches 100 pounds.
In this preferred method, 50 fluid ounces of an alkaline detergent made in this fashion is added to compartments 14 a through liquid supply system 38. This yields a first liquid in the form of an aqueous alkaline detergent solution with a detergent concentration of about 0.04 to 0.06 mass percent (the remainder being water), a pH of about 10.5 to 11.0, an alkalinity of about 0.02 to 0.03 percent sodium oxide, and a temperature of slightly less than the water input temperature. The agitation means of machine 12 is operated continuously so that the contents of compartments 14 a-d are moderately agitated. Agitation is accomplished throughout the rotation of the horizontal washer cylinder. As the cylinder turns the materials are lifted up and out of the water. The rotation speed is controlled so that a portion of the load is lifted to a point near the top of the cylinder, then allowed to drop back into the cleaning (and/or disinfecting) solution. This lifting and dropping action forces washing solution into and through all areas of the medical waste being processed.
During this agitation procedure, the detergent solution removes loose particles and water soluble materials from the infectious medical waste. The action is one of sequestering metallic salts in water and waste, saponifying and emulsifying greases and oils, and suspending them so that they can be removed by rinsing.
Upon completion of the first cycle, which takes about 10.5 minutes to complete, drainage valve 50 opens to discharge the bulk alkaline detergent solution from compartments 14 a-d. When the flow rate of the alkaline detergent solution from the compartment and through drains 46 has dropped essentially to zero, drainage valve 50 is closed. At this stage, a certain amount of alkaline detergent solution remains in the medical waste within the compartments. For example, cellulosic materials typically retain moisture. The dry weight of the medical waste originally placed in the compartment may have been 400 pounds, whereas the weight of the medical waste in the compartment after drainage, and including the residual alkaline detergent solution remaining with the waste, may be over 1000 pounds.
Typically during this first alkaline detergent treatment step, approximately 90% of the pathogens are removed from the waste by detergency, by solubilizing water-soluble materials into the solution, and by physically carrying such particles and matter away from the waste materials. These removed pathogens are carried away with the bulk portion of the solution that is drained from the compartment.
The preferred method further includes a second step of wetting the infectious medical waste with a disinfecting liquid to disinfect the infectious medical waste. This second step preferably, but not necessarily, is carried out upon completion of the first step as described above. In this second step, the second input means inputs the liquid disinfectant as a bleaching agent solution, and preferably an aqueous bleaching agent solution. The substantial removal of soils in the first step and the continual break up and dissolution of the fats and oils in the second step wash water prevents in the main the chlorine from creating chlorinated organics as the sodium hypochlorite enters the process in the second step.
A number of disinfecting liquids may be employed in the practice of the invention. For example, the disinfecting liquid may comprise bleaching agents, alcohols (e.g., ethanol), peroxides, and persulfates such as potassium persulfates.
Bleaching agents often are segregated into two classes—oxidizing bleaching agents and reducing bleaching agents—and both are amenable to use in practicing the invention. Examples of suitable oxidizing bleaching agents include sodium hypochlorite and hydrogen peroxide. The oxidizing bleaching agent may be used alone or, more preferably, may be used in solution. Solvents useful in such bleaching agent solutions include water. As a general rule of thumb, the bleach concentration in the solution preferably ranges from 100 to 500 ppm on a solution basis. The disinfecting liquid preferably will have a pH of about 8 to 10.
In the preferred method of the invention, the liquid disinfectant comprises an aqueous solution of sodium hypochlorite. Superior results have been obtained where the sodium hypochlorite that is added to the water is a 12% to 14% by mass aqueous solution. Approximately 64 fl. oz. of the 12% to 14% sodium hypochlorite is added to compartments 14 a-d through liquid supply system 38 to yield an available concentration in the compartments of about 340-350 ppm. The resulting aqueous sodium hypochlorite mixture has a pH of about 8-10, and a temperature in the range of 155 to 165° F. All throughout this second cycle, which lasts for about 10.5 minutes, the machine agitates to impart mechanical action to the materials in the compartment, as described above. Upon the completion of the second cycle, drainage valve 50 opens to drain the aqueous sodium hypochlorite solution from the compartments.
After the bulk liquid has been drained from the compartments, doors 28 are opened and the mesh bags are removed from the compartments by gravity as machine 12 raises and turns its opened doorways down towards a large container. The mesh bags and their contents are raised by an overhead hoist (not shown) and placed in an extractor (also not shown), and an extraction process carried out to substantially reduce the moisture content in the waste materials. As an example, the mesh bags might be centrifuged in a commercially-available extractor such as an Ellis Economizer centrifugal extractor, marketed by Ellis Corporation, Itasca, Ill. The extractor would operate at about 750 revolutions per minute (rpm) for about 5 minutes, which would be expected to reduce the liquid content of the waste to a water retention by weight of about 35% of the dry waste material weight.
After the extraction process, the mesh bags may be opened and their contents removed. The mesh bags have been cleaned and disinfected in the process, and are therefore ready for additional rounds of use and reuse. Having thus been disinfected, the waste materials removed from the mesh bags, which at the beginning of the treatment process constituted infectious medical waste, now constitute sanitized, unregulated waste that may be disposed of in a sanitary land fill. This treatment process yields waste that does not have objectionable physical appearance.
The presently preferred apparatus and method of the invention involve batch-type processing in which the infectious medical waste is treated one batch, or one group of batches, at a time. It should be appreciated, however, that the invention is quite well suited to other types of operations. For example, the invention could be advantageously employed using continuous or semi-continuous treatment operations. As an example of a semi-continuous operation, a machine such as a tunnel washing machine could be used to treat multiple batches of infectious medical waste. Operations based on washer-extractor machine designs also are useable to practice the invention. Continuous flow systems that use tunnel conveyors and the like also could be useful for the practice of the invention. Batch-type operations involving immersion or immersion baths, such as that described in Example 4 above, also are amenable.
Having now described the invention and its preferred embodiments and methods, additional advantages and modifications will readily occur to those skilled in the art. For example, the number of cycles is not necessarily limiting. Greater numbers of cycles than those disclosed above certainly could be used. The duration of treatment also is not necessarily limiting. Depending upon the circumstances, it may be advantageous to treat a given batch of mechanical waste, e.g., for one, two, four, 24 hours, or even longer. Therefore, the invention in its broader aspects is not limited to the specific details, representative devices, and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.
What is claimed is:
1. A method of batch treating infectious medical waste, said method comprising the steps of: placing infectious medical waste into a container having a wall member forming a concavity and having a perimeter, the wall member being permeable to water, a protective surface coupled to the wall member perimeter for protecting workers from potentially injurious contact with medical wastes when medical wastes are in the container, and an access for selectively opening and closing the container; and washing the container and infectious medical waste with agitation to convert the infectious medical waste into noninfectious medical waste.
2. The method according to claim 1 wherein the step of placing infectious medical waste into the container includes placing prepackaged receptacles of infectious medical waste into the container.
3. The method according to claim 1 wherein the step of placing infectious medical waste into the container includes enclosing the infectious medical waste within the container through use of a zipper.
4. The method according to claim 1 wherein the step of washing the container includes washing the container in a substantially liquid tight volume.
5. The method according to claim 1, wherein the wall member comprises a mesh having a mesh size permeable to water yet sufficiently small to retain particles of the infectious medical waste.
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UDP Broadcast to All IPs not working in some networks
There is a lot of other answers related to this issue, however I believe this is specific.
I am using Delphi XE2 and Indy 10.5.8 and TIdUDPServer
In my local development network I have everything on the same network ip subrange and all connected to the very same Access Point (LinkSys)
I have Androids sendind UDP Broadcast to <IP_ADDRESS> to request the server ip address that is written in Delphi listening using TIdUDPServer on the port 44444.
The requests get there fine and I can answer back no problem. Works exactly as expected.
However I have noted that in some networks it does not work! It is always simple networks based on an access point, I am not sure but seems that where the problem happens the server PC is connect to the LAN port while the devices are using the wifi, all in the same access point.
Could be the case that the access points do not broadcast the UDP packet by the both LAN and wifi? I know that this kind of broadcast is very limited, but I have not found any information that tell me that in the same access point there is limitations like that.
Is there are ways to test, or workaround?
This solution needs to be strong enough to deal with the many AP out there.
EDIT: For those that want to get the source code for retrieving more information from the network including the broadcast ip as mentioned on the answer below follow this solution, it is all there.
http://www.code10.info/index.php?option=com_content&view=article&id=54:articleretrieve-network-adapter-information&catid=47:cat_coding_algorithms_network&Itemid=78
<IP_ADDRESS> is not the best option for sending UDP broadcasts, and some routers/firewalls do block it unless configured otherwise. The better option is to use the NIC's actual subnet broadcast IP instead. For example, if a UDP socket is bound to local IP <IP_ADDRESS> with a subnet mask of <IP_ADDRESS>, then the broadcast IP for that subnet is <IP_ADDRESS>.
Most platforms have OS-specific APIs for retrieving a NIC's actual broadcast IP, such as getifaddrs() on POSIX systems, or at least for retrieving the NIC's subnet mask, such as GetAdaptersInfo() and GetAdaptersAddresses() on Windows, so you can calculate the broadcast IP manually.
Retrieving the local broadcast IP(s) may be added to Indy in a future version.
Thanks, I am new on these things, One of the reasons of using this broadcast is because my server software is running in a PC that has IP dynamically assigned, and should be like that to be simple for the customer. You mean that the broadcast IP is going to change always for that subnet according to the server binding IP? And sending UDP message to this broadcast IP is like the same sending to all the ips in that subnet?
It doesn't matter if the locally bound IP is dynamic or not. You can still retrieve the current subnet info for the NIC that the IP is assigned to and determine the current broadcast IP (it is very simple to calculate manually if needed). Even though the bound IP may change dynamically, the IP range and mask for the subnet is not likely to change very often, since they are part of the router's subnet configuration that would affect all clients on that subnet. Yes, sending a packet to the broadcast IP delivers the packet to all IPs within the subnet (hence why it is a broadcast IP).
Fantastic!, I have tested and works very nicely, do you mind pointing to a solution in delphi to make the calculation if it is at hand? I have changed my android app to send to <IP_ADDRESS> (for my test case) and my server received the UDP broadcast and could answer. That will is helping a lot you cant imagine.
Let me answer my question with your answer: http://embarcadero.newsgroups.archived.at/public.delphi.internet.winsock/201002/1002273322.html if you have any other link to a ready solution I appreciate. Thanks
Here is a link to a complete solution in delphi with all the network information needed: http://www.code10.info/index.php?option=com_content&view=article&id=54:articleretrieve-network-adapter-information&catid=47:cat_coding_algorithms_network&Itemid=78
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Thread:YingHe/@comment-33896974-20190910053923/@comment-44010099-20191022020711
And the coin just came back without anyone flinging it back.Weird.
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Talk:Gummierung
philately?
Why this label? It's by far not restricted to stamps. Fytcha (talk) 17:35, 23 November 2021 (UTC)
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<?php
namespace AuraIsHere\LaravelMultiTenant\Traits;
use AuraIsHere\LaravelMultiTenant\Contracts\LoftyScope;
use AuraIsHere\LaravelMultiTenant\Exceptions\TenantModelNotFoundException;
use AuraIsHere\LaravelMultiTenant\TenantQueryBuilder;
use AuraIsHere\LaravelMultiTenant\TenantScope;
use Illuminate\Database\Eloquent\Model;
use Illuminate\Database\Eloquent\ModelNotFoundException;
use Illuminate\Database\Eloquent\SoftDeletingScope;
use Illuminate\Support\Facades\App;
use Illuminate\Support\Facades\Config;
/**
* Class TenantScopedModelTrait.
*
*
* @method static void addGlobalScope(\Illuminate\Database\Eloquent\ScopeInterface $scope)
* @method static void creating(callable $callback)
*/
trait TenantScopedModelTrait
{
public static function bootTenantScopedModelTrait()
{
$tenantScope = App::make('AuraIsHere\LaravelMultiTenant\TenantScope');
// Add the global scope that will handle all operations except create()
static::addGlobalScope($tenantScope);
// Add an observer that will automatically add the tenant id when create()-ing
static::creating(function (Model $model) use ($tenantScope) {
$tenantScope->creating($model);
});
}
/**
* Returns a new builder without the tenant scope applied.
*
* $allUsers = User::allTenants()->get();
*
* @return \Illuminate\Database\Eloquent\Builder
*/
public static function allTenants()
{
return with(new static())->newOriginalQueryWithoutScope(new TenantScope());
}
/**
* Get the name of the "tenant id" column.
*
* @return string
*/
public function getTenantColumns()
{
return isset($this->tenantColumns) ? $this->tenantColumns : Config::get('tenant.default_tenant_columns');
}
/**
* Prepare a raw where clause. Do it this way instead of using where()
* to avoid issues with bindings when removing.
*
* @param $tenantColumn
* @param $tenantId
*
* @return string
*/
public function getTenantWhereClause($tenantColumn, $tenantId)
{
return "{$this->getTable()}.{$tenantColumn} = '{$tenantId}'";
}
/**
* Override the default findOrFail method so that we can rethrow a more useful exception.
* Otherwise it can be very confusing why queries don't work because of tenant scoping issues.
*
* @param $id
* @param array $columns
*
* @throws TenantModelNotFoundException
*/
public static function findOrFail($id, $columns = ['*'])
{
try {
return parent::query()->findOrFail($id, $columns);
} catch (ModelNotFoundException $e) {
throw with(new TenantModelNotFoundException())->setModel(get_called_class());
}
}
/**
* Get a new query builder with nested where for the model's table.
*
* @return AuraIsHere\LaravelMultiTenant\TenantQueryBuilder
*/
public function newQuery()
{
$tenant_builder = $this->newTenantQueryWithoutScopes();
//Create a normal query first, allowing the (interfaced)
// scope to use the whereRaw from the non-overridden
// Eloquent\Query
$tenant_builder->setQuery($this->newOriginalQuery()->getQuery());
return $tenant_builder;
}
/**
* Get a new query builder for the model's table.
* without the nesting behaviour.
*
* @return \Illuminate\Database\Eloquent\Builder
*/
public function newOriginalQuery()
{
$builder = $this->newQueryWithoutScopes();
return $this->applyGlobalScopes($builder);
}
/**
* Get a new query instance without a given scope.
* and without nesting behaviour.
*
* @param \Illuminate\Database\Eloquent\ScopeInterface $scope
*
* @return \Illuminate\Database\Eloquent\Builder
*/
public function newOriginalQueryWithoutScope($scope)
{
$this->getGlobalScope($scope)->remove($builder = $this->newOriginalQuery(), $this);
return $builder;
}
/**
* Get a new query builder with nested where
* without global scopes.
*
* @return AuraIsHere\LaravelMultiTenant\TenantQueryBuilder|static
*/
public function newTenantQueryWithoutScopes()
{
$builder = $this->newEloquentTenantBuilder(
$this->newBaseQueryBuilder()
);
// Once we have the query builders, we will set the model instances so the
// builder can easily access any information it may need from the model
// while it is constructing and executing various queries against it.
return $builder->setModel($this)->with($this->with);
}
/**
* Create a new Eloquent Tenant query builder for the model.
*
* @param \Illuminate\Database\Query\Builder $query
*
* @return AuraIsHere\LaravelMultiTenant\TenantQueryBuilder|static
*/
public function newEloquentTenantBuilder($query)
{
return new TenantQueryBuilder($query);
}
/**
* Apply all of the global scopes to an Eloquent builder
* or it's nested.
*
* @param \Illuminate\Database\Eloquent\Builder $builder
*
* @return \Illuminate\Database\Eloquent\Builder
*/
public function applyGlobalScopes($builder)
{
foreach ($this->getGlobalScopes() as $scope) {
if ($scope instanceof LoftyScope || $scope instanceof SoftDeletingScope) {
$scope->apply($builder, $this);
} else {
$nestable = $this->newQueryWithoutScopes();
$scope->apply($nestable, $this);
$builder->addNestedWhereQuery($nestable->getQuery());
}
}
return $builder;
}
/**
* Handle dynamic method calls into the model.
*
* @param string $method
* @param array $parameters
*
* @return mixed
*/
public function __call($method, $parameters)
{
if (in_array($method, ['increment', 'decrement'])) {
return call_user_func_array([$this, $method], $parameters);
}
$query = $this->newQuery();
return call_user_func_array([$query, $method], $parameters);
}
}
|
Q: Test for natural language inference.
Premise: "A man dressed in brown holding a light saber."
Hypothesis: "A man dressed as a jedi holding a light saber."
Is the hypothesis entailed by the premise?
Options:
- yes
- no
- it is not possible to tell
A: Dressed in brown holding a light saber is the means to be dressed as a Jedi.
The answer is yes.
QUESTION: Premise: "A person climbing a rock while others are standing around and watching."
Hypothesis: "A person is breaking a rock climbing speed record while his friends cheer him on."
Is the hypothesis entailed by the premise?
Options:
- yes
- it is not possible to tell
- no
Let's solve it slowly: The rock climbing speed record is not mentioned in the first sentence and the others are not described so you wouldnt know that they are his friends.
The answer is it is not possible to tell.
[QUESTION] Given the sentence "Person riding a racing motorcycle while other people watch." is it true that "People are watching a motorcyclist."?
A person riding a racing motorcycle is a kind of motorcyclist.
The answer is yes.
Q: Can we conclude from "Two woman with black purses are looking at the back of a camera." that "Two people are about to take a picture."?
Options:
- yes
- no
- it is not possible to tell
A: Two woman with black purses are looking at the back of a camera does not necessary that they are about to take a picture.
The answer is it is not possible to tell.
QUESTION: Can we conclude from "Little girl in bare feet sitting in a circle." that "A girl is waiting for her mother to pick her up."?
Options:
- yes
- no
- it is not possible to tell
Let's solve it slowly: A girl sitting in a circle does not imply the girl is waiting for her mother to pick her up.
The answer is it is not possible to tell.
QUESTION: Test for natural language inference.
Premise: "A guy works on a building."
Hypothesis: "The worker works on the building."
Is the hypothesis entailed by the premise?
Options:
- yes
- no
- it is not possible to tell
Let's solve it slowly:
A guy is a worker who is working on a building.
The answer is yes.
|
Category:Northwest Territories/Wikipedia
category=Published category=Canada notcategory=Disputed count=5 addfirstcategorydate=true namespace=main
Note. This page is used by the Wikinews Importer Bot to update the Wikipedia Northwest Territories Portal news.
|
Frank Roberts (Australian footballer)
Frank George Roberts (27 February 1912 – 26 June 1989) was an Australian rules footballer who played for St Kilda and Melbourne in the Victorian Football League (VFL).
While at St Kilda Roberts played as a forward, kicking 24 goals in his debut season and another 20 the following year. He spent 1935 playing back home at Sandhurst and in 1936 returned to the league with Melbourne. This time he was used as a defender and was on the half back flank in Melbourne's 1939 premiership team. He played at fullback on Richmond star Jack Titus in the 1940 Grand Final and again finished on the winning side.
|
30 THE EASTERN HUNTEKS.
some other sense, for which we have no name, con veyed to his practised intelligence as frequently appears to be the case an impression that the tiger was there, I cannot say ; but he was evidently not satisfied, and ordered the patch to be beaten through more closely.
Violent gesticulations, and only partially sup pressed howls of excitement from one of the look outs in the trees, announced that Kugonauth's acuteness had not deceived him. Though the man elongated his skinny arm and finger to the utmost, and pointed to a certain part of the cover, the game remained invisible to the hunters ; and it shortly appeared to have passed from the fellow's observa tion, as he ceased his movements, and contented himself with peering through the leaves of the tree in which he was posted. In about half a minute, however, Norman's quick eye lighted for a single second on a rufous coloured mass, stealthily sneaking along through the jungle ; but almost as soon as seen, it disappeared. Another brief space, and again he caught a glimpse of it right in front of him, and distant about a hundred yards. It stopped for a moment to listen, but its meditations were quickly brought to an end by the crack of Norman's rifle. It was a rapid snap shot, but the bullet if ineffectual must have whizzed pretty close, for, with
|
/*******************************************************************************
* Copyright (c) 2017 by JoyLau. All rights reserved
******************************************************************************/
package cn.joylau.echarts;
import cn.joylau.echarts.code.DataZoomType;
import cn.joylau.echarts.code.FilterMode;
import cn.joylau.echarts.code.Orient;
import cn.joylau.echarts.style.TextStyle;
import java.util.Date;
/**
* 数据区域缩放。与toolbox.feature.dataZoom同步，仅对直角坐标系图表有效
*
* @author JoyLau
*/
public class DataZoom extends Basic<DataZoom> implements Component {
/**
* 类型
*/
private DataZoomType type;
/**
* 显示label的小数精度。默认根据数据自动决定
*/
private String labelPrecision;
/**
* 显示的label的格式化器
*/
private String labelFormatter;
/**
* 是否在 dataZoom-silder 组件中显示数据阴影。数据阴影可以简单得反应数据走势
*/
private String showDataShadow;
/**
* 文字样式
*/
private TextStyle textStyle;
/**
* 数据窗口范围的起始数值。如果设置了 dataZoom-slider.start 则 startValue 失效
*/
private Object startValue;
/**
* 数据窗口范围的结束数值。如果设置了 dataZoom-slider.end 则 endValue 失效
*/
private Object endValue;
/**
* 设置 dataZoom-inside 组件控制的 角度轴
*/
private Object angleAxisIndex;
/**
* 设置 dataZoom-inside 组件控制的 半径轴
*/
private Object radiusAxisIndex;
/**
* dataZoom 的运行原理是通过 数据过滤 来达到 数据窗口缩放 的效果
*/
private FilterMode filterMode;
/**
* 设置触发视图刷新的频率。单位为毫秒（ms）。一般不需要更改这个值
*/
private Integer throttle;
/**
* 布局方式，默认为水平布局，可选为：'horizontal' | 'vertical'
*/
private Orient orient;
/**
* 默认#ccc，数据缩略背景颜色
*/
private String dataBackgroundColor;
/**
* 默认值rgba(144,197,237,0.2)，选择区域填充颜色
*/
private String fillerColor;
/**
* 默认值rgba(70,130,180,0.8)，控制手柄颜色
*/
private String handleColor;
/**
* 控制手柄大小
*/
private Integer handleSize;
/**
* 当不指定时默认控制所有横向类目，可通过数组指定多个需要控制的横向类目坐标轴Index，仅一个时可直接为数字
*/
private Object xAxisIndex;
/**
* 当不指定时默认控制所有纵向类目，可通过数组指定多个需要控制的纵向类目坐标轴Index，仅一个时可直接为数字
*/
private Object yAxisIndex;
/**
* 数据缩放，选择起始比例，默认为0（%），从首个数据起选择
*/
private Integer start;
/**
* 数据缩放，选择结束比例，默认为100（%），到最后一个数据选择结束
*/
private Integer end;
/**
* 缩放变化是否实时显示，建议性能较低的浏览器或数据量巨大时不启动实时效果
*/
private Boolean realtime;
/**
* 数据缩放锁，默认为false，当设置为true时选择区域不能伸缩，即(end - start)值保持不变，仅能做数据漫游
*/
private Boolean zoomLock;
/**
* 缩放变化是否显示定位详情
*/
private Boolean showDetail;
public String labelPrecision() {
return this.labelPrecision;
}
public DataZoom labelPrecision(String labelPrecision) {
this.labelPrecision = labelPrecision;
return this;
}
public String labelFormatter() {
return this.labelFormatter;
}
public DataZoom labelFormatter(String labelFormatter) {
this.labelFormatter = labelFormatter;
return this;
}
public String showDataShadow() {
return this.showDataShadow;
}
public DataZoom showDataShadow(String showDataShadow) {
this.showDataShadow = showDataShadow;
return this;
}
public TextStyle textStyle() {
if (this.textStyle == null) {
this.textStyle = new TextStyle();
}
return this.textStyle;
}
public DataZoom textStyle(TextStyle textStyle) {
this.textStyle = textStyle;
return this;
}
public Object startValue() {
return this.startValue;
}
public DataZoom startValue(Object startValue) {
this.startValue = startValue;
return this;
}
public DataZoom startValue(String startValue) {
this.startValue = startValue;
return this;
}
public DataZoom startValue(Integer startValue) {
this.startValue = startValue;
return this;
}
public DataZoom startValue(Date startValue) {
this.startValue = startValue;
return this;
}
public Object endValue() {
return this.endValue;
}
public DataZoom endValue(Object endValue) {
this.endValue = endValue;
return this;
}
public DataZoom endValue(Integer endValue) {
this.endValue = endValue;
return this;
}
public DataZoom endValue(String endValue) {
this.endValue = endValue;
return this;
}
public DataZoom endValue(Date endValue) {
this.endValue = endValue;
return this;
}
public String getLabelPrecision() {
return labelPrecision;
}
public void setLabelPrecision(String labelPrecision) {
this.labelPrecision = labelPrecision;
}
public String getLabelFormatter() {
return labelFormatter;
}
public void setLabelFormatter(String labelFormatter) {
this.labelFormatter = labelFormatter;
}
public String getShowDataShadow() {
return showDataShadow;
}
public void setShowDataShadow(String showDataShadow) {
this.showDataShadow = showDataShadow;
}
public TextStyle getTextStyle() {
return textStyle;
}
public void setTextStyle(TextStyle textStyle) {
this.textStyle = textStyle;
}
public Object getStartValue() {
return startValue;
}
public void setStartValue(Object startValue) {
this.startValue = startValue;
}
public Object getEndValue() {
return endValue;
}
public void setEndValue(Object endValue) {
this.endValue = endValue;
}
public DataZoomType type() {
return this.type;
}
public DataZoom type(DataZoomType type) {
this.type = type;
return this;
}
public Object angleAxisIndex() {
return this.angleAxisIndex;
}
public DataZoom angleAxisIndex(Object angleAxisIndex) {
this.angleAxisIndex = angleAxisIndex;
return this;
}
public DataZoom angleAxisIndex(Integer angleAxisIndex) {
this.angleAxisIndex = angleAxisIndex;
return this;
}
public DataZoom angleAxisIndex(Integer... angleAxisIndex) {
this.angleAxisIndex = angleAxisIndex;
return this;
}
public Object radiusAxisIndex() {
return this.radiusAxisIndex;
}
public DataZoom radiusAxisIndex(Object radiusAxisIndex) {
this.radiusAxisIndex = radiusAxisIndex;
return this;
}
public DataZoom radiusAxisIndex(Integer radiusAxisIndex) {
this.radiusAxisIndex = radiusAxisIndex;
return this;
}
public DataZoom radiusAxisIndex(Integer... radiusAxisIndex) {
this.radiusAxisIndex = radiusAxisIndex;
return this;
}
public FilterMode filterMode() {
return this.filterMode;
}
public DataZoom filterMode(FilterMode filterMode) {
this.filterMode = filterMode;
return this;
}
public Integer throttle() {
return this.throttle;
}
public DataZoom throttle(Integer throttle) {
this.throttle = throttle;
return this;
}
public DataZoomType getType() {
return type;
}
public void setType(DataZoomType type) {
this.type = type;
}
public Object getAngleAxisIndex() {
return angleAxisIndex;
}
public void setAngleAxisIndex(Object angleAxisIndex) {
this.angleAxisIndex = angleAxisIndex;
}
public Object getRadiusAxisIndex() {
return radiusAxisIndex;
}
public void setRadiusAxisIndex(Object radiusAxisIndex) {
this.radiusAxisIndex = radiusAxisIndex;
}
public FilterMode getFilterMode() {
return filterMode;
}
public void setFilterMode(FilterMode filterMode) {
this.filterMode = filterMode;
}
public Integer getThrottle() {
return throttle;
}
public void setThrottle(Integer throttle) {
this.throttle = throttle;
}
/**
* 获取handleSize值
*/
public Integer handleSize() {
return this.handleSize;
}
/**
* 设置handleSize值
*
* @param handleSize
*/
public DataZoom handleSize(Integer handleSize) {
this.handleSize = handleSize;
return this;
}
/**
* 获取orient值
*/
public Orient orient() {
return this.orient;
}
/**
* 设置orient值
*
* @param orient
*/
public DataZoom orient(Orient orient) {
this.orient = orient;
return this;
}
/**
* 获取dataBackgroundColor值
*/
public String dataBackgroundColor() {
return this.dataBackgroundColor;
}
/**
* 设置dataBackgroundColor值
*
* @param dataBackgroundColor
*/
public DataZoom dataBackgroundColor(String dataBackgroundColor) {
this.dataBackgroundColor = dataBackgroundColor;
return this;
}
/**
* 获取fillerColor值
*/
public String fillerColor() {
return this.fillerColor;
}
/**
* 设置fillerColor值
*
* @param fillerColor
*/
public DataZoom fillerColor(String fillerColor) {
this.fillerColor = fillerColor;
return this;
}
/**
* 获取handleColor值
*/
public String handleColor() {
return this.handleColor;
}
/**
* 设置handleColor值
*
* @param handleColor
*/
public DataZoom handleColor(String handleColor) {
this.handleColor = handleColor;
return this;
}
/**
* 获取xAxisIndex值
*/
public Object xAxisIndex() {
return this.xAxisIndex;
}
/**
* 设置xAxisIndex值
*
* @param xAxisIndex
*/
public DataZoom xAxisIndex(Object xAxisIndex) {
this.xAxisIndex = xAxisIndex;
return this;
}
/**
* 获取yAxisIndex值
*/
public Object yAxisIndex() {
return this.yAxisIndex;
}
/**
* 设置yAxisIndex值
*
* @param yAxisIndex
*/
public DataZoom yAxisIndex(Object yAxisIndex) {
this.yAxisIndex = yAxisIndex;
return this;
}
/**
* 获取start值
*/
public Integer start() {
return this.start;
}
/**
* 设置start值
*
* @param start
*/
public DataZoom start(Integer start) {
this.start = start;
return this;
}
/**
* 获取end值
*/
public Integer end() {
return this.end;
}
/**
* 设置end值
*
* @param end
*/
public DataZoom end(Integer end) {
this.end = end;
return this;
}
/**
* 获取realtime值
*/
public Boolean realtime() {
return this.realtime;
}
/**
* 设置realtime值
*
* @param realtime
*/
public DataZoom realtime(Boolean realtime) {
this.realtime = realtime;
return this;
}
/**
* 获取zoomLock值
*/
public Boolean zoomLock() {
return this.zoomLock;
}
/**
* 设置zoomLock值
*
* @param zoomLock
*/
public DataZoom zoomLock(Boolean zoomLock) {
this.zoomLock = zoomLock;
return this;
}
/**
* 获取showDetail值
*/
public Boolean showDetail() {
return this.showDetail;
}
/**
* 设置showDetail值
*
* @param showDetail
*/
public DataZoom showDetail(Boolean showDetail) {
this.showDetail = showDetail;
return this;
}
/**
* 获取orient值
*/
public Orient getOrient() {
return orient;
}
/**
* 设置orient值
*
* @param orient
*/
public void setOrient(Orient orient) {
this.orient = orient;
}
/**
* 获取dataBackgroundColor值
*/
public String getDataBackgroundColor() {
return dataBackgroundColor;
}
/**
* 设置dataBackgroundColor值
*
* @param dataBackgroundColor
*/
public void setDataBackgroundColor(String dataBackgroundColor) {
this.dataBackgroundColor = dataBackgroundColor;
}
/**
* 获取fillerColor值
*/
public String getFillerColor() {
return fillerColor;
}
/**
* 设置fillerColor值
*
* @param fillerColor
*/
public void setFillerColor(String fillerColor) {
this.fillerColor = fillerColor;
}
/**
* 获取handleColor值
*/
public String getHandleColor() {
return handleColor;
}
/**
* 设置handleColor值
*
* @param handleColor
*/
public void setHandleColor(String handleColor) {
this.handleColor = handleColor;
}
/**
* 获取xAxisIndex值
*/
public Object getxAxisIndex() {
return xAxisIndex;
}
/**
* 设置xAxisIndex值
*
* @param xAxisIndex
*/
public void setxAxisIndex(Object xAxisIndex) {
this.xAxisIndex = xAxisIndex;
}
/**
* 获取yAxisIndex值
*/
public Object getyAxisIndex() {
return yAxisIndex;
}
/**
* 设置yAxisIndex值
*
* @param yAxisIndex
*/
public void setyAxisIndex(Object yAxisIndex) {
this.yAxisIndex = yAxisIndex;
}
/**
* 获取start值
*/
public Integer getStart() {
return start;
}
/**
* 设置start值
*
* @param start
*/
public void setStart(Integer start) {
this.start = start;
}
/**
* 获取end值
*/
public Integer getEnd() {
return end;
}
/**
* 设置end值
*
* @param end
*/
public void setEnd(Integer end) {
this.end = end;
}
/**
* 获取realtime值
*/
public Boolean getRealtime() {
return realtime;
}
/**
* 设置realtime值
*
* @param realtime
*/
public void setRealtime(Boolean realtime) {
this.realtime = realtime;
}
/**
* 获取zoomLock值
*/
public Boolean getZoomLock() {
return zoomLock;
}
/**
* 设置zoomLock值
*
* @param zoomLock
*/
public void setZoomLock(Boolean zoomLock) {
this.zoomLock = zoomLock;
}
/**
* 获取showDetail值
*/
public Boolean getShowDetail() {
return showDetail;
}
/**
* 设置showDetail值
*
* @param showDetail
*/
public void setShowDetail(Boolean showDetail) {
this.showDetail = showDetail;
}
/**
* 获取handleSize值
*/
public Integer getHandleSize() {
return handleSize;
}
/**
* 设置handleSize值
*
* @param handleSize
*/
public void setHandleSize(Integer handleSize) {
this.handleSize = handleSize;
}
}
|
using System;
using System.Collections.Generic;
using System.Security.Cryptography;
namespace Pulsar4X.ECSLib
{
/// <summary>
/// most of this is adapted from: https://crackstation.net/hashing-security.htm#aspsourcecode
/// https://github.com/defuse/password-hashing
/// Original Author: Taylor Hornby "defuse"
/// BSD 2-clause "Simplified" License
/// </summary>
public static class AuthProcessor
{
// The following constants may be changed without breaking existing hashes.
public const int SALT_BYTE_SIZE = 24;
public const int HASH_BYTE_SIZE = 24;
public const int PBKDF2_ITERATIONS = 500;
public const int ITERATION_INDEX = 0;
public const int SALT_INDEX = 1;
public const int PBKDF2_INDEX = 2;
/// <summary>
/// Initializes this Processor.
/// </summary>
internal static void Initialize()
{
}
/// <summary>
/// Validates a password given a hash of the correct one.
/// </summary>
/// <param name="factionEntity">the factionEntity</param>
/// <param name="password">The password to check.</param>
public static bool Validate(Entity factionEntity, string password)
{
AuthDB authDB = factionEntity.GetDataBlob<AuthDB>();
// Extract the parameters from the hash
char[] delimiter = { ':' };
string[] split = authDB.Hash.Split(delimiter);
int iterations = Int32.Parse(split[ITERATION_INDEX]);
byte[] salt = Convert.FromBase64String(split[SALT_INDEX]);
byte[] hash = Convert.FromBase64String(split[PBKDF2_INDEX]);
byte[] testHash = PBKDF2(password, salt, iterations, hash.Length);
return SlowEquals(hash, testHash);
}
/// <summary>
/// Stores a password as a salted hash in a factions AuthDB.
/// </summary>
/// <param name="game"></param>
/// <param name="factionEntity"></param>
/// <param name="password"></param>
public static void StorePasswordAsHash(Game game, Entity factionEntity, string password)
{
//Entity factionEntity = game.GlobalManager.GetEntityByGuid(factionGuid);
AuthDB authDB;
if (factionEntity.HasDataBlob<AuthDB>())
authDB = factionEntity.GetDataBlob<AuthDB>();
else
{
authDB = new AuthDB();
factionEntity.SetDataBlob<AuthDB>(authDB);
}
authDB.Hash = CreateHash(password);
}
/// <summary>
/// Creates a salted PBKDF2 hash of the password.
/// </summary>
/// <param name="password">The password to hash.</param>
/// <returns>The hash of the password.</returns>
public static string CreateHash(string password)
{
// Generate a random salt
RNGCryptoServiceProvider csprng = new RNGCryptoServiceProvider();
byte[] salt = new byte[SALT_BYTE_SIZE];
csprng.GetBytes(salt);
// Hash the password and encode the parameters
byte[] hash = PBKDF2(password, salt, PBKDF2_ITERATIONS, HASH_BYTE_SIZE);
return PBKDF2_ITERATIONS + ":" +
Convert.ToBase64String(salt) + ":" +
Convert.ToBase64String(hash);
}
/// <summary>
/// Computes the PBKDF2-SHA1 hash of a password.
/// </summary>
/// <param name="password">The password to hash.</param>
/// <param name="salt">The salt.</param>
/// <param name="iterations">The PBKDF2 iteration count.</param>
/// <param name="outputBytes">The length of the hash to generate, in bytes.</param>
/// <returns>A hash of the password.</returns>
private static byte[] PBKDF2(string password, byte[] salt, int iterations, int outputBytes)
{
Rfc2898DeriveBytes pbkdf2 = new Rfc2898DeriveBytes(password, salt);
pbkdf2.IterationCount = iterations;
return pbkdf2.GetBytes(outputBytes);
}
/// <summary>
/// Compares two byte arrays in length-constant time. This comparison
/// method is used so that password hashes cannot be extracted from
/// on-line systems using a timing attack and then attacked off-line.
/// </summary>
/// <param name="a">The first byte array.</param>
/// <param name="b">The second byte array.</param>
/// <returns>True if both byte arrays are equal. False otherwise.</returns>
private static bool SlowEquals(byte[] a, byte[] b)
{
uint diff = (uint)a.Length ^ (uint)b.Length;
for (int i = 0; i < a.Length && i < b.Length; i++)
diff |= (uint)(a[i] ^ b[i]);
return diff == 0;
}
}
}
|
Giuseppe Riva
Giuseppe Riva (4 November 1834 in Ivrea – 10 November 1916 ) was an Italian lawyer and painter, known for portraits, historical paintings, and landscapes.
He was a resident of Milan. One of his masterworks was While reading a memorial plaque for the Calvinist Covenanters, Catherine de' Medici surprised by Mary Stuart, sent to the Exhibition of Fine Arts at Turin in 1883. To the same exhibition in 1887, he sent Ultimi istanti di Cola di Rienzi, last Tribune of Rome. In 1872 at the Milan Exposition, he displayed, several half-figure portraits. At the 1883 Rome Exposition, he displayed Two Shepherdesses, an Idyll. Another painter of the same name was active in Bergamo and Rome (1861–1941).
|
LexV2BotLocaleIds = "ko_KR" don't work
Describe the bug
When I set the LexV2BotLocaleIds variable to "ko_KR", stack deployment fail.
Status Reason : Received response status [FAILED] from custom resource : Message returned : "ko_KR"
To Reproduce
Set LexV2BotLocaleIds variable to value including "ko_KR" like "en_US,es_US,fr_CA,ko_KR".
Deploy qnabot stack.
Expected behavior
Successful deployment.
Please complete the following information about the solution:
[ ] Version: 5.1.0 ( (SO0189) QnABot with admin and client websites - (Master v5.1.0) )
To get the version of the solution, you can look at the description of the created CloudFormation stack. For example, "(SO0189) QnABot [...] v0.0.1".
[ ] Region: us-east-1
[ ] Was the solution modified from the version published on this repository? No
[ ] If the answer to the previous question was yes, are the changes available on GitHub?
[ ] Have you checked your service quotas for the sevices this solution uses? Yes
[ ] Were there any errors in the CloudWatch Logs? No
Screenshots
If applicable, add screenshots to help explain your problem (please DO NOT include sensitive information).
Additional context
Setting LexV2BotLocaleIds as "ja_JP", stack is successfully deployed.
I think this may cause the problem:
https://github.com/aws-solutions/aws-qnabot/blob/main/lambda/lexv2-build/handler.py
When lambda "QnABot-Lexv2BotLambda" runs to translate slot type values, there're no ko_KR defines like in LEXV2_BOT_LOCALE_VOICE
LEXV2_BOT_LOCALE_VOICES = { "de_DE": "Hans", "en_AU": "Nicole", "en_GB": "Amy", "en_US": "Joanna", "es_419": "Mia", "es_ES": "Conchita", "es_US": "Lupe", "fr_CA": "Chantal", "fr_FR": "Mathieu", "it_IT": "Bianca", "ja_JP": "Mizuki" }
Do not put ko_KR in LexV2BotLocaleIds variable and deploy with just default settings. Then go to content designer website and change setting - "enable_multi_language_support" parameter to "true". If you refresh client page then lex will understand Korean by using Amazon Translate.
Lex occurred a build failure in Korean languate version. I think some utterances that type is AMAZON.AlphaNumeric caused internal build failure in CloudFormation Template.
https://docs.aws.amazon.com/lexv2/latest/dg/howitworks-builtins-slots.html
AMAZON.AlphaNumeric type supported all locales except Korean
Thanks for raising this issue, and sorry for the delay in responding back.
This was addressed as part of QnABot release 5.1.1.
More details: https://github.com/aws-solutions/aws-qnabot/blob/main/CHANGELOG.md#511---2022-02-04
Closing this issue.
|
<?php
namespace App;
use Illuminate\Support\Facades\DB;
use Illuminate\Database\Eloquent\Model;
use Yajra\DataTables\Facades\DataTables;
use Illuminate\Database\Eloquent\SoftDeletes;
class tb_pedidos_oracao extends Model
{
use SoftDeletes;
//use Notifiable;
protected $table = 'tb_pedidos_oracao';
protected $primaryKey = 'id_pedidos_oracao';
protected $fillable = [];
protected $hidden = [];
protected $softDelete = true;
const CREATED_AT = 'dhs_cadastro';
const UPDATED_AT = 'dhs_atualizacao';
const DELETED_AT = 'dhs_exclusao_logica';
public function getDtPedidosOracao(){
$objReturn = DB::table('tb_pedidos_oracao')
->get();
return DataTables::collection($objReturn)
->addColumn('dhs_acadastro', function($model){
return date('d/m/Y', strtotime($model->dhs_cadastro));
})
->make(true);
}
public function getPedidosAtivos(){
return DB::table('tb_pedidos_oracao')
->whereNull('tb_pedidos_oracao.dhs_exclusao_logica')
->get();
}
}
|
Joseph OLSON, Monica Olson, Javier Vargas, Plaintiffs-Appellants, v. UNITED STATES of America, Defendant-Appellee.
No. 03-15141.
United States Court of Appeals, Ninth Circuit.
Jan. 6, 2006.
Thomas G. Cotter, Haralson, Miller, Pitt, Feldman & McAnally, P.L.C., for the plaintiffs-appellants.
Peter D. Keisler, Mark B. Stern, and Dana J. Martin, Civil Division, Department of Justice, and Paul K. Charlton, United States Attorney, for the defendant-appellee.
Before B. FLETCHER and REINHARDT, Circuit Judges, and RESTANI, Chief Judge, United States Court of International Trade.
The Honorable Jane A. Restani, Chief Judge, United States Court of International Trade, sitting by designation.
ORDER
This case is remanded to the district court for further proceedings consistent with the decision of the United States Supreme Court in United States v. Olson, —U.S.-, 126 S.Ct. 510, 163 L.Ed.2d 306 (2005). The plaintiffs-appellants shall be allowed to conduct discovery on whether the United States can be held liable under the Federal Tort Claims Act pursuant to a “private persons” analogy.
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7th Michigan Infantry Regiment
The 7th Michigan Infantry Regiment was an infantry regiment that served in the Union Army during the American Civil War.
Service
The 7th Michigan Infantry was organized at Monroe, Michigan and mustered into Federal service for a three-year enlistment on August 22, 1861. Among the ranks was future brigadier general Henry Baxter, who was captain of Company C.
The 7th was assigned to the Army of the Potomac soon after it was formed and served in the 3rd Brigade, 2nd Division, 2nd Corps, for the duration of the war. Sister regiments in the 3rd Brigade included the 19th Massachusetts, 20th Massachusetts, 42nd New York, and 59th New York.
Infantryman William Rufus Shafter was wounded at the Battle of Fair Oaks; he later received the Medal of Honor for heroism during the battle.
Sergeant Alonzo Smith of Company C received the Medal of Honor on December 1, 1864 for his actions at the Battle of Boydton Plank Road on October 27, 1864.
The 7th was one of the first regiments to cross the Rappahannock River on Dec. 11th, 1862 while under fire from Confederate sharpshooters hidden in the buildings of Fredericksburg, the first opposed riverine assault in American military history.
In July 1862, Norman J. Hall, a Regular Army artilleryman assumed command of the regiment and led it until he was promoted to brigade command before the Battle of Gettysburg.
The regiment was mustered out on July 5, 1865.
Total strength and casualties
The regiment suffered 11 officers and 197 enlisted men who were killed in action or mortally wounded and 3 officers and 186 enlisted men who died of disease, for a total of 397 fatalities.
Commanders
* Major Sylvanus W. Curtiss
* Colonel Norman J. Hall
* Lt Colonel Amos Steele
* Lt Colonel George W. LaPoint
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Breathing mask with sticky edge
ABSTRACT
Breathing assist kits having a cranio-cervical extension device and a strapless breathing mask are presented. The mask comprises a seal that couples the mask to a victim's face preferably using an adhesive gasket that ensures the mask does not become dislodged while a care giver provides assisted breathing. The mask can also include, among other enhancements, a window or a light gathering lens to provide a view into the victim's mouth.
This application claims the benefit of priority to U.S. Provisional Application having Ser. No. 60/992,863 filed on Dec. 6, 2007. These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
FIELD OF THE INVENTION
The field of the invention is assisted breathing technologies.
BACKGROUND
There are numerous reasons why a victim having breathing problems would require assistance breathing including suffering from sleep apnea, requiring anesthesia during surgery, or having a cardiopulmonary emergency. Previously known breathing assist kits include mask to support assisted breathing. However, such kits can be difficult to use, require specialized training, are bulky, or otherwise unusable by a care provider lacking sufficient training.
One issue with previously known breathing assist kits is that a tube could be required for intubation to ensure a victim's trachea is open to air flow. Intubation is well beyond the skill level of an ordinary person attempting to provide care to the victim. However, not all breathing kits require intubation, but rather include a simple mask to provide air flow while a care provider tilts the victim's head into a proper position. Unfortunately, the care provider again could lack sufficient training to place a victim in a position having proper capital cervical extension for improved air flow.
Another issue with breathing kits is that provided breathing masks have to be strapped to a victim's head to prevent the mask from becoming dislodged. For example, U.S. Patent Application Publication to Lurie et al. 2003/0062040 describes an emergency ventilation system having a strapped mask as is the trend in the market for such systems. Unfortunately, it takes time to put the breathing mask on a victim's face which can cost valuable time especially during a life or death emergency. Additionally, should the victim vomit, the mask should be removed quickly so that the victim's mouth can be cleaned to prevent choking. Furthermore, known masks including those described by Lurie lack features that allow a care provider, especially untrained providers, to determine if the victim's airway is indeed clear.
What has not yet been appreciated is that a strapless mask, counter to current trends in breathing assist kits, can be used in conjunction with a cranio-cervical extension device to overcome the limitations of know kits. Preferably masks also include additional features to view into the mask to allow a care provider to ensure a victim's airway is clear. A strapless mask can be removed quickly in the event the victim is in respiratory distress. U.S. Patent Application Publication 2005/0056286 to Huddart et al. describes respiratory masks that could be strapless. However, Huddart fails to address the need for features to view into the mask or including such a mask with a breathing assist kit.
Thus, there is still a need for providing a breathing assist kit that ensures a victim's air passage is open and that includes a mask that can be easily coupled to a victim's face and or removed if necessary.
SUMMARY OF THE INVENTION
The present invention provides apparatus, systems and methods in which a breathing assist kit can be used to ensure a victim's trachea is open for air flow and that includes a mask which can be quickly put on or taken off of a victim's face. Preferred masks have a window or a light source for viewing into the victim's airway.
A preferred breathing assist kit comprises a cranio-cervical extension device and a strapless breathing mask. When placed beneath a victim's neck, the cranio-cervical extension device properly causes capital extension of the victim's car nio-cervical area to improve air flow into and out of the lungs. The breathing mask couples to the victim's face without requiring straps. In preferred embodiments, the mask comprises a sticky or tacky gasket to prevent the mask from moving from a desired location on the victim's face.
Preferred masks include a light source for illumination the interior of the mask or the interior of the victim's air passage. Contemplated light sources can include a window, a light gathering lens, or electrical lights (e.g., light emitting diodes).
Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which like numerals represent like components.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 a is a schematic of a breathing assist kit having a cranio-cervical extension device and a strapless breathing mask.
FIG. 1 b is a left side perspective view of the cranio-cervical extension device of FIG. 1 a.
FIG. 1 c is a perspective view of the strapless breathing mask of FIG. 1 a.
FIG. 1 d is a right side perspective view of the cranio-cervical extension device of FIG. 1 a.
FIG. 2 a is a front perspective view of a strapless breathing mask.
FIG. 2 b is a back perspective view of the strapless breathing mask of FIG. 2 a.
FIG. 3 is an exploded view of the cranio-cervical extension device of FIG. 2 a, with a strapless breathing mask disposed in a cavity of the device
DETAILED DESCRIPTION
In FIGS. 1 a-1 d, a breathing assist kit 120 comprises a cranio-cervical extension device 110 and a strapless breathing mask 100. Example device 110 has mask 100 enclosed within a cavity of device 110. Example device 130 illustrates a scenario where provider has removed mask 100 from an interior cavity of device 130.
Kit 120 is preferably configured for emergency use and can be mounted on a wall of a high traffic area where people congregate. For example, an emergency kit 120 can be placed within a school, a mall, a church, an airport, or other areas.
In a preferred embodiment, a cranio-cervical extension device 130 comprises a saddle shaped pillow that cradles the neck and provides proper capital extension for assisted breathing. When the pillow is positioned beneath a victim's neck, the victim's trachea has improved air flow during assisted breathing. Although a preferred kit 120 includes a saddle shaped pillow, all other cranio-cervical extension devices are also contemplated including a wedge that raises the victim's thoracic spine and provides proper capital extension of the victim's car nio-cervical area for improved air flow. An example of a suitable car nio-cer vial extension device having a saddle shaped pillow is described in co-owned U.S. patent application having Ser. No. 12/327,363 titled “Cranio-Cervical Extension Pillow with Dual Arcs”.
A preferred cranio-cervical extension device 320 also has a cavity that is defined by the shell of the device, as shown in FIG. 3. It is contemplated that the kit's mask 300 is packaged within the cavity to reduce the overall volume of the kit. An example of a suitable car nio-cer vial extension device is described in co-owned U.S. Patent Application having Ser. No. 12/327,363 titled “CPR System with Feedback Instructions”.
The kit's mask 100 preferably comprises a ventilation mask, a resuscitation mask, or a respiration mask shaped to cover a wide variety of victims' nose and mouth region. However, it is contemplated that mask 100 could also cover only the nose or only the mouth.
In FIGS. 2 a and 2 b, a strapless breathing mask 200 comprises a seal 230 that couples mask 200 to a victim's face. A preferred breathing mask 200 includes one or more light sources 210 or 220 that provide illumination to the interior of mask 200 or the victim's airways.
Seal 230 associated with strapless mask 200 allows the mask to stay affixed to the victim's face during potentially violent maneuvers including CPR compression strokes. Seal 230 does not necessarily have to be air tight, but rather substantially adheres to the victim's face using a suitable adhesive, preferably a bio-compatible adhesive. Preferably the seal is a sticky or tacky gasket that can be replaced after use. By providing a sticky gasket, the mask can be placed on a victim quickly and removed quickly should a need arise. Examples of suitable hypoallergenic, bio-compatible adhesives include those offered by Polymer Science, Inc. or Dow-Corning comprising silicon gel or polyurethane.
In yet other embodiments mask 200 also includes one or more advantageous features. Preferably mask 200 includes at least one passive light source 210 or 220. Mask 200 can include a window 220 that provides a care provider a clear view into the interior of mask 200 or the victim's airway. This allows the care giver to make sure the airway is unobstructed. Mask 200 can additionally include a light gathering lens 210 to provide additional light within the mask. Especially preferred lenses include a Fresnel lens that both gathers light and magnifies the view within the mask. Lens 210 provides an inexpensive means for passively illuminating the interior of mask 200. However, it is also contemplated that active light sources can also be incorporated mask 200, possibly an electrical light. An example of a cost effective active light source include battery powered light emitting diodes (LEDs). LEDs are compact, have low power usage, and can be integrated easily into mask 200.
Mask 200 can also including a gas port 240 that can couple to a gas source. Port 200 allows air, or other gas, to enter a victim's trachea during assisted breathing. Contemplated gas sources include pressurized containers, gas tanks, pumps, air bags, humans, or other sources of gas. Port 240 is also contemplated to include one or more valves that control the flow of gas into and out of the mask.
It is also contemplated that the mask 200 can include a bite block 260 (see FIG. 2 b) or a tongue depressor (not shown) to keep the victim's mouth unobstructed. In preferred embodiments gas port 240 comprises a lumen through which gas can travel through the bite block 260 to the trachea of the victim.
It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.
What is claimed is:
1. A breathing assist kit comprising: a cranio-cervical extension device comprising a pillow having a shape that opens a victim's air passage when the device pillow is disposed under the victim's neck; a strapless breathing mask having a gas port, a bite block, a seal, and a lens; wherein the gas port has a lumen coupled with a lumen of the bite block; wherein the seal provides an adhesive force that is adapted to allow the mask to stay affixed to a user's face during administration of CPR to the user; and wherein the lens is configured to gather light and magnify an interior view of the mask for illuminating a user's airway.
2. The kit of claim 1, wherein the pillow is saddle shaped.
3. The kit of claim 1, wherein the cranio-cervical extension device comprises a cavity.
4. The kit of claim 3, wherein the mask is packaged within the cavity.
5. The kit of claim 1, wherein the seal comprises a sticky gasket.
6. The kit of claim 1, wherein the mask covers at least one of a mouth area and a nose area of the victim's face.
7. The kit of claim 1, wherein the lens comprises a Fresnel lens.
8. The kit of claim 1, wherein the strapless mask further includes an electrical light.
9. The kit of claim 8, wherein the electrical light comprises LEDs.
10. The kit of claim 1, further comprising a mesh bag having an interior space that is sized and dimensioned to store the cranio-cervical extension device and the strapless breathing mask.
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Lappeenrannan NMKY
Lappeenrannan NMKY (also known as LrNMKY for short or as Team Lappeenranta in European competitions) is a basketball club based in a city of Lappeenranta, Finland. It was formed in 1951 as a Young Men's Christian Association (NMKY in Finnish) and has won two Finnish Championships (2005, 2006) and two Finnish Cups (2005, 2006).
Team Lappeenranta participated in Fiba EuroCup challenge in 2005/2006 finishing in semi-finals. In 2007/2008 season team is willing to make another run in Europe, this time in FIBA EuroCup. After the 2013–14 season, NMKY left the Korisliiga because of financial problems.
Honours
* Korisliiga
* Winners (2): 2005, 2006
* Finnish Cup
* Winners (4): 2005, 2006, 2007, 2008
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#pragma once
#include "SpellShapeProcessor.hpp"
// A common class for processing a spell, whether by direct-cast, a wand,
// etc.
class SpellcastingProcessor
{
public:
// Returns true if the spell was identified as a result of casting it.
bool process(SpellShapeProcessor* spell_processor, CreaturePtr caster, MapPtr current_map, const Coordinate& caster_coord, const Direction spell_direction, const Spell& spell, const int bonus, const ItemStatus effect_status, const AttackType attack_type) const;
};
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replaced TextView with HtmlTextView, collapse not working
hi
i just replaced the TextView in the lib with this HtmlTextView which extends TextView itself. expanding works fine but collapsig wont work.
where is the problem?
thanks in advance
String strHtml = "<b>"content with html</b>"
Spanned spanned = Html.fromHtml(strHtml );
// sample code snippet to set the text content on the ExpandableTextView
ExpandableTextView expTv1 = (ExpandableTextView) view.findViewById(R.id.expand_text_view)
.findViewById(R.id.expand_text_view);
// IMPORTANT - call setText on the ExpandableTextView to set the text content to display
expTv1.setText(Html.fromHtml(String.valueOf(spanned)));
Hope to help you.
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Talk:How NOT to write a Creepypasta/@comment-3194815-20130711183804/@comment-5849201-20130721170342
Feel free to contribute, but without killing the original story.
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above derived also hold good.
The constructions there given are applicable here also, if we suppose the cones of rays transposed from the left half of the circle to the right half, so that the points where the refractions take place again fall in the periphery. "We get, therefore,
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module LootSystem
class Chest
# List of all the drops that have a chance to be spawned in this chest
attr_reader :potential_droplist
def initialize
@potential_droplist = Droplist.new
end
# Adds a drop to the potential drops pool.
def add(params)
@potential_droplist.add(Drop.new(params))
end
# List of the drops that will be spawned.
def items_found
@final_droplist ||= generate_final_droplist
end
private
# Get a list of all drops that will be spawned by the chest
def generate_final_droplist
@final_droplist = Droplist.new
@potential_droplist.each { |drop| process_potential_drop(drop) }
return @final_droplist
end
# Check if the potential drop will be given to the player after opening a chest
def process_potential_drop(drop)
@final_droplist.add(drop) if drop.successful?
end
end
end
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chore: remove <Layout> props
These show up as undefined props in our .tsx files nowadays.
I think these were removed upstream in https://github.com/facebook/docusaurus/pull/6925
How do those pages get their HTML <title> elements if not from those props?
It also looks like those props are still documented in the Docusaurus docs: https://docusaurus.io/docs/creating-pages#add-a-react-page
Weirdly, it doesn't. I guess these are needed after all. Let me investigate further about why I'm getting intellisense issues, then.
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Method of increasing the leak tightness of a mechanical connector
ABSTRACT
The invention relates to a method of increasing the leak tightness of a mechanical connector of an extracorporeal blood treatment machine, wherein the mechanical connector has a pair of connection parts which have corresponding sealing surfaces, with at least one of the two sealing surfaces being wetted at least sectionally by a viscous fluid before the joining together of the connection parts and/or with the connection gap between the connection parts being covered by a sheath after their joining together. The invention further relates to an extracorporeal blood treatment machine comprising a mechanical connector which has a pair of connection parts which have corresponding sealing surfaces, with the sealing surfaces being at least sectionally wetted with a viscous liquid with a closed connector and/or with the connection gap between the connection parts of the closed connector being covered by a sheath.
The invention relates to a method of increasing the leak tightness of a mechanical connector of an extracorporeal blood treatment machine as well as to an extracorporeal blood treatment unit.
Microbubbles are gas bubbles having a diameter of a few μm which are as a rule no longer visible due to their small size. They arise at different points and under different conditions in extracorporeal blood circuits, inter alia by the discharge of blood-soluble gases or by air entry at very small leaks in the vacuum region of the extracorporeal circuit, and adhere to the inner surface of the extracorporeal hose system or of the blood treatment unit. Their effects when they are detached and infuse into the human body are greater than previously presumed. For example, micro bubbles were found in vital organs such as the lung, heart and brain of dialysis patients.
There are indications that, for example, the Luer-Lock connection between an arterial cannula and the arterial hose, a connection of two conical hard plastic parts which is located in the vacuum region of the blood pump, is not always airtight and small amounts of air are sucked into the blood hose. In a Luer-Lock connector, a hard plastic is pressed against a hard plastic, with the sealed region of such an areal pressing being limited to a cone. Investigations have confirmed that micro bubble numbers and volumes are much larger downstream of a Luer-Lock connector than upstream of a Luer-Lock connector in the vacuum region.
Microbubbles can only be separated with limitations in the venous chamber due to their small size and their small buoyancy and can only be conditionally recognized by the prescribed protection system for avoiding air infusion.
It is the underlying object of the invention to reduce the number of micro bubbles in extracorporeal blood circuits or to reduce the infusion of micro bubbles into the patient's body.
Against this background, the invention relates to a method of increasing the leak tightness of a mechanical connector of a medical device, wherein the mechanical connector has a pair of connection parts which have corresponding sealing surfaces, with at least one of the two sealing surfaces being wetted at least sectionally by a viscous liquid before the joining together of the connection parts and/or with the connection gap between the connection parts being covered by a sheath after their joining together. In accordance with the invention, the above-named object is achieved by an increase in the leak tightness of existing connectors.
The viscous liquid can, for example, have a dynamic viscosity of between 10² and 10⁵, preferably of between 10³ and 10⁴ mPa·s at 25° C. A liquid having such viscosity values is particularly suitable for a simple handling in the application and for an effective seal. The liquid is preferably sterile and has a high biocompatibility.
The viscous liquid should in particular be blood-compatible, i.e. an entry of a small quantity of the liquid into the blood of a patient should not be harmful.
The connection gap extends along the outer periphery of the contact surface between the two connection parts. The joining together comprises a contacting of the two sealing surfaces as well as a locking of the two connection parts in a manner such that the contact of the two sealing surfaces is maintained and the sealing surfaces preferably lie on one another with a certain contact pressure. This can be achieved, for example, by a screwing or latching of the two connection parts. The connection parts and in particular the contact surfaces of the connection parts can be produced from hard plastic in an embodiment.
In an embodiment, one connection part has a male cone and one connection part has a female cone, with at least one section of the outer jacket surface of the male cone and at least one section of the inner jacket surface of the female cone forming the sealing surfaces. The two respective sections contact one another after a joining together of the parts and seal the plug-in connection.
In an embodiment, the mechanical connector is a Luer-Lock connection. In this respect, it is a standardized connector for medical engineering, with one connection part of the connector having a male cone and the other connection pair having a female cone. To secure the connections against a release in an optimum manner, the Luer-Lock connector furthermore has a system for screwing the connection parts in addition to the cones, with an external thread at the connection part in which the female cone is located engaging into an internal thread which is seated at the connection part having the male cone.
In an embodiment, only the sealing surface of a connection part is wetted with the viscous liquid before the joining together of the connection parts. Provision can, for example, be made that only the jacket surface of the male cone is wetted with the viscous liquid. This is sufficient for establishing an air-impermeable connection and the outer jacket surface of the male cone is more easily accessible than the inner jacket surface of the female cone.
In an embodiment, the medical device is an extracorporeal blood treatment machine. The extracorporeal blood treatment machine can be a dialyzer. Furthermore, the extracorporeal blood treatment machine can, for example, be an ultrafiltration device or a heart-lung machine. The use of the method in accordance with the invention is also conceivable in other medical devices. For example, the use is also conceivable in endoscopic abdominal surgery or in gynecology, with an artificial surgical cavity being able to be created with the aid of gases. A gas-impermeable connection would be very meaningful here to limit the introduced quantity of gas.
In an embodiment, the connector at which the method is implemented connects a liquid-conducting circuit to an inflow line or outflow line. For example, the liquid-conducting circuit can be an extracorporeal blood circuit. This circuit is typically configured as a disposable, with the disposable having different interfaces which are connected to lines, cannula, etc. at the machine side with the aid of a connector. Examples for inflow lines and outflow lines in the sense of this embodiment comprise the arterial port, a pre dilution line, a post dilution line, a heparin line, a port for blood removal, a port for medication dispensing, a drain or the venous port. Provision can in particular be made that the connector at which the method is implemented connects an arterial cannula to the arterial line of an extracorporeal blood circuit (arterial port).
In an embodiment, the liquid-conducting circuit comprises at least one pump and the method is implemented at a connector which is arranged on the intake side of this at least one pump. Air can in particular enter into the circuit at the intake side of the pump since there is a vacuum with respect to the environment. For example, the liquid-conducting circuit is an extracorporeal blood circuit and the method is implemented at a connector which is arranged on the intake side of the blood pump.
In an embodiment, the sheath is a flexible sheath. A tighter-sealing connection can be achieved with the aid of a flexible sheath than with a rigid protector since the sheath can nestle against the outer surface of the connector. Furthermore, a flexible sheath can be manufactured less expensively and can be simply slipped over the connector after the joining together of the connection parts. Provision can be made that the sheath is slipped over the connector such that it completely covers the region of the connection gap.
In an embodiment, the sheath is a flexible hose piece or a ring. These shapes make it possible simply to push the sheath over the connector after the joining together of the connection parts.
In an embodiment, the sheath is produced from a silicone elastomer. This material has proved particularly suitable due to the good properties with respect to elasticity, biocompatibility and leak tightness.
The invention further relates to a medical device comprising a mechanical connector which has a pair of connection parts which have corresponding sealing surfaces, with the sealing surfaces being at least sectionally wetted with a viscous liquid with a closed connector and/or with the connection gap between the connection parts of the closed connector being covered by a sheath.
The closed connector designates the state of the connector after the joining together of the connection parts. The medical device can be an extracorporeal blood treatment machine. The extracorporeal blood treatment machine can be a dialyzer. Furthermore, the extracorporeal blood treatment machine can, for example, be an ultrafiltration device or a heart-lung machine. It is furthermore conceivable that the medical device is, for example, an instrument of endoscopic abdominal surgery or of gynecology. Further medical devices at which a gas-impermeable connection of two connection parts is meaningful are also covered by the present invention.
In an embodiment, one connection part has a male cone and one connection part has a female cone, with at least one section of the outer jacket surface of the male cone and at least one section of the inner jacket surface of the female cone forming the sealing surfaces. In an embodiment, the mechanical connector is a Luer-Lock connection.
In an embodiment, the connector connects a liquid-conducting circuit, in particular an extracorporeal blood circuit, to an inflow line and an outflow line. For example, the liquid-conducting circuit can be an extracorporeal blood circuit. This circuit is typically configured as a disposable, with the disposable having different interfaces which are connected to lines, cannula, etc. at the machine side with the aid of a connector. Examples for inflow lines and outflow lines in the sense of this embodiment comprise the arterial port, a pre dilution line, a post dilution line, a heparin line, a port for blood removal, a port for medication dispensing, a drain or the venous port. In an embodiment, the liquid-conducting circuit comprises at least one pump and the connector is arranged on the intake side of this at least one pump. The liquid-conducting circuit is, for example, an extracorporeal blood circuit and the connector is arranged on the intake side of the blood pump. Provision can in particular be made that the connector connects an arterial cannula to the arterial line of an extracorporeal blood circuit (arterial port).
In an embodiment, the sheath is a flexible sheath, with provision preferably being made that this sheath completely covers the region of the connection gap. The sheath can be a flexible hose piece or a ring. The sheath can be produced from a silicone elastomer.
Further details and advantages result from the FIGURES and embodiments described in the following. The only FIGURE shows an illustration of a Luer-Lock connector in cross-section.
The connector has a first connection part 1 having a male cone 2 and an internal thread 3. The second connection part 4 has a female cone 5 and a projection 6 which engages into the internal thread. On the joining together of the connection parts 1 and 4, they are screwed and the outer jacket surface of the male cone 2 and the inner jacket surface of the female cone 5 are pressed against one another.
Provision is made in accordance with the invention that the outer jacket surface of the male cone 2 is wetted with a viscous, sterile and biocompatible liquid before the joining together of the two connection parts 1 and 4.
Provision is alternatively or additionally made in accordance with the invention that a hose piece of a flexible silicone elastomer is slipped over the connector after the joining together of the connection parts 1 and 4. The inner diameter of the flexible hose piece or of the ring is selected somewhat smaller than the outer diameter of the connector to achieve an airtight termination. The hose piece should in particular completely cover the connection gap which arises at the point marked by reference numeral 7 between the two connection parts after their joining together.
1. A method of increasing the leak tightness of a mechanical connector of a medical device, in particular of a dialysis machine, wherein the mechanical connector has a pair of connection parts which have corresponding sealing surfaces, characterized in that at least one of the two sealing surfaces is at least sectionally wetted with a viscous liquid before the joining together of the connection parts; and/or in that the connection gap between the connection parts is covered by a sheath after their joining together.
2. A method in accordance with claim 1, characterized in that one connection part has a male cone and one connection part has a female cone, with at least one section of the outer jacket surface of the male cone and at least one section of the inner jacket surface of the female cone forming the sealing surfaces.
3. A method in accordance with claim 1, characterized in that the mechanical connector is a Luer-Lock connector.
4. A method in accordance with claim 1, characterized in that only the sealing surface of a connection part, preferably only the jacket surface of the male cone, is wetted with the viscous liquid before the joining together of the connection parts.
5. A method in accordance with claim 1, characterized in that the connector at which the method is implemented connects a liquid-conducting circuit, in particular an extracorporeal blood circuit, to an inflow or to an outflow.
6. A method in accordance with claim 5, characterized in that the liquid-conducting circuit comprises at least one pump and the method is implemented at a connector which is arranged on the intake side of this at least one pump.
7. A method in accordance with claim 1, characterized in that the sheath is a flexible sheath, with provision preferably being made that this sleeve is slipped over the connector such that it completely covers the region of the connection gap.
8. A method in accordance with claim 1, characterized in that the sheath is a flexible hose piece or a ring.
9. A method in accordance with claim 1, characterized in that the sheath is produced from a silicone elastomer.
10. A medical device, in particular a dialysis machine, comprising a mechanical connector which has a pair of connection parts which have corresponding sealing surfaces, characterized in that the sealing surfaces are wetted with a viscous liquid at least sectionally with a closed connector; and/or in that the connection gap between the connection parts of the closed connector is covered by a sheath.
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Thread:Noodleofbowl/@comment-22439-20160820023227
Good day fellow Admiral! Welcome to !
If you are a new player, you may want to read up on the tutorials to get you started:
Thank you and have a good sortie run!
( This is an automated message )
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Blob storage link dead
The link in Challenge 2 - Task 1 - Step 4 is dead.
https://logsbenchmark00.blob.core.windows.net/logsbenchmark-onegb/2014/?sp=rl&st=2022-08-18T00:00:00Z&se=2030-01-01T00:00:00Z&spr=https&sv=2021-06-08&sr=c&sig=5pjOow5An3%2BTs5mZ%2FyosJBPtDvV7%2FXfDO8pLEeeylVc%3D
Found there's another link.
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Portal:Trains/Anniversaries/November 17/More
19th century
* 1880 – Ministerial approval is given for construction of what would become Gare d'Oyonnax in France.
* 1885 – The first through train from Chicago via Santa Fe lines arrives in San Diego.
20th century
* 1941 – The Prospector multiple unit passenger trainset debuts on the Denver and Rio Grande Western Railroad in operating between Denver, Colorado, and Salt Lake City, Utah.
* 1979 – The first stage of Brisbane Suburban electrification between Ferny Grove and Darra is commissioned by the Queensland Premier Joh Bjelke-Petersen. It is the first section of electrification in Australia to use 25,000 volts 50 Hz overhead power supply.
21st century
* 2012 – The first revenue-earning use of an E5 Series Shinkansen takes place as a special Joetsu Shinkansen 30th Anniversary (上越新幹線開業30周年号」) service from Niigata to Tokyo.
Births
* 1857 – William Benson Storey, president of Atchison, Topeka and Santa Fe Railway 1920-1933, is born (d. 1940).
Deaths
* 1920 – T. Jefferson Coolidge, president of Atchison, Topeka and Santa Fe Railway 1880-1881, dies (b. 1831).
* 1927 – Charles Sanger Mellen, president of Northern Pacific Railway 1897-1903, New Haven Railroad 1903-1913 and Maine Central Railroad 1910-1914, dies (b. 1852).
|
Earth is the third planet from the Sun and is the largest of the terrestrial planets. The Earth is the only planet in our solar system not to be named after a Greek or Roman deity. The Earth was formed approximately 4.54 billion years ago and is the only known planet to support life.
| Name | Radius | Average Temperature |
|---|---|---|
| Mars | 3,403km | -46°c |
| Venus | 6,052km | 462°c |
|
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Home Calendar Topics Just Charlestown About Us
Thursday, March 31, 2022
The arrogance of Rhode Island’s payday loan industry
Legal loan sharks get what they want
By Steve Ahlquist for UpRiseRI
That's far-right Charlestown Senator Elaine Morgan sitting near the center
It’s been a while since in person committee meetings were held by the Rhode Island Senate in rooms with no cameras, meaning that these hearings are not being live-streamed, or even recorded, beyond a pretty bad audio recording.
UpriseRI brought a camera into room 310 last night to cover the Senate Committee on Commerce which was taking up, for perhaps the twelfth time in a dozen years, legislation that would repeal the provisions of the general laws allowing deferred deposit providers, also known as “payday lenders.”
The bill, S2166, is sponsored by Senator Ana Quezada (Democrat, District 2, Providence), who was unable to introduce her bill in person. See some earlier reporting on the issue of payday lending here:
The General Assembly continues to be complicit in the evil of payday loans
How predatory payday lenders keep customers hooked
Repeal of this provision is supported by a vast array of community, faith, social service and advocacy organizations and activists, but opposed by a small clique of powerful, wealthy and politically connected lobbyists and corporation who make literally millions of dollars via their predatory lending practices.
In her testimony in favor of the bill to resign in payday lending, Margaux Morisseau, Deputy Director of the Rhode Island Coalition to End Homelessness, spoke about doing social service work in Woonsocket in 2009 and realizing that “hard working families were going to be homeless” because payday lender Advance America had opened a store in the middle of the neighborhood she worked in, and was targeting residents.
After working with legislators at the General Assembly to craft the first payday lending reform bill – which would have capped the interest lenders could charge at 36% annually, the bill was removed from consideration minutes before a scheduled floor vote “because of one phone call made by a powerful lobbyist,” said Morisseau.
A bill to stop payday lending has never reached the House or Senate floor for a vote.
The protection payday lenders are given by General Assembly leadership, protections that include holding hearings in rooms without cameras, has bred arrogance among those who lobby for – and profit from – payday lending.
Lobbyists Stephen Alves (paid $40k annually by predatory lender Access Financial) and and former Speaker of the House William Murphy (a bargain at $30k annually from Purpose Financial, doing business as Advance America) don’t even bother deliver oral testimony to the committee – at least when there are cameras rolling – they instead submit written testimony.
Murphy does one better than Alves – his name isn’t even on the testimony he provides. Instead, he delivered a letter from Purpose Financial’s Senior Policy Counsel Julie Townsend. You can read their testimony at the bottom of this report.
These lobbyists don’t testify at hearings because they don’t need to. They have the State House firmly in their grip. The hearings on their bills are done in camera-less rooms. They kill bills with last minute phone calls and at chummy fundraisers. They see inequality and human suffering not as an evil, but as an opportunity.
But the arrogance award for those testifying against the payday loan reform legislation at last night’s committee hearing has to go to Bill Staderman, President of the Rhode Island Association of Financial Service Centers, who owns and operates payday loan companies throughout the state. Staderman presented himself as a clever, charming monster, but he is neither clever nor charming.
Staderman began by saying that payday loans were “simple” things that people enter into willingly, of their own free will. Playing to the privilege he sensed in the room, Staderman said, “I’m sure that most people here, probably everybody here, has not gotten a payday loan themselves…”
Getting these payday loans is “quick, and it’s a small amount – the most in Rhode Island is $500,” said Staderman, which brought a challenge from Senator Tiara Mack (Democrat, District 6, Providence) during questioning.
Noting her personal experience of growing up low-income, “I’ve seen the financial cost, I’ve seen the generational cost and I’ve also experienced the generational cost of predatory payday loans,” said Senator Mack. “We can call them small amounts [but] $500 to someone who does not make $500 a week … is a large chunk of money.”
Staderman could not help but respond to Mack’s powerful, factual and emotive testimony with anything but condescension. “I appreciate you agreeing with what I said,” said Staderman. “Here is the reason why: $500, I said before, might be a small amount – I grew up in a tenement in Brooklyn…” Then putting on some sort of undefinable accent, Staderman continued, “$500. It’s a lot of money fuggedaboutit.” Sensing he was going to be interrupted by Senator Mack, Staderman interrupted, “Let me finish please Senator…”
Staderman then babbled on for minutes, and took questions from Senators Cynthia Mendes (Democrat, District 18, East Providence) and Kendra Anderson (Democrat, District 31, Warwick, Cranston). Finally, Senator Mack spoke up again, trying to explain her family’s struggle with generational poverty. As tears came to Senator Mack, Staderman said interrupted her with, “I grew up with that, I’m sorry.”
“I’m not done,” said Senator Mack. “I’m crying, but not done. It’s hard to…”
“My father didn’t have a car until he was 40 years old,” said Staderman, interrupting again. “Or a license.”
“Yes,” said Senator Mack, who patiently explained more of her lived experience. She also spoke about the working people who cannot afford the time away from work to testify at the State House on issues of vital importance.
As Senator Mack left the room to compose herself, Staderman still needed the final word. He was cut off by Committee Chair Susan Sosnowski (Democrat, District 37, Block Island, South Kingstown). “No that’s enough,” she said.
What none of the testimony provided by the payday loan industry does is try to refute the arguments made against the practice by the advocates testifying against them.
State Treasurer Seth Magaziner, for instance, noted that Rhode Island remains the only state in New England that still allows this usurious practice. Treasurer Magaziner added that the United States Congress outlawed payday lending in and around military bases because they are seen as a threat to military preparedness.
Alan Krinsky, testifying on behalf go the Economic Progress Institute, brought up several key points, backed by research and data, about the negative impacts of payday loans on both borrowers and the community.
• the average payday loan consumer gets caught in a cycle of debt, taking out 10 loans per year due to an inability to pay of the balance and constantly growing fees;
• payday lenders increase wealth disparities by disproportionately targeting people of color; and
• nationally, 18 states and the District of Columbia enforce laws with rate caps of 36 percent or lower. Rhode Island is the only New England state.
Also submitting testimony in favor of reigning in payday lenders were the United Way of Rhode Island, Rhode Island Kids Count and the Capital Good Fund.
|
Do Do NOT
i . Utilize sunlight when that i . Do not use artificial light is available, for it is the bright- in a region where bright sun est light to be had on our planet light is constantly available. ( 232).
|
AutoMapper MapperConfiguration(config => c.AddMaps(assembly)) overload not registering closed generic maps
I'm working on a .Net 8.0 project based on Jason Taylor's Clean Architecture template. In this project, AutoMapper is used throughout to map between domain entities and data-transfer objects (DTOs). In the application's unit test project, there are tests defined to validate the defined mappings. I recently introduced some DTOs that make use of generics to set the datatype for the Id property on a lookup object. After introducing the generics (and their associated maps), my AutoMapper tests began to fail, but only for the generics and only where the AddMaps overload is used. If the maps are explicitly added using the MapperConfiguration(config => c.CreateMap(src,dest) overload, the maps work as expected.
Believing this to be an AutoMapper bug, I opened an issue in the AutoMapper repo, but I was directed here instead.
I created a .NET Fiddle demonstrating the issue in browser, and the full reproduction code is below. Is there a configuration oversight, feature limitation, or is this an AutoMapper bug?
public class MyClass
{
public int Id { get; set; }
public string Name { get; set; } = null!;
}
public record MyClassDto
{
public int Id { get; init; }
public string Name { get; init; } = null!;
private class Mapping : Profile
{
public Mapping() => CreateMap<MyClass, MyClassDto>();
}
}
public record MyGenericClassDto<T>
{
public T Id { get; init; } = default!;
public string Name { get; init; } = null!;
private class Mapping : Profile
{
public Mapping() => CreateMap<MyClass, MyGenericClassDto<int>>();
}
}
public class MappingTests
{
[Test]
// Will succeed
[TestCase(typeof(MyClass), typeof(MyClassDto))]
// Will fail
[TestCase(typeof(MyClass), typeof(MyGenericClassDto<int>))]
public void ShouldSupportMappingFromSourceToDestination_AddMaps(
Type source,
Type destination)
{
var instance = GetInstanceOf(source);
var configuration = new MapperConfiguration(config =>
config.AddMaps(Assembly.GetAssembly(typeof(MyClass))));
var mapper = configuration.CreateMapper();
mapper.Map(instance, source, destination);
}
[Test]
// Will succeed
[TestCase(typeof(MyClass), typeof(MyClassDto))]
// Will succeed
[TestCase(typeof(MyClass), typeof(MyGenericClassDto<int>))]
public void ShouldSupportMappingFromSourceToDestination_CreateMap(
Type source,
Type destination)
{
var instance = GetInstanceOf(source);
var config = new MapperConfiguration(cfg =>
cfg.CreateMap(source, destination));
var mapper = new Mapper(config);
mapper.Map(instance, source, destination);
}
private static object GetInstanceOf(Type type) =>
type.GetConstructor(Type.EmptyTypes) != null
? Activator.CreateInstance(type)! :
// Type without parameterless constructor
RuntimeHelpers.GetUninitializedObject(type);
}
I would try with Net 7.0. I've seen a number of cases where Net 8.0 is broken.
@jdweng I made a .Net 7.0 reproduction of the problem: https://dotnetfiddle.net/4Y0YmU
Approach 1: Patch AutoMapper
After messing around with this a bit more, I started investigating the AutoMapper source code in an effort to track down this behavior discrepancy, and I have identified the affected code. I've copied the method below, removing the irrelevant bits.
private void AddMapsCore(IEnumerable<Assembly> assembliesToScan)
{
var autoMapAttributeProfile = new Profile(nameof(AutoMapAttribute));
foreach (var type in assembliesToScan.Where(a => !a.IsDynamic && a != typeof(Profile).Assembly).SelectMany(a => a.GetTypes()))
{
if (typeof(Profile).IsAssignableFrom(type) && !type.IsAbstract && !type.ContainsGenericParameters)
{
AddProfile(type);
}
foreach (var autoMapAttribute in type.GetCustomAttributes<AutoMapAttribute>())
{
// Scans assemblies with AutoMap attribute. Irrelevant to the question.
}
}
AddProfile(autoMapAttributeProfile);
}
You'll notice just inside the foreach loop, AutoMapper explicitly ignores any Profile classes that are generic (or that are nested within a generic). If you follow that AddProfile(type) call, you'll see why:
public void AddProfile(Type profileType) => AddProfile((Profile)Activator.CreateInstance(profileType));
The method simply instantiates a new instance of the class implementing Profile. For generic classes, it's necessary to provide the type arguments to the class being instantiated. With that in mind, we can create a few new methods, and we can update the AddMapsCore method to leverage them for generic classes.
public void AddGenericProfile(Type profileType) => AddProfile((Profile)GetInstanceOfGeneric(profileType));
private static object GetInstanceOfGeneric(Type genericType)
{
var typeArgs = GetGenericArguments(genericType);
var constructedType = genericType.MakeGenericType(typeArgs);
return Activator.CreateInstance(constructedType);
}
private static Type[] GetGenericArguments(
Type genericType) =>
genericType.GetGenericArguments()
.Select(_ => typeof(object))
.ToArray();
private void AddMapsCore(IEnumerable<Assembly> assembliesToScan)
{
var autoMapAttributeProfile = new Profile(nameof(AutoMapAttribute));
foreach (var type in assembliesToScan.Where(a => !a.IsDynamic && a != typeof(Profile).Assembly).SelectMany(a => a.GetTypes()))
{
if (typeof(Profile).IsAssignableFrom(type) && !type.IsAbstract)
{
if (type.ContainsGenericParameters)
{
AddGenericProfile(type);
}
else
{
AddProfile(type);
}
}
foreach (var autoMapAttribute in type.GetCustomAttributes<AutoMapAttribute>())
{
// Scans assemblies with AutoMap attribute. Irrelevant to the question.
}
}
AddProfile(autoMapAttributeProfile);
}
With these updates, our tests as written in the OP will pass. To validate this, I forked and updated AutoMapper with the changes and introduced the new tests to cover these scenarios. All existing tests and the new ones passed. The Pull Request for the change is here, but unfortunately it was closed by the maintainers without much/any feedback.
Approach 2: Move Nested Maps Outside of Generics
If efforts to resolve the AutoMapper usage inconsistency through a PR are unsuccessful, it is possible to work around this issue by moving the nested Profile to its own class outside of the generic class it was originally defined in. The use case in the OP can be solved by updating the code to the following:
public record MyGenericClassDto<T>
{
public T Id { get; init; } = default!;
public string Name { get; init; } = null!;
}
internal class GenericMapping : Profile
{
public GenericMapping() => CreateMap<MyClass, MyGenericClassDto<int>>();
}
This usage is not fully synonymous with that in the OP as it leaks details of the mapping class outside of its original containing class, whereas the non-generic version can be fully encapsulated. More importantly, it creates an inconsistency when it comes to implementation details. Source generators, analyzers, and other tools designed to help abstract away some of these details will need to be aware of this discrepancy.
|
Page:The Fall of Constantinople.djvu/71
Rh who had held their position from a time preceding the Christian era ; and, so far as the north of the peninsula is concerned, this fact may be taken as the starting-point in any examination of the settlement of the peoples who subsequently swarmed into the empire. That position, before the time with which I am concerned, had been successively disturbed by the inroads of various races. In view, however, of the importance which has always attached to the Slav element in the Balkan peninsula, and of the influence of its members upon the various populations with which they came in contact, it is necessary to remember that they were among the first and most widespread of the races which inhabited that great district within historical times. It has been contended that the Slavs had even settled so far south as the Peloponnesus itself, and the evidence in support of this theory has been the statement of the Byzantine writers that in the west of that peninsula there were people, called Slavs by these writers, who were of a different race and who spoke a different language from the Greeks. It has, however, I think, been clearly shown that while in the capital these settlers were regarded as hostile, their Greek neighbors of the same period looked upon them as brothers and as liberators. In all probability they were Albanians — a people who in the Middle Ages were regarded as the descendants of the Macedonian race to which Alexander the Great belonged.
The Huns
The Huns, a Turanian people, had in the fifth century invaded the territory of the Slavs, devastating whole provinces and creating wastes for their cattle. They formed part of the great Asiatic race which was destined to give so much trouble to the empire and finally to overthrow it. The Byzantine writers correctly called them Turks. Like all of their race, they were a nomadic people. During the ninth and tenth centuries they overran Illyria and Macedonia, and had devastated Attica. After the many incursions of these and other similar races, we continually find that, while
|
Thread:<IP_ADDRESS>/@comment-22439-20130920011505
Hi, welcome to ! Thanks for your edit to the Doppelganger page.
Please leave me a message if I can help with anything!
|
package net.modificationstation.stationapi.impl.item;
import net.mine_diver.unsafeevents.listener.EventListener;
import net.mine_diver.unsafeevents.listener.ListenerPriority;
import net.minecraft.item.ItemBase;
import net.modificationstation.stationapi.api.block.BlockMiningLevel;
import net.modificationstation.stationapi.api.event.item.IsItemEffectiveOnBlockEvent;
import net.modificationstation.stationapi.api.event.item.ItemStrengthOnBlockEvent;
import net.modificationstation.stationapi.api.item.tool.OverrideIsEffectiveOn;
import net.modificationstation.stationapi.api.item.tool.ToolLevel;
import net.modificationstation.stationapi.api.mod.entrypoint.Entrypoint;
import net.modificationstation.stationapi.api.mod.entrypoint.EventBusPolicy;
@Entrypoint(eventBus = @EventBusPolicy(registerInstance = false))
public class ToolEffectivenessImpl {
@EventListener(priority = ListenerPriority.HIGH)
private static void isEffective(IsItemEffectiveOnBlockEvent event) {
ItemBase item = event.itemInstance.getType();
if (item instanceof ToolLevel) {
event.effective =
((BlockMiningLevel) event.block).getToolTypes(event.meta, event.itemInstance) != null &&
((BlockMiningLevel) event.block).getToolTypes(event.meta, event.itemInstance).stream().anyMatch(entry -> entry != null && entry.isInstance(event.itemInstance.getType())) &&
((ToolLevel) item).getToolLevel() >= ((BlockMiningLevel) event.block).getBlockLevel(event.meta, event.itemInstance);
if (item instanceof OverrideIsEffectiveOn)
event.effective = ((OverrideIsEffectiveOn) item).overrideIsEffectiveOn((ToolLevel) item, event.block, event.meta, event.effective);
}
}
@EventListener(priority = ListenerPriority.HIGH)
private static void getStrength(ItemStrengthOnBlockEvent event) {
if (
event.itemInstance.getType() instanceof ToolLevel &&
((BlockMiningLevel) event.block).getBlockLevel(event.meta, event.itemInstance) <= ((ToolLevel) event.itemInstance.getType()).getToolLevel() &&
((BlockMiningLevel) event.block).getBlockLevel(event.meta, event.itemInstance) != -1 &&
((BlockMiningLevel) event.block).getToolTypes(event.meta, event.itemInstance) != null &&
((BlockMiningLevel) event.block).getToolTypes(event.meta, event.itemInstance).stream().anyMatch((toolLevel) -> toolLevel != null && toolLevel.isInstance(event.itemInstance.getType()))
)
event.strength = ((ToolLevel) event.itemInstance.getType()).getMaterial().getMiningSpeed();
}
}
|
using SignalRChat.Common.ApiClient;
using SignalRChat.DomainManager.Interfaces;
using SignalRChat.Dtos;
using SignalRChat.Models;
using System;
using System.Collections.Generic;
using System.Linq;
using System.Web;
namespace SignalRChat.DomainManager
{
public class UserManager : IUserManager
{
private readonly IHttpClient httpClient;
public UserManager(IHttpClient httpClient)
{
this.httpClient = httpClient;
}
public Models.UserViewModel GetViewModel(string userName)
{
var userDto = httpClient.GetAsync<UserDto>(string.Format("Api/User/Name/{0}", userName));
return userDto.ToViewModel();
}
public Models.UserViewModel GetViewModel(int userId)
{
var userDto = httpClient.GetAsync<UserDto>(string.Format("Api/User/{0}", userId));
return userDto.ToViewModel();
}
public Models.UserViewModel AddUser(Models.UserViewModel user)
{
if (user == null) { throw new ArgumentNullException("user"); }
var userDto = httpClient.PostAsJsonAsyncWithReturnValue("api/User", user.ToDtoModel());
return userDto.ToViewModel();
}
public string GetAccessToken(string userID, string password, out UserViewModel user)
{
var token = GetAccessToken(userID, password, out user, "");
return token;
}
private string GetAccessToken(string userID, string password, out UserViewModel user, string clientId = "")
{
UserDto userDto;
var token = httpClient.GetAccessToken(userID, password, out userDto, clientId);
user = userDto.ToViewModel();
return token;
}
public Models.UserViewModel UpdateUserOnLineStatus(Models.UserViewModel user)
{
var userDto = user.ToDtoModel();
httpClient.PutAsJsonAsync("api/User/OnLine", user, true);
return user;
}
public List<Models.UserViewModel> GetOnlineUser()
{
var userList = httpClient.GetAsync<List<UserDto>>(string.Format("Api/User/GetOnLineUser"));
List<Models.UserViewModel> result = new List<Models.UserViewModel>();
userList.ForEach(a => result.Add(a.ToViewModel()));
return result;
}
}
}
|
/*
* Copyright 2012-2018 Chronicle Map Contributors
*
* Licensed under the Apache License, Version 2.0 (the "License");
* you may not use this file except in compliance with the License.
* You may obtain a copy of the License at
*
* http://www.apache.org/licenses/LICENSE-2.0
*
* Unless required by applicable law or agreed to in writing, software
* distributed under the License is distributed on an "AS IS" BASIS,
* WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
* See the License for the specific language governing permissions and
* limitations under the License.
*/
package net.openhft.chronicle.hash.impl.util;
import java.io.IOException;
import java.nio.ByteBuffer;
import java.nio.channels.FileChannel;
public final class FileIOUtils {
private FileIOUtils() {
}
public static void readFully(FileChannel fileChannel, long filePosition, ByteBuffer buffer)
throws IOException {
int startBufferPosition = buffer.position();
while (buffer.remaining() > 0 &&
buffer.position() < fileChannel.size()) {
int bytesRead = fileChannel.read(buffer,
filePosition + buffer.position() - startBufferPosition);
if (bytesRead == -1)
break;
}
}
public static void writeFully(FileChannel fileChannel, long filePosition, ByteBuffer buffer)
throws IOException {
int startBufferPosition = buffer.position();
while (buffer.remaining() > 0) {
fileChannel.write(buffer, filePosition + buffer.position() - startBufferPosition);
}
}
}
|
import React, { Component } from 'react';
import PropTypes from 'prop-types';
import { Wrapper, NameHolder } from './Item.style';
class Item extends Component {
static propTypes = {
name: PropTypes.oneOfType([PropTypes.string, PropTypes.element]).isRequired,
}
render() {
const { name } = this.props;
return (
<Wrapper>
<NameHolder>{name}</NameHolder>
</Wrapper>
);
}
}
export default Item;
|
Changed Linear to rename In and Out to avoid conflicting names (fixes #53)
It's a pretty small pull request. The only changes are to src/haliax/nn/linear.py and an added test at tests/test_nn.py.
The change in linear.py is to make Linear rename the In axis name(s) to end in "_in" and the Out axis name(s) to end in "_out" when creating the weight/bias tensors. When Linear is called, it now renames the inputs and outputs accordingly.
The added test in test_nn.py makes sure that a Linear module can be created with conflicting axes' names in In and Out.
fixes #53
thanks for doing this. I kind of think the way it was originally specced is the correct way, b/c of sharding, but I'm willing to be convinced
|
Add support for changing always finish activities.
Adds a setAlwaysFinishActivitiesState() method to control the state of the "always finish activities" setting.
Looks good to me, thanks!
|
package com.marsrover;
import static org.junit.Assert.assertEquals;
import org.junit.Test;
/**
* Test class for Position.
*
* @author Shiva
*/
public class PositionTest {
/**
* +VE: Testing the constructor and assigning the value
*/
@Test
public void testPositionConstructor() {
int x = 5;
int y = 6;
int orientation = 1;
Position pos = new Position(x, y, orientation);
assertEquals(x, pos.getX());
assertEquals(y, pos.getY());
assertEquals(orientation, pos.getOrientation());
}
}
|
Talk:New Year's Party Hat/@comment-34877689-20180604205902
Will anyone take my list for orange party hat? The two wrists are a decent short collar
|
package com.androidnerds.tictactoe.game;
import androidx.annotation.NonNull;
import com.androidnerds.tictactoe.game.model.Cell;
import com.androidnerds.tictactoe.game.model.CellState;
import com.androidnerds.tictactoe.game.model.EvaluationResult;
import java.util.ArrayList;
import java.util.List;
import static com.androidnerds.tictactoe.game.BoardUtils.isIndexInPrincipalDiagonal;
import static com.androidnerds.tictactoe.game.BoardUtils.isIndexOnBoard;
import static com.androidnerds.tictactoe.game.BoardUtils.isIndexOnCornerOfBoard;
import static com.androidnerds.tictactoe.game.BoardUtils.isIndexOnSecondaryDiagonal;
/**
* Utility class that performs the rules validation for the Tic-Tac-Toe game.
*/
public final class GameStatusEvaluator {
private GameStatusEvaluator() {
//empty constructor
}
/**
* Method evaluates whether the user has won the game after the current move.
* For evaluation, it applies the below set of rules.
* Rule#1 - User has selected all the cells in the row as the current selected cell.
* Rule#2 - User has selected all the cells in the column as the current selected cell.
* Rule#3 - User has selected all the cells in the principal diagonal.
* Rule#4 - User has selected all the cells in the secondary diagonal.
* Rule#5 - User has selected all the cells on the four corners of the board.
* Rule#6 - User has selected a 2x2 box on the board.
*
* @param board - 2d array representation of the tic-tac-toe board
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
* @return {@link EvaluationResult} object which contains:
* 1. Status as whether the user has won or not
* 2. If won, then contains list of cells that were matched.
*/
@NonNull
protected static EvaluationResult evaluate(@NonNull CellState[][] board, int row, int column) {
EvaluationResult evaluationResult = hasPlayerWonTheRow(board, row, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
evaluationResult = hasPlayerWonTheColumn(board, row, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
evaluationResult = hasPlayerWonThePrincipalDiagonal(board, row, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
evaluationResult = hasPlayerWonTheSecondaryDiagonal(board, row, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
evaluationResult = hasPlayerWonAllCorners(board, row, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
return hasPlayerWonASquare(board, row, column);
}
/**
* Method returns whether the player has selected all the cells in the current row.
*
* @param board - 2d array representation of the tic-tac-toe board
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
*/
@NonNull
private static EvaluationResult hasPlayerWonTheRow(@NonNull CellState[][] board, int row, int column) {
CellState currentPlayerState = board[row][column];
EvaluationResult evaluationResult = new EvaluationResult();
List<Cell> matchedCells = new ArrayList<>();
int columnIndex = 0;
while (columnIndex < board[row].length && board[row][columnIndex] == currentPlayerState) {
matchedCells.add(new Cell(row, columnIndex, true, currentPlayerState));
columnIndex++;
}
if (columnIndex == board[row].length) {
evaluationResult.setWin(true);
evaluationResult.setMatchedCells(matchedCells);
} else {
evaluationResult.setWin(false);
}
return evaluationResult;
}
/**
* Method returns whether the player has selected all the cells in the current column.
*
* @param board - 2d array representation of the tic-tac-toe board
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
*/
@NonNull
private static EvaluationResult hasPlayerWonTheColumn(@NonNull CellState[][] board, int row, int column) {
CellState currentPlayerState = board[row][column];
EvaluationResult evaluationResult = new EvaluationResult();
List<Cell> matchedCells = new ArrayList<>();
int rowIndex = 0;
while (rowIndex < board.length && board[rowIndex][column] == currentPlayerState) {
matchedCells.add(new Cell(rowIndex, column, true, currentPlayerState));
rowIndex++;
}
if (rowIndex == board.length) {
evaluationResult.setWin(true);
evaluationResult.setMatchedCells(matchedCells);
} else {
evaluationResult.setWin(false);
}
return evaluationResult;
}
/**
* Method returns whether the player has selected all the four corners of the board.
*
* @param board - 2d array representation of the tic-tac-toe board
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
*/
@NonNull
private static EvaluationResult hasPlayerWonAllCorners(@NonNull CellState[][] board, int row, int column) {
EvaluationResult evaluationResult = new EvaluationResult();
if (isIndexOnCornerOfBoard(board, row, column)) {
CellState playerState = board[row][column];
if (board[0][0] == playerState &&
board[0][board.length - 1] == playerState &&
board[board.length - 1][0] == playerState &&
board[board.length - 1][board.length - 1] == playerState) {
evaluationResult.setWin(true);
List<Cell> matchedCells = new ArrayList<>();
matchedCells.add(new Cell(0, 0, true, playerState));
matchedCells.add(new Cell(0, board.length - 1, true, playerState));
matchedCells.add(new Cell(board.length - 1, 0, true, playerState));
matchedCells.add(new Cell(board.length - 1, board.length - 1, true, playerState));
evaluationResult.setMatchedCells(matchedCells);
}
} else {
evaluationResult.setWin(false);
}
return evaluationResult;
}
/**
* Method returns whether the player has selected all the cells in the secondary diagonal.
*
* @param board - 2d array representation of the tic-tac-toe board
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
*/
@NonNull
private static EvaluationResult hasPlayerWonTheSecondaryDiagonal(@NonNull CellState[][] board, int row, int column) {
EvaluationResult evaluationResult = new EvaluationResult();
if (isIndexOnBoard(board, row, column) && isIndexOnSecondaryDiagonal(row, column, board.length)) {
int rowIndex = 0;
int columnIndex = board.length - 1;
List<Cell> matchedCells = new ArrayList<>();
while (rowIndex < board.length &&
columnIndex >= 0 &&
board[rowIndex][columnIndex] == board[row][column]) {
matchedCells.add(new Cell(rowIndex, columnIndex, true, board[row][column]));
rowIndex++;
columnIndex--;
}
if (rowIndex == board.length && columnIndex == -1) {
evaluationResult.setWin(true);
evaluationResult.setMatchedCells(matchedCells);
}
}
return evaluationResult;
}
/**
* Method returns whether the player has selected all the cells in the principal diagonal.
*
* @param board - 2d array representation of the tic-tac-toe board
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
*/
@NonNull
private static EvaluationResult hasPlayerWonThePrincipalDiagonal(@NonNull CellState[][] board, int row, int column) {
EvaluationResult evaluationResult = new EvaluationResult();
if (isIndexOnBoard(board, row, column) && isIndexInPrincipalDiagonal(row, column)) {
int rowIndex = 0;
int columnIndex = 0;
List<Cell> matchedCells = new ArrayList<>();
while (rowIndex < board.length &&
columnIndex < board.length &&
board[rowIndex][columnIndex] == board[row][column]) {
matchedCells.add(new Cell(rowIndex, columnIndex, true, board[row][column]));
rowIndex++;
columnIndex++;
}
if (rowIndex == board.length && columnIndex == board.length) {
evaluationResult.setWin(true);
evaluationResult.setMatchedCells(matchedCells);
}
}
return evaluationResult;
}
@NonNull
private static EvaluationResult validateSquare(@NonNull CellState[][] board, CellState playerState, int rowStart, int columnStart, int rowEnd, int columnEnd) {
int columnIndex;
int rowIndex;
boolean playerWon = true;
EvaluationResult evaluationResult = new EvaluationResult();
List<Cell> matchedCells = new ArrayList<>();
for (columnIndex = columnStart; columnIndex <= columnEnd; columnIndex++) {
for (rowIndex = rowStart; rowIndex <= rowEnd; rowIndex++) {
if (!isIndexOnBoard(board, rowIndex, columnIndex) || board[rowIndex][columnIndex] != playerState) {
playerWon = false;
break;
} else {
matchedCells.add(new Cell(rowIndex, columnIndex, true, playerState));
}
}
if (playerWon && rowIndex == rowEnd + 1 && columnIndex == columnEnd) {
evaluationResult.setWin(true);
evaluationResult.setMatchedCells(matchedCells);
}
}
return evaluationResult;
}
/**
* Method evaluates whether the player has formed a 2x2 box after the current move.
* The selected cell can possibly form 4 2x2 box with the adjacent elements on the board.
* 1. Top Left box.
* 2. Top Right box.
* 3. Bottom Left box.
* 4. Bottom Right box.
* If the user forms a 2x2 box on, then the player has won the game.
*
* @param board - 2d array representation of the tic-tac-toe board.
* @param row - rowIndex of the selected cell on the board.
* @param column - columnIndex of the selected cell on the board.
*/
@NonNull
private static EvaluationResult hasPlayerWonASquare(@NonNull CellState[][] board, int row, int column) {
//Top Left
EvaluationResult evaluationResult = validateSquare(board, board[row][column], row - 1, column - 1, row, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
//TopRight
evaluationResult = validateSquare(board, board[row][column], row - 1, column, row, column + 1);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
//BottomLeft
evaluationResult = validateSquare(board, board[row][column], row, column - 1, row + 1, column);
if (evaluationResult.hasWon()) {
return evaluationResult;
}
//BottomRight
return validateSquare(board, board[row][column], row, column, row + 1, column + 1);
}
}
|
Jasperson and others, Appellants, vs. Industrial Commission and others, Respondents.
March 9
April 11, 1939.
For the appellants there was a brief by Quarles, Spence & Quarles, attorneys, and Kenneth Grubb and Henry S. Reuss of counsel, all of Milwaukee, and oral argument by Mr. Grubb.
For the respondent Industrial Commission there was a brief by the Attorney General and Mortimer Levitan, assistant attorney general, and oral argument by Mr. Levitan.
For the respondents Hardware Mutual Casualty Company and James Peterson there was a brief by Richmond, Jack-man, Wilkie & Toebaas of Madison, and oral argument by W. L. Jackman and H. M. Wilkie.
Fowler, J.
The plaintiffs are partners doing business under the name of Whittlesey Cranberry Company and their compensation insurer. The defendants are James Peterson, a trucker, his compensation insurer, and Emmerick, who1 was an employee of either the Cranberry Company or Peterson and was injured in the course of his employment. The commission found that. Emmerick was an employee of the Cranberry Company (hereinafter referred to as the “company”), and ordered compensation to be paid by them and their insurer. The circuit court confirmed the award. Plaintiffs claim that he was an employee of Peterson, and the compensation should have been ordered paid by him and his insurer.
Emmerick was hauling- sapd. in his truck to be spread upon the cranberry marsh of the company. His contract of hire was made upon the marsh the day he began work in the presence of Peterson and Damme. Damme had charge of the spreading of the sand for the company with power to- hire and discharge employees for them. The plaintiffs claim that Emmerick’s contract of hire must be determined entirely from what was said in the conversation in which these men then and there participated, and that there is no conflict in the testimony of these three men as to’ what was said. Perhaps if there were no such conflict the plaintiffs’ contention should be upheld. But examination of the record discloses a conflict in important particulars. According to the testimony of Emmerick and Damme as to- what was said a clear case of a contract of hire between Peterson and Emmerick was made. That contract was that Emmerick was to haul sand for Peterson at $1 an hour, and was to haul at least eight loads a day. The men were working eight hours a day. Peterson had three of his own trucks hauling sand and he was being paid $1 a load. Thus, if Emmerick hauled more than eight loads a day, and he said he could haul at least ten loads in eight hours, Peterson would, under the testimony of Damme and Emmerick, be making $1 a load on all that Emmerick hauled over eight loads a day. Certain other items of evidence strongly indicate that all three persons present during the conversation at the time considered that Emmerick was an employee of Peterson. Peterson on the day of the injury notified his insurer of it. Damme, who made out the cards showing the number of loads hauled, made out a card showing delivery of twenty-six loads on the day Emmerick worked, and the testimony is undisputed that Peterson’s trucks hauled only twenty-four loads that day. Emmerick sent a bill to Peterson for either $2 or $3 for his services hauling sand on that day, without stating either the number of hours worked or the number of loads hauled. He also laid his claim before the commission as against Peterson. But PetersÓn testified that he told Emmerick he (Emmerick) was to' receive $1 for each load hauled. He also testified that “I told him [Emmerick] he could get the same as we were getting, a dollar per load.” Peterson also testified that Damme said that Emmerick could get gas from him, and Emmerick asked about paying for it, and Damme said he would take it out of his check. Peterson admits it was he who> told Emmerick he could go to work, but also testified that when he made his agreement with Mr. Jasperson, of the company, for hauling sand, Jasperson asked him if he could get other trucks besides his own, and he told Jasperson that he “would do that for him.” He also procured another trucker besides Emmerick to' put two trucks on the job, but before telling him he could go* to work he told Jasperson that this trucker wanted $1.50 a load for one of his trucks which held a yard more than the other and $1 for the other which Jasperson agreed to pay, and told him to “get the trucks for him,” and he so told the trucker. After the injury and his adjuster had talked to him about it, Peterson told Jasperson that he didn’t want anything more to do with hiring trucks.
Besides this conflict of testimony between Peterson on the one hand and Emmerick and Damme on the other, Emmerick and Damme signed two statements before an adjuster of the insurer of the company, and all of these statements would support the inference that Emmerick was an employee of the company. The statements of both are in conflict with their testimony on the stand in important particulars. Damme’s statements are to the effect that he understood that he had hired Emmerick for the company, and that the company was to pay him and deduct the gas furnished him from his pay. In the written statement Emmerick stated that he was to' be paid $1 a load, and that he didn’t know who was to' pay him. Damme claimed on the hearing that his written statements, so far as contradictory with his testimony, were false, and that he knew they were false when he made them, but he made them because he wanted Emmerick to get compensation, and that he knew the company carried insurance. The company claims these statements are hearsay, and cannot be taken as evidence of the contract of hire. But if this be so, they impeach the testimony of both Emmerick and Damme, and they were proper for consideration in determining whether Peterson’s version of the conversation constituting the contract of hire or that of Emmerick and Damme was correct. In view of the statutory rule that the commission’s findings are conclusive, they must be sustained if there is evidence to support them. See sec. 102.23, Stats. We cannot say, in view of Peterson’s testimony, that there is no evidence to support the finding that Emmerick was an employee of the company. Nor can we say Peterson’s evidence is not “substantial” evidence under the requirement of the rule of Consolidated Edison Co. v. National L. R. Board, 305 U. S. 197, 229, 59 Sup. Ct. 206, 83 L. Ed. 126, where it is said of a like statutory provision in the National Labor Relations Act:
“ ‘The findings of the board as to- the facts, if supported by evidence, shall be conclusive,’ means supported by substantial evidence. . . . Substantial evidence is more than a mere scintilla. It means such relevant evidence as a reasonable mind might accept as adequate to support a conclusion.”
By the Court. — The judgment of the circuit court is affirmed.
|
Jacob J. Brown et al., Respondents, v. Dennis W. Neely et al., Appellants.
Appeal by defendants from a judgment in favor of plaintiffs, in an action for an injunction restraining defendants from interfering with the natural flow of a brook running through the properties of plaintiffs and defendants, which interference, plaintiffs claim, caused damage to their house. Judgment unanimously affirmed, with costó. No opinion. Present — Nolan, P. J., Johnston, Adel, Wenzel and MacCrate, JJ. [197 Misc. 173.] [See 278 App. Div. 569.]
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incendiar
Verb
* 1) to set fire to, set on fire
* 2) to burn down maliciously
Etymology
From.
Verb
* 1) to set something on fire
Etymology
, from.
Adjective
* 1) incendiary
Etymology
From, or Latin incendō.
Verb
* 1) to set fire to
|
In re COMBUSTION, INC.
Action No. 94MDL4000.
United States District Court, W.D. Louisiana, Lafayette-Opelousas Division.
Jan. 5, 1995.
Calvin Fayard, plaintiffs’ Steering Committee, Denham Springs, LA.
Thomas McNamara, defendants’ Steering Committee, Liskow & Lewis, Lafayette, LA; and Charles Seabolt, McDermott, Inc., New Orleans, LA.
PARTIAL MEMORANDUM RULING
TYNES, United States Magistrate Judge.
Pending discovery motions raise numerous issues related to the application and interpretation of various privileges and doctrines, including the attorney-client and joint defense privileges and the work product doctrine. The threshold issue which must be determined before these various issues can be analyzed individually is whether state or federal law governs. This issue is addressed independent of the remaining issues raised in plaintiffs’ discovery motions.
In general terms, this class action, toxic tort litigation involves the damage claims filed by the original plaintiffs in state court, which raise issues of state law; the third party tort indemnity claims, which raise issues of state law; the third party federal tort claim indemnity issues, which raise issues of federal law; and finally, the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA), 42 U.S.C. 9601, et seq. (1987) claims, which raise issues of federal law. The question of whether state or federal privilege law applies in this case is determined by Fed.R.Evid. 501 and interpretive jurisprudence.
Fed.R.Evid. 501 provides:
Except as otherwise required by the Constitution of the United States or provided by Act of Congress or in rales prescribed by the Supreme Court pursuant to statutory authority, the privilege of a witness, person, government, State, or political subdivision thereof shall be governed by the principles of the common law as they may be interpreted by the courts of the United States in the light of reason and experience. However, in civil actions and proceedings, with respect to an element of a claim or defense as to which State law supplies the rule of decision, the privilege of a witness, person, government, State, or political subdivision thereof shall be determined in accordance with State law. (emphasis added).
The Plaintiffs’ Steering Committee (“PSC”) contends that state law governs all privilege issues, citing Fed.R.Evid. 501. The Defendants’ Steering Committee (“DSC”) states, on the other hand, that the issue is a “red herring” inasmuch as it is “well settled that federal law governs the work product doctrine in federal courts” and inasmuch as Louisiana statutory law and federal common law both expressly recognize the joint defense privilege.
While the issue of which law controls the work product doctrine is not actually well settled, a significant number of trial courts across the country have held that federal law, i.e. F.R.Civ.P. 26(b)(3) and interpretive jurisprudence, governs the application of the work product doctrine. See Shipes v. BIC Corp., 154 F.R.D. 301, 305 n. 2 (M.D.Ga. 1994); EDO Corp. v. Newark Ins. Co., 145 F.R.D. 18, 21 (D.Conn.1992); Harper v. Auto-Owners Ins. Co., 138 F.R.D. 655, 658 (S.D.Ind.1991); Auto-Owners Ins. Co. v. Totattape, Inc., 135 F.R.D. 199, 201 (M.D.Fla. 1990); Fine v. Facet Aerospace Prods. Co., 133 F.R.D. 439 (S.D.N.Y.1990); Airheart v. Chicago and North Western Transp. Co., 128 F.R.D. 669, 670 (D.S.D.1989); Rinaldi’s Fast Foods, Inc. v. Great American Ins. Cos., 123 F.R.D. 198 (M.D.N.C.1988); Railroad Salvage of Conn., Inc. v. Japan Freight Consolidators, Inc., 97 F.R.D. 37 (E.D.N.Y.1983).
The rationale underlying the conclusion reached by these courts is that the work product doctrine is not a substantive privilege within the meaning of Rule 501; instead, it is a device providing qualified immunity from discovery. In the words of the Rinaldi court:
“Work product is not a privilege within the meaning of Rule 501 which protects the sanctity of confidential communications. Rather, it is a tool of judicial administration, borne out of concerns over fairness and convenience and designed to safeguard the adversarial system, but not having an intrinsic value in itself outside the litigation arena.” (citations omitted).
123 F.R.D. at 201; see also, 8 C. Wright & A. Miller & R. Marcus, Federal Practice and Procedure Section 2025, at 212 (1970) (work product may be more accurately described as providing an immunity as opposed to a privilege for confidential communications).
I join the courts cited above in holding that the work product doctrine is not a privilege within the meaning of Rule 501. Therefore, federal law provides the decisional framework for all work product issues raised in this litigation.
I turn now to a discussion of the attorney client and joint defense privileges. As a preliminary matter, the DSC’s arguments suggest that a ruling on whether state or federal privilege law applies is unnecessary inasmuch as both Louisiana statutory law and federal common law expressly recognize the joint defense and attorney client privileges. I do not find the DSC’s position on this issue persuasive for several reasons.
First, the Louisiana statutory law recognizing the joint defense privilege did not become effective until January 1, 1993. La. C.E. Art. 506 B.(3) (1993). The discovery issues raised by plaintiffs’ motions have been pending since 1992 when motions were first filed in state court. Although the DSC contends in its’ brief that Article 506 B.(3) merely codified earlier jurisprudence, the DSC cited no jurisprudence to support this statement, and indeed, this Court’s computer aided legal research revealed no Louisiana cases directly recognizing the joint defense privilege, either prior or subsequent to La.C.E. Art. 506 B.(3). Thus, the question of whether the joint defense privilege was cognizable under Louisiana law at the relevant time remains open. In addition to questions concerning when the joint defense privilege was first recognized by Louisiana law, and whether Article 506 B. (3) applies retrospectively, there is the possibility that state courts will interpret and apply the joint defense privilege differently during the future pendency of this litigation than the federal courts have in the past. Finally, there are, more likely than not, nuances between state and federal interpretations of the attorney-client privilege.
For these reasons, and in the interests of certainty and order, the question of whether state or federal privilege law governs the interpretation of attorney-client and joint defense privilege issues must be addressed.
The language of Rule 501 raises more questions, than it provides answers. In fact, the Senate Judiciary Committee anticipated the uncertainties created by the version of Rule 501 which was enacted. The Notes of Committee On the Judiciary Senate Report No. 93-1277 observe quite accurately that “[t]he formulation adopted by the House is pregnant with litigious mischief.” Rule 501, as adopted, provides that federal common law applies to issues of privilege except in civil proceedings “with respect to an element of a claim or defense as to which state law supplies the rule of decision ...” In sum, Rule 501 makes it clear that state privilege law will apply in diversity cases, and that federal privilege law will apply in federal question cases. However, in federal question cases where pendent state law claims have been asserted, the rule is equivocal.
The Senate Committee Notes to Senate Report No. 93-1277 expressly anticipated several of the problems with which the courts have grappled in applying Rule 501, as follows:
“Another problem not entirely avoidable is the complexity or difficulty the Rule introduces into the trial of a federal case containing a combination of federal and state claims and defenses ... two different bodies of privilege law would need to be consulted. It may even develop that the same witness-testimony might be relevant on both counts and privileged as to one but not the other ...” Notes of Committee On the Judiciary, Senate Report No. 93-1277.
Nor does the legislative history shed much light upon congressional intent concerning the problem of conflicting federal and state privilege law. American Civil Liberties Union of Mississippi, Inc. v. Finch, 638 F.2d 1336, 1343 n. 13 (5th Cir.Unit A March 1981); Walker v. Lewis, 127 F.R.D. 466, 468 (W.D.N.C.1989); Perrignon v. Bergen Brunswig Corp., 77 F.R.D. 455, 458-59 (N.D.Cal. 1978).
With little guidance from the text of the rule or the legislative history, the courts have decided the issue of conflicting federal and state privilege law for themselves. A number of courts that have confronted the issue in the context of the discoverability of evidence have held that the federal law of privilege governs where the evidence sought is relevant to both federal and state claims. von Bulow v. von Bulow, 811 F.2d 136, 140 (2nd Cir.1987); Wm. T. Thompson Co. v. General Nutrition Corp., 671 F.2d 100, 104 (3rd Cir.1982); Memorial Hosp. for McHenry County v. Shadur, 664 F.2d 1058, 1061 n. 3 (7th Cir.1981); Perrignon v. Bergen Brunswig Corp., supra; Cary v. Soileau, 125 F.R.D. 432 (W.D.La.1989); Pinkard v. Johnson, 118 F.R.D. 517 (M.D.Ala.1987); First Federal Savings & Loan v. Oppenheim, Appel, Dixon, 110 F.R.D. 557, 560 (S.D.N.Y. 1986); Sirmans v. City of South Miami, 86 F.R.D. 492, 494-95 (S.D.Fla.1980).
The Sixth and Eleventh Circuit Courts of Appeals joined and broadened this trend in 1992 holding in sweeping language that the federal law of privilege governs determination of all privilege issues raised in a federal question case involving pendent state law claims, even where the evidence sought is relevant to a pendent state law claim to which a contrary state privilege law would otherwise apply. Hancock v. Hobbs, 967 F.2d 462 (11th Cir.1992); Hancock v. Dodson, 958 F.2d 1367 (6th Cir.1992). See also, Puricelli v. Borough of Morrisville, 136 F.R.D. 393, 396 (E.D.Pa.1991).
Although the Fifth Circuit has never directly addressed the pending issue, it did address a similar issue in American Civil Liberties Union of Mississippi, Inc. v. Finch. In Finch, the Fifth Circuit held that compelling federal interests in a ease arising under 42 U.S.C. § 1983 permitted the federal court to assess the applicability of a claimed state-law privilege independent of the judgment of the Mississippi state legislature and state courts, 638 F.2d at 1343. Presumably, then, Finch advises trial courts to balance competing state and federal interests in determining what weight shall be given to state privilege law. In the case at hand, federal question jurisdiction is substantively based on CERCLA and the Federal Tort Claims Act, 28 U.S.C. 1346, both of which invoke exclusive federal jurisdiction. Clearly, federal interests here are strong. Thus, Finch seems to provide further support for the application of federal privilege law as well.
Finally, the general policies of the federal rules favoring uniformity and simplicity support the approach taken by the Hancock decisions. An approach consistent with the literal language of Rule 501 would dictate that every question posed at a deposition and every written interrogatory or request for production propounded be evaluated to determine whether it sought evidence solely related to state issues, solely related to federal issues, or related to both federal and state issues and then if it appeared that the evidence related to both federal and state issues, under Finch, one would then weigh the competing federal and state interests to determine which privilege law would ultimately apply. Such an approach would be unworkable in most cases, and would create chaos in a case of proportions as massive as this one. See Hancock v. Hobbs, 967 F.2d at 466; Wm. T. Thompson Co., 671 F.2d at 104 expressly acknowledging the impracticality of such an approach.
In accord with the foregoing, I further hold that the federal law of privilege provides the rule of decision with respect to privilege issues affecting the discoverability of evidence in this federal question case involving pendent state law claims.
The Clerk of Court is directed to provide facsimile notice of this partial ruling to all liaison counsel.
. This partial ruling addresses one significant issue raised in the motions filed by the plaintiffs docketed as Official Documents 926 and 965. The remaining issues will be resolved by separate ruling.
. It would be extremely burdensome, if not actually impossible, to conduct a comprehensive evaluation at this point to determine whether any conflicts exist between federal and stat'e interpretations of the attorney-client privilege. Experience leads me to believe that there are nuances, if not actual conflicts, between federal and state interpretations.
. See the following commentators for discussion of the interpretation difficulties created by Rule 501: 23 C. Wright & K. Graham, Federal Practice and Procedure, Section 5434 at 861-864 (1980); 2 J. Weinstein & M. Berger, Weinstein’s Evidence, P 501(02) at 501-22 to -23 (1980).
. The deadline for appeal of this partial ruling is January 16, 1995. (U.L.L.R. 19.09W allows the Court to modify the time period for appeal).
|
Umm Khurayzah
Um Khreizeh (أم خريزة) is a Syrian village located in Sabburah Subdistrict in Salamiyah District, Hama. According to the Syria Central Bureau of Statistics (CBS), Um Khreizeh had a population of 250 in the 2004 census.
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Sprite animation wobbly / jumping in IE11
I built a semi-transparent sprite png which can be found at https://www.srf.ch/static/srf-data/test_sprite.png
It is a 17280px high and 910px wide png (30 * 576 = 17280) - everything seems correct.
I now want to loop through each frame (it should be a map of slightly moving points) with CSS3 keyframes using background-position.
I adapted an example from https://builtvisible.com/3-logical-alternatives-to-animated-gifs/ (see http://codepen.io/tombennet/pen/oxmaLd)
I can reproduce that example, when I try to adapt it to my needs (i.e. my sprite), I get this: http://codepen.io/anon/pen/rmwwMG
Now: On IE11, Windows 7, this thing is jumping very slightly up and down. In FF or Chrome it is displaying correctly.
What startles me is that the Ninja guy from the initial example doesn't jump, he seems to work fine. I wonder what the difference in my sprite/css is.
On IE11, Windows 7, this thing is jumping very slightly up and down. In FF or Chrome it is displaying correctly.
My guess is that the issue is caused by the way different browsers handle the rounding of floating point numbers. IE11 (as well as Edge) truncates after the second decimal place which makes the positioning sometimes imprecise. There have been lot of complaints about this behaviour as for example the building of a layout grid (columns) with percentage values requires some additional hacks to add the columns' width to 100%.
In Firefox (53) and Chrome (57) the number is rounded to at least the fourth decimal. That makes a visible difference in your example. If we have 29 steps, each step moves the background-image by 3.448...%, so after 6 steps the value should be at 20.6896...%. I took this specific step as here we get the biggest difference between the actual value and the visible value shown in IE11 (20.68%). This results in an inaccuracy of ~1.67px. On average we have an inaccuracy of 0.86px in this example.
The reason why it does not flicker in the ninja example is that the inaccuracy is lower (due to a smaller image height of 752px compared to your 17280px; note that the image's height gets multiplied by the percentage value so that a higher pixel value has a greater impact). The maximum difference between the actual value and the rendered value is only 0.0752px and on average we only have an inaccuracy of 0.0376px in the ninja example.
How to solve the issue
It's really as simple as not to rely on floating point numbers in this scenario. We move the background-image by 576px for 30 steps:
.test {
width: 910px;
height: 576px;
background: url('https://i.sstatic.net/Tsw6G.png') no-repeat 0 0%;
animation: sprite 1s steps(30) infinite;
}
@keyframes sprite {
from { background-position: 0 0px; }
to { background-position: 0 -17280px; }
}
<div class="test"></div>
Absolutely great answer - it makes things clear and presents a working solution, thanks a lot. I actually experimented with setting pixel values but I still used 29 steps (instead of 30) and I wrongly specified background-position: 0 17280px instead of the negative number. If I assume correctly one uses negative values because that's how the pixel coordinate system works?
I am glad that you like it :) I enjoy debugging and answering these kind of edge cases. Regarding the question with the negative value: if you want the image to move to the top you have to pull it up. I've quickly created a fiddle for demonstrating how positioning works (just imagine that the background placeholder is one frame of your image).
I now tried to adapt an example from https://www.sitepoint.com/responsive-sprite-animations-imagemagick-greensock/ in order to make it responsive. There (I guess) I had to use percentages. Now the image even jumps in Chrome et al., depending on the window width: http://codepen.io/anon/pen/QvQWJP. Interestingly, at 910px it works great (probably because that's the original size). The article also talks about EMs and setting the base font size, maybe that would be a possibility (instead of percentages?). Quick help would be greatly appreciated!
I tried several things but couldn't figure out what's the reason for this behaviour here, sorry :(
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List Candidates in Election class
@hadryyassine hello yassine we're seeing redundancy in the candidate data because each candidate object contains a nested JSON object representing the election, which also includes the list of candidates ...
Hello Hossam, I think you're right. Thanks for the remark.
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This text is missing some spaces, please add them: ShowfamilytreestartingwithAneLarsen
Show family tree starting with Ane Larsen
This text is missing some spaces, please add them: Largeapartmentforupto8peoplewith5m2privateterracenexttoSagradaFamília,Barcelona
Large apartment for up to 8 people with 5 m2 private terrace next to Sagrada Família, Barcelona
This text is missing some spaces, please add them: Inseconds,youwilllearnexactlywhattypeofMVDfileyouhave,thesoftwareprogramassociatedwithyourfile,thepublisherwhocreatedit,itssecuritysafetystatus,andavarietyofotherusefulinformation.
In seconds, you will learn exactly what type of MVD file you have, the software program associated with your file, the publisher who created it, its security safety status, and a variety of other useful information.
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Please create TypeScript definition file for VueRouter
At the moment, only "vue" is available at http://definitelytyped.org/tsd/.
+1
It's in the repo but for some reason not showing up in search. https://github.com/DefinitelyTyped/DefinitelyTyped/blob/41f8573534b0fff88707d0a4cb870456b50cd43b/vue-router/vue-router.d.ts
Sorry to ask, but will it be included in the npm like vue and vue-class-component ?
It is so nice to be able to write vue code without typings :-)
@druppy yeah, we will likely do that in the future.
For whatever reason, @yyx990803, the one in DefinitelyTyped doesn't have a router.map() function. Am I following outdated docs or is this an issue?
@20zinnm If you're moving to Vue 2.x, router.map() has been deprecated in vue-router@next (VueJS 2.x) It has been replaced by the routes option.
Oh. Thanks :). Never would've guessed there was a new router.
On Sep 20, 2016 9:05 PM, "jeffcj"<EMAIL_ADDRESS>wrote:
@20zinnm https://github.com/20zinnm If you're moving to Vue 2.x,
router.map() has been deprecated in vue-router@next (VueJS 2.x) It has
been replaced by the routes option.
—
You are receiving this because you were mentioned.
Reply to this email directly, view it on GitHub
https://github.com/vuejs/vue-router/issues/372#issuecomment-248490465,
or mute the thread
https://github.com/notifications/unsubscribe-auth/AF2RQn-SAGMvhUayYE3ouxB9gvgC0ENsks5qsJDfgaJpZM4HZnin
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|
var vm = require('vm');
var fs = require('fs');
// this is the context in which all of our test code will run
var initSandbox = {
console: console,
require: require,
setTimeout: setTimeout,
clearTimeout: clearTimeout,
Buffer: Buffer
};
var context = vm.createContext(initSandbox);
if(!process.argv[2]) {
console.log('No makeCouch.js specified');
process.exit(1);
}
else {
console.log('Using ' + process.argv[2]);
}
fs.readFile('../browser/qunit/qunit/qunit.js', 'utf-8', function(err, data) {
if(err) {
throw err;
}
vm.runInContext(data, context);
fs.readFile('../../sag.js', 'utf-8', function(err, data) {
if(err) {
throw err;
}
vm.runInContext(data, context);
fs.readFile(process.argv[2], 'utf-8', function(err, data) {
vm.runInContext(data, context);
fs.readFile('../sag-tests.js', 'utf-8', function(err, data) {
if(err) {
throw err;
}
vm.runInContext(data, context);
});
});
});
});
|
<?php
class Epg
{
public $validEpg = false;
public $epgSource;
public $from_cache = false;
function __construct($result, $set = false)
{
$this->LoadEpg($result, $set);
}
public function getData()
{
$output = array();
foreach ($this->epgSource->channel as $item) {
$channel_id = trim((string) $item->attributes()->id);
$display_name = !empty($item->{'display-name'}) ? trim((string) $item->{'display-name'}) : '';
if (array_key_exists($channel_id, $output)) {
continue;
}
$output[$channel_id] = array();
$output[$channel_id]['display_name'] = $display_name;
$output[$channel_id]['langs'] = array();
}
foreach ($this->epgSource->programme as $item) {
$channel_id = trim((string) $item->attributes()->channel);
if (!array_key_exists($channel_id, $output)) {
continue;
}
$title = $item->title;
foreach ($title as $data) {
$lang = (string) $data->attributes()->lang;
if (!in_array($lang, $output[$channel_id]['langs'])) {
$output[$channel_id]['langs'][] = $lang;
}
}
}
return $output;
}
public function getProgrammes($epg_id, $streams)
{
global $ipTV_db;
$list = array();
foreach ($this->epgSource->programme as $item) {
$channel_id = (string) $item->attributes()->channel;
if (!array_key_exists($channel_id, $streams)) {
continue;
}
$desc_data = $data = '';
$start = strtotime(strval($item->attributes()->start));
$stop = strtotime(strval($item->attributes()->stop));
if (empty($item->title)) {
continue;
}
$title = $item->title;
if (is_object($title)) {
$epg_lang_check = false;
foreach ($title as $data) {
if ($data->attributes()->lang == $streams[$channel_id]['epg_lang']) {
$epg_lang_check = true;
$desc_data = base64_encode($data);
break;
}
}
if (!$epg_lang_check) {
$desc_data = base64_encode($title[0]);
}
} else {
$desc_data = base64_encode($title);
}
if (!empty($item->desc)) {
$desc = $item->desc;
if (is_object($desc)) {
$epg_lang_check = false;
foreach ($desc as $data) {
if ($data->attributes()->lang == $streams[$channel_id]['epg_lang']) {
$epg_lang_check = true;
$data = base64_encode($data);
break;
}
}
if (!$epg_lang_check) {
$data = base64_encode($desc[0]);
}
} else {
$data = base64_encode($item->desc);
}
}
$channel_id = addslashes($channel_id);
$streams[$channel_id]['epg_lang'] = addslashes($streams[$channel_id]['epg_lang']);
$date_start = date('Y-m-d H:i:s', $start);
$date_stop = date('Y-m-d H:i:s', $stop);
$list[] = '(\'' . $ipTV_db->escape($epg_id) . '\', \'' . $ipTV_db->escape($channel_id) . '\', \'' . $ipTV_db->escape($date_start) . '\', \'' . $ipTV_db->escape($date_stop) . '\', \'' . $ipTV_db->escape($streams[$channel_id]['epg_lang']) . '\', \'' . $ipTV_db->escape($desc_data) . '\', \'' . $ipTV_db->escape($data) . '\')';
}
return !empty($list) ? $list : false;
}
public function LoadEpg($result, $set)
{
$errors = pathinfo($result, PATHINFO_EXTENSION);
if (($errors == 'gz')) {
$content = file_get_contents($result);
$epgSource = simplexml_load_string($content, 'SimpleXMLElement', LIBXML_COMPACT | LIBXML_PARSEHUGE);
$content = gzdecode(file_get_contents($result));
$epgSource = simplexml_load_string($content, 'SimpleXMLElement', LIBXML_COMPACT | LIBXML_PARSEHUGE);
}
else if ($errors == 'xz') {
$content = shell_exec("wget -qO- \"{$result}\" | unxz -c");
$epgSource = simplexml_load_string($content, 'SimpleXMLElement', LIBXML_COMPACT | LIBXML_PARSEHUGE);
}
if ($epgSource !== false) {
$this->epgSource = $epgSource;
if (empty($this->epgSource->programme)) {
ipTV_lib::SaveLog('Not A Valid EPG Source Specified or EPG Crashed: ' . $result);
} else {
$this->validEpg = true;
}
} else {
ipTV_lib::SaveLog('No XML Found At: ' . $result);
}
$epgSource = $content = null;
}
}
?>
|
Maria Gatland
Maria Gatland (born Maria McGuire 1948, Dublin, Ireland) is a councillor in the London Borough of Croydon for the Conservative Party, who has represented the South Croydon ward since 2018. From 2002 to 2018 she was a Conservative councillor for the Croham ward. She is also a former Croydon Council cabinet member for education, a post she resigned after controversy due to being a former member of the Provisional Irish Republican Army.
Early life
Gatland was born Maria McGuire, to a middle-class family, and lived in the suburb of Churchtown, Dublin. She was one of four siblings (two brothers and a sister). She was educated at St. Anne's Primary and Secondary Schools, Milltown, Dublin and then University College Dublin, where she studied English language and literature.
IRA involvement
McGuire became a member of the Provisional IRA in July 1971. Three months later, in October 1971, she accompanied Dáithí Ó Conaill (a leader of the Republican movement, also known as David O'Connell) to the European continent on an arms buying expedition. The expedition came to nothing because it was reported in the British press, and Ó Conaill and McGuire abandoned the mission. She and Ó Conaill had an affair during this trip, although he was married. She, Ó Conaill, and Ó Conaill's wife had a difficult discussion about the issues involved shortly after they returned to Ireland.
Provisional IRA bomb attacks in Belfast, on 21 July 1972 (known as Bloody Friday) killed seven civilians and two soldiers, and left 130 injured. McGuire subsequently decided to leave the Provisional IRA. She was told by the British authorities that if she did so she would receive Special Branch protection. In the late summer of 1972 she appeared in London and wrote a series of articles for The Observer, went into hiding, and wrote a book about her experiences in the organisation called To Take Arms: A Year in the Provisional IRA which was published in 1973. In it she was hostile to Seán Mac Stíofáin, IRA Chief of Staff at the time she was a member, but remained sympathetic to Ó Conaill, and wished him success in internal struggles against Mac Stíofáin in the Republican movement.
Political career
In 2002, Maria Gatland was elected as a member of the Conservative Party to Croydon Council as a councillor for Croham ward and in 2006 became Croydon's cabinet member for education. She says that she never hid her IRA past. When the Conservative Party found out about her past she stepped down as a council cabinet member in early December 2008. At the same time Gatland was suspended by the party, but in following month, January 2009, she was accepted back to the Tory fold. She was subsequently re-elected as a councillor in the 2010 and 2014 local elections.
Personal life
Around 1986 McGuire moved to Croydon where she met and married her late husband Mervyn Gatland, who ran a garden maintenance business. Gatland has said that at this time she was very fragile and had two breakdowns.
|
- Leu Gly Lys Lys 20 - (2) INFORMATION FOR SEQ ID NO:139: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 22 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:139: #Leu Thr Trp Gln Pro Ileeu Ile Arg Gln Leu #15 - Leu Gln Tyr Ile Leu Gln 20 - (2) INFORMATION FOR SEQ ID NO:140: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:140: #Thr Trp Leu Phe Ser Asneu Ile Arg Leu Leu #15 - Cys Arg Thr Leu Leu Ser Glu Val Tyr # 25 - (2) INFORMATION FOR SEQ ID NO:141: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 21 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:141: #Thr Trp Leu Phe Ser Asneu Ile Arg Leu Leu #15 - Cys Arg Thr Leu Leu 20 - (2) INFORMATION FOR SEQ ID NO:142: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 21 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:142: #Thr Leu Leu Arg Ile Leu sn Ser Phe Leu Trp #15 - Gln Arg Ile Leu Phe 20 - (2) INFORMATION FOR SEQ ID NO:143: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:143: #Thr Trp Leu Phe Pro Asneu Ile Arg Leu Leu #15 - Cys Arg Thr Leu Leu Ser Arg Val Tyr # 25 - (2) INFORMATION FOR SEQ ID NO:144: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:144: #Ser Phe Leu Trp Thr Leu eu Thr Arg Cys Asn #15 - Leu Arg Ile Leu Gln Arg Ile Leu Phe # 25 - (2) INFORMATION FOR SEQ ID NO:145: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:145: #Thr Trp Leu Phe Ser Asneu Ile Lys Leu Leu #15 - Cys Lys Thr Leu Leu Ser Lys Val Tyr # 25 - (2) INFORMATION FOR SEQ ID NO:146: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 23 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:146: #Ala Ser Arg Gln Leu Ile le Arg Gln Leu Thr #15 - Pro Gln Leu Ile Gln Tyr Val 20 - (2) INFORMATION FOR SEQ ID NO:147: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 23 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:147: #Ala Ser Arg Gln Leu Ile le Arg Gln Leu Thr #15 - Pro Gln Leu Ile Gln Tyr Val 20 - (2) INFORMATION FOR SEQ ID NO:148: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 27 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:148: #Pro Ile Leu Gln Arg Leu yr Gln Ile Leu Gln #15 #Leu er Ala Thr Leu Gln Ala Ile Arg Glu Val # 25 - (2) INFORMATION FOR SEQ ID NO:149: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:149: #Pro Ile Leu Gln Arg Leu yr Gln Ile Leu Gln #15 #Leu Arg la Thr Leu Gln Arg Ile Arg Glu Val # 25 - (2) INFORMATION FOR SEQ ID NO:150: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:150: #Ala Ser Leu Arg Gln Leu le Arg Gln Leu Thr #15 #Leu L euro Gln Leu Ile Gln Tyr Val Arg Ser # 25 - (2) INFORMATION FOR SEQ ID NO:151: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:151: #Pro Ile Leu Gln Lys Leu yr Gln Ile Leu Gln #15 #Leu Lys la Thr Leu Gln Lys Ile Lys Glu Val # 25 - (2) INFORMATION FOR SEQ ID NO:152: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:152: #Arg Thr Leu Leu Ser Arg eu Phe Ser Asn Cys #15 - Val Tyr Gln Ile Leu Gln Pro Ile Leu # 25 - (2) INFORMATION FOR SEQ ID NO:153: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:153: #Arg Thr Leu Leu Ser Arg eu Phe Ser Asn Arg #15 - Val Tyr Gln Ile Leu Gln Glu Ile Leu # 25 - (2) INFORMATION FOR SEQ ID NO:154: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 22 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:154: #Leu Ser Arg Val Tyr Gl neu Arg Arg Thr Leu #15 - Ile Leu Gln Glu Ile Leu 20 - (2) INFORMATION FOR SEQ ID NO:155: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 29 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:155: #Ala Val Ile Ile Arg Cy sly Leu Arg Gly Leu #15 #Ile Ile Argrg Gly Leu Asn Leu Ile Phe Glu # 25 - (2) INFORMATION FOR SEQ ID NO:156: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 29 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:156: #Ala Val Ile Ile Arg Ilely Leu Arg Gly Leu #15 #Ile Ile Argrg Gly Leu Asn Leu Ile Phe Glu # 25 - (2) INFORMATION FOR SEQ ID NO:157: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 30 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:157: #Ala Val Ile Pro Arg Arg ly Leu Arg Gly Leu #15 #Glu Ile Ile Argrg Gly Leu Asn Leu Ile Phe # 30 - (2) INFORMATION FOR SEQ ID NO:158: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 29 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:158: #Arg Ile Cys Ile Arg Ile le Leu Asn Leu Gly #15 #Ala Ile Arg la Leu Gly Arg Leu Gly Tyr Gly # 25 - (2) INFORMATION FOR SEQ ID NO:159: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 29 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:159: #Ala Val Ile Ile Lys Ilely Leu Lys Gly Leu #15 #Ile Ile Ly sys Gly Leu Asn Leu Ile Phe Glu # 25 - (2) INFORMATION FOR SEQ ID NO:160: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:160: #Arg Ala Ile Arg His Ileal Gln Gly Ala Cys #15 #Ile Leurg Arg Ile Arg Gln Gly Leu Arg Arg # 25 - (2) INFORMATION FOR SEQ ID NO:161: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:161: #Arg Ala Ile Glu His Ileal Gln Gly Ala Cys #15 #Ile Leurg Arg Ile Glu Gln Gly Leu Glu Arg # 25 - (2) INFORMATION FOR SEQ ID NO:162: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:162: #Arg Ala Ile Glu His Ileal Gln Gly Ala Cys #15 #Ile Leurg Arg Ile Arg Gln Gly Leu Glu Arg # 25 - (2) INFORMATION FOR SEQ ID NO:163: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:163: #Arg Ala Ile Arg His Ileal Gln Gly Ala Cys #15 #Ile Leurg Arg Ile Glu Gln Gly Leu Glu Arg # 25 - (2) INFORMATION FOR SEQ ID NO:164: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:164: #Arg Ala Ser Arg His Ileal Gln Gly Ala Cys #15 #Ile Leurg Arg Ile Arg Gln Gly Leu Glu Arg # 25 - (2) INFORMATION FOR SEQ ID NO:165: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:165: #Arg Ala Ile Arg His Ileal Gln Gly Ala Cys #15 #Ile Leurg Arg Ser Arg Gln Gly Leu Glu Arg # 25 - (2) INFORMATION FOR SEQ ID NO:166: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:166: #Thr Trp Leu Phe Ser Asneu Ile Glu Leu Leu #15 - Cys Arg Thr Leu Leu Ser Glu Val Tyr # 25 - (2) INFORMATION FOR SEQ ID NO:167: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:167: #Pro Ile Leu Gln Glu Leu yr Gln Ile Leu Gln #15 #Leu Arg la Thr Leu Gln Arg Ile Arg Glu Val # 25 - (2) INFORMATION FOR SEQ ID NO:168: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 21 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:168: #Val Thr Arg Ile Val Glusp Leu Leu Leu Ile #15 - Leu Leu Gly Arg Glu 20 - (2) INFORMATION FOR SEQ ID NO:169: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 28 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: None - (xi) SEQUENCE DESCRIPT - #ION: SEQ ID NO:169: #Arg Ala Ile Arg His Ileal Gln Gly Ala Tyr #15 #Ile Leurg Arg Ile Arg Gln Gly Leu Glu Arg # 25 __________________________________________________________________________
We claim:
1. A peptide selected from the group consisting of;a) RVIRVVQGACRAIRHIPRRIR (SEQ ID NO: 10), b) RVIRVVRGACRAIRHIPRRIR (SEQ ID NO: 38), c) LWETLRRGGRWILAIPRRIR (SEQ ID NO: 73), d) LWETLRRGCRWILAIPRRIR (SEQ ID NO: 92) and, e) LWRLLRRGGRWILAIPRRIR (SEQ ID NO: 97).
2. The peptide of claim 1 having the amino acid sequence:RVIRVVQGACRAIRHIPRRIR (SEQ ID NO: 10).
3. A composition comprising the peptide of claim 2 and a carrier.
4. The peptide of claim 1 having the amino acid sequence:RVIRVVRGACRAIRHIPRRIR (SEQ ID NO: 38).
5. A composition comprising the peptide of claim 4 and a carrier.
6. The peptide of claim 1 having the amino acid sequence:LWETLRRGGRWILAIPRRIR (SEQ ID NO: 73).
7. A composition comprising the peptide of claim 6 and a carrier.
8. The peptide of claim 1 having the amino acid sequence:LWETLRRGCRWILAIPRRIR (SEQ ID NO: 92).
9. A composition comprising the peptide of claim 8 and a carrier.
10. The peptide of claim 1 having the amino acid sequence:LWRLLRRGGRWILAIPRRIR (SEQ ID NO: 97).
11. A composition comprising the peptide of claim 10 and a carrier.
12. A composition comprising one or more peptides of claim 1 and a carrier.
13. A disulfide-linked dimerized peptide wherein the monomeric peptide has the amino acid sequence: RVIRVVQGACRAIRHIPRRIR (SEQ ID NO:10).
14. A composition comprising the disulfide-linked dimerized peptide of claim 13 and a carrier.
|
Alicia Vikander
Movies
* Tomb Raider (2018)
* Tomb Raider 2 - Upcoming
Trivia
* Vikander and her predecessor, Angelina Jolie, are both Academy Award winners.
|
/* global describe beforeEach it */
// const {expect} = require('chai')
// const db = require('../index')
// const User = db.model('user')
// const {UserData} = require('../../../script/seed')
// describe('User model', () => {
// beforeEach(() => {
// return db.sync({force: true})
// })
// describe('instanceMethods', () => {
// describe('correctPassword', () => {
// let cody
// beforeEach(async () => {
// cody = await User.create({
// email: UserData[0].email,
// password: UserData[0].password,
// firstName: UserData[0].firstName,
// lastName: UserData[0].lastName,
// phoneNumber: UserData[0].phoneNumber,
// address: UserData[0].email.address
// })
// })
// it('returns true if the password is correct', () => {
// expect(cody.correctPassword('mypassword')).to.be.equal(true)
// })
// it('returns false if the password is incorrect', () => {
// expect(cody.correctPassword('apassword')).to.be.equal(false)
// })
// }) // end describe('correctPassword')
// }) // end describe('instanceMethods')
// }) // end describe('User model')
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With the exception of a few Malayan and Chinese species, the true Passifloras are natives of tropical America. Many of them are cultivated as curiosities, and some of them for the beauty of their flowers and for their festooning foliage. The leaves are either digi tately lobed or angled or perfectly entire. The large, showy flowers are solitary in the axils or on axillary racemes. The fruit is oblong or globular and usually fleshy or berry-like, .S-carpeled but 1-loculed, the seeds being borne on parietal placentae. The fruit is allied to the pepo of the Cvicurbitacese. The ovary is supported on a long stalk which is inclosed in or usually united with the tube formed by the union of the bases of the filaments. The structure of the fruit is well shown in Pig. 1650; the remains of the fioral envelopes have broken from the attachment on the torus and rest on the fruit. The petals are borne on the throat of the calyx, but in some species they are absent. Nearly or quite a dozen Passifloras are native to the U. S., and one of them, P. Intea, grows naturally as far north as southern Pa. and Illinois. From Virginia south, the Maypop, P. incarnuta, is a very common plant in fields and waste places. Both these species are herbaceous perennials. The fruit of some Passifloras is edible.
In cultivation, the Passifloras have been considerably hybridized, and they are also confused with Tacsonia. In 1871 Masters enumerated 184 species (Trans. Linn. Soc. 26), and a number of species have been discovered since that time.
PASSIFLORA
Most of the Passion-flowers are yellow or green in color of envelopes, but there are fine reds in P. rai-f- mosa, P. Baddiana, P. coccinea, P. alatu, P. vitifoliu, and two or three others. l H. B.
P. carulea and Constance Elliott are both hardy at Washington. In summer time we use P. faetida (raised from seed annually), and during the last two seasons, P. Uolimensis, for trellis work. The last named is a good thing for this line of work ; the fls. are white, purple center, about 2 in. in diameter; native of Mexico; very easy to prop, from cuttings of soft wood. Not many of the tender species and hybrids are grown to any great extent in this country. P. alata and P. quadnriKjiihiris are desirable climbers for a roomy, warm greenhouse. P. quadrangularis, var. ancubifo'lia, seems to flower quite as freely as the green-leaved one. Passifloras are prop, from cuttings of the half-ripened growth, with bottom heat. P. racemosa and P. lyoudoni are a trifle difficult to root from cuttings; the growths should be as ripe as possible for this purpose. Keep the under sur face of the leaves flat on the sand while rooting. The native P. im-iirnutd grows very freely at Washington, becoming more or less of a weed and hard to eradicate. G. W. Oliver.
B. Fls. apetalows, nsnally with no bracts.
1. grficilia, Jacq. Slender annual: Ivs. rather small, broadly deltoid-ovate, very shallowly and bluntly :i lobed: fls. solitary, pale green of whitish, considerably surpassed by the Ivs., the calyx-lobes oblong or lanceo late, the filiform rays of the corona in a single row and equal; seeds with 6 elevated ridges. Brazil. B.R. 11:870. — Fl. about 1 in. across. Easily grown either indoors or in the open, as a garden annual.
2. aden6poda, Moc. & Sess. [P. acerifdlia, Cham. & Schlecht.). Lvs. glabrous, cordate, 3-nerved and 5 lobed, the lobes ovate-acuminate and somewhat serrate: bracts cut-serrate. Mex. to S. Amer. — Once advertised by Saul.
3. H41imi, Mast. Tall, glabrous climber, with very slender terete branches; lvs. ovate, peltate at base, strongly 3-ncrved and each of the side nerves ending in a tooth, but the leaf -margin otherwise entire but bearing minute red glands: stipules kidney-shaped, dentate, purplish, nearly or quite 1 in. across: fl. -bracts 2, en tire: fl. about 3 in. across, solitary, whitish, the corona shorter than the envelopes, the outer filaments being orange. Mex. B.M. 7052. R.H. 1809, p. 430 (as J)is emma Hahnii).^ G.C. II. 12:504.
4. trifasci&ta, Lem. Lvs. 3-lobed to one-third or one half their depth, the margins entire, with an irregular reddish purple band along each of the three midribs: fls. yellowish, fragrant, small. Brazil. I.H. 15:544.— Interesting for its ornamental foliage.
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package comparators.dom;
import java.util.ArrayList;
import java.util.List;
import comparators.DomAbstractComparator;
public class DomComparators {
static List<DomAbstractComparator> domComparators = new ArrayList<DomAbstractComparator>();
public static List<DomAbstractComparator> getComparators() {
domComparators.add(new RTEDComparator());
//domComparators.add(new APTEDComparator());
domComparators.add(new DOMLevenshteinComparator());
domComparators.add(new SimHashComparator());
domComparators.add(new ContentHashComparator());
return domComparators;
}
}
|
Talk:Steven Universe (character)/@comment-39748580-20191120043552/@comment-44032435-20191206032911
Imagine it turns out the fusion's name is "Larven" XD
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ONIONS. 81
the drill was rigid, if there was any deviation from a straight line in any of the drills, there would be the same deviation in all of them, and if we could avoid cutting up the plants in one row, we should also avoid doing so in the other three rows. Such a drill and cultivator combined would be not only very useful for sowing and cultivating onions, but for many other farm and garden crops, such as turnips, beets, parsnips, carrots, etc.
Until we have such a machine, we must do the best we can with the tools we now have. In fact, a farmer who undertakes to raise onions for the first time as a field crop, could hardly use such a machine as I have proposed, he would require to have his land much cleaner and smoother, and freer from stones than would likely to be the case on any ordinary farm. Land for onions has to be made to order. Onions do better on old onion land than when they are raised on any ordinary soil for the first time. Nearly all other crops do better in rotation, than when grown year after year on the same land. It is not clear why onions should be an exception. I think chemistry and plant food have far less to do with it than the mechanical state of the land. If a man or a boy would bestow the necessary amount of labor in preparing and enriching the land, I see no reason to doubt that he could get just as good a crop the first year, as he could the second, third, or tenth year; but no man will do it; per haps a boy may. There was some excuse for men in years gone by they had not the necessary tools. With our modern implements we can place land in wonderfully fine condition, at comparatively little expense, the first year; but much of the work ought to be done in the autumn. Suppose you try how rich, and mellow, you can make an acre of land this fall. It does not make very much difference how you do it. The first thing, however, is to get off all the stones, and stumps, and rubbish. If a harrow will do it any good, harrow it; if
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/**
* String to Milliseconds.
* Author: Brian Barnett, [email protected], http://brianbar.net/ || http://3kb.co.uk/
* Date: 2014-06-02
**/
(function(exports) {
'use strict';
/**
* Returns array of strings that match the regex which checks for (h)our, (m)inutes & (s)econds - case insensitive & will accept a single space between the number & the time measurement. Will also allow decimal values (0.5, 0.25 etc). Will return an empty array if no matches (which will result in a 0 second value as the delay.).
* @param {String} str [ e.g. '2h30m', '1 Hour, 25 Minutes', '0.5h 20sec' ]
* @return {Array} [ e.g ['2h', '30m'] ]
*/
function _getMatches(str) {
str = str || '';
return str.toLowerCase().match(/((\d+|)\.\d+|\d+)( [smhd]|[smhd])/g) || [];
}
/**
* Takes the arary from the _getMatches function and converts the h,m,s value to its equivalent value in milliseconds, the total value is returned as the delay to use.
* @param {[Array]} matches [Array as returned from the _getMatches function]
* @return {[Number]} [Output of the total in milliseconds]
*/
function _getDelay(matches) {
var delay = 0;
matches.forEach(function(i) {
if (i.indexOf('d') !== -1) {
delay += +i.split('d')[0] * 864e5;
}
if (i.indexOf('h') !== -1) {
delay += +i.split('h')[0] * 36e5;
}
if (i.indexOf('m') !== -1) {
delay += +i.split('m')[0] * 6e4;
}
if (i.indexOf('s') !== -1) {
delay += +i.split('s')[0] * 1e3;
}
});
return delay; //in milliseconds
}
exports.ms = function(str) {
return _getDelay(_getMatches(str));
};
})(typeof exports === 'undefined' ? this['str'] = {} : exports);
|
User:That Hairy Canadian
I am just another bag of mostly water living on this planet we call, Earth.
I am no different than anybody else, besides those limited little things that make us 'Unique Beings'.
I eventually earned a Bachelor of Science in Mathematics, with Honours - way back in 1992 (when I finally got around to caring whether I had a piece of paper that confirmed my qualifications for the many High Tech jobs which I have performed since my earliest teenaged years). I have lived and worked internationally in a wide variety of industries due to the varied nature of Computer Science. I have studied in such varied subject areas as; Medieval History, Medical Science, Veterinary Medicine, Mathematics, Astrophysics, Law, Telecommunications, Special Needs Education, Develpmental Disabilities, Linguistics, et. al.
I have personal tastes, knowledge, and views based upon my life experience that may; enlighten you, annoy you, or otherwise cause you distress. I apologise for any of those possible outcomes which you may experience.
If you have anything to add to or edit within my contributions to Wikipedia (or elsewhere), please try to remain civil. [Sometimes that can be a difficult thing to do]
Please keep in mind that I live a very full and vibrant life, so I may not respond to some inquiries, may take a long time to respond, or may not care enough to bother at all.
If you really wish to hound me about anything specific then you may be able to find me via the following link... That Hairy Canadian, or you could just email me directly at wikimail - That Hairy Canadian.
* Remember to be happy, it annoys those who wish you ill. James Theodore Randall, passed away May 25, 2015 at the age of 49 years.
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Rounded connective tissue- cells are rare among the Gnathobdel lidee, Trocheta being the only genus which presents such. They occur in masses, and are also very generally arranged in rows, pro bably prior to their conversion into botryoidal tissue.
gated, and it may be branched and formed fibres.
It is possible to trace this process ; a slightly irregular cell elon gates more and more, and its processes become drawn out, so that ultimately little cell-substance is left and a long very fine fibre is produced, the cell all the time doubtless adding to matrix.
singly.
3. Ect-entoplastic metamorphosis — the cell developes pigment. a. The cells take no part in the formation of a vascular system. This series of modifications is well seen in Pontobdella. Indifferent
Such cells either remain rounded or they divide into irregular groups, and afterwards become much branched. The process may be shortened, the cell branching, and forming pigment simultaneously.
A set of modifications similar to those just described takes place, but intracellular vacuolation taking place at the same time vascular spaces are formed. These come in communication with the capillaries of the true vascular system on the one hand, and with the sinuses on the other.
the vacuolation of indifferent connective tissue cells.
It may be noticed here that in forms where no canalisation of pig mented cells has occurred, the blood is always colourless, while in forms with red blood such canalisation of pigmented tissue has occurred in the formation of the vascular system.
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1952 Air France SNCASE Languedoc crash
The 1952 Air France SNCASE Languedoc crash occurred on 3 March 1952 when a SNCASE SE.161/P7 Languedoc aircraft of Air France crashed on take-off from Nice Airport for Le Bourget Airport, Paris, killing all 38 people on board. The cause of the accident was that the aileron controls had jammed, which in itself was contributed to by a design fault. The accident was the third-deadliest in France at the time and is the deadliest involving the SNCASE Languedoc.
Aircraft
The accident aircraft was a SNCASE SE.161/P7 Languedoc, msn 43, registration F-BCUM. The aircraft was powered by four 1220 hp Pratt & Whitney R-1830 SIC-3-G engines.
Accident
Shortly after take-off from Nice Airport on a scheduled domestic passenger flight to Le Bourget Airport, Paris, the aircraft was seen to bank to the left, roll onto its back and crash about 1 km north of the airport. All four crew and 34 passengers on board were killed. The flight had originated in Tunis, Tunisia. The accident was the third deadliest in France at the time and is the deadliest involving the SNCASE Languedoc.
Thirteen of the victims were British, including shipowner John Emlyn-Jones and his wife. Amongst the other victims were the French actresses Lise Topart and Michèle Verly and the American actress and ballet dancer Harriet Toby. A Frenchwoman was initially reported to have survived the crash seriously injured, but she died later in hospital, bringing the total to 38 deaths.
Investigation
An investigation found that the cause of the accident was that the co-pilot's aileron controls had jammed due to a chain slipping off its sprocket. The difficulty of setting and inspecting the chains in the dual control columns was cited as a contributory factor in the accident.
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How to off the Bootstrap Loading Button while text is still Empty
Please help
I want to off the bootstrap loading button while my textbox is still empty
the user need to fill all textbox before the submit button will change the text to loading
whats happening to me is that when the user click the submit button the bootstrap is loading too even the page textbox still has a required field.
this my current code
<link rel="stylesheet" href="http://netdna.bootstrapcdn.com/bootstrap/3.0.3/css/bootstrap.min.css">
<link rel="stylesheet" href="http://netdna.bootstrapcdn.com/bootstrap/3.0.3/css/bootstrap-theme.min.css">
<script type="text/javascript" src="http://code.jquery.com/jquery.min.js"></script>
<script src="http://netdna.bootstrapcdn.com/bootstrap/3.0.3/js/bootstrap.min.js"></script>
<script type="text/javascript">
$(function() {
$(".btn").click(function(){
$(this).button('loading').delay(1000).queue(function() {
$(this).button('reset');
$(this).dequeue();
});
});
});
</script>
<form class="form-horizontal" action="button.php" method="post" style=" width:450px;">
<label class="control-label col-xs-3">Username Name :</label>
<input type="textbox" class="form-control" name="txtfirst" placeholder="First Name"required>
<label class="control-label col-xs-3">Password :</label>
<input type="textbox" class="form-control" name="txtlast" placeholder="Last Name"required>
<button type="submit" class="btn btn-primary" data-loading-text=" Loading... ">Login</button>
</form>
Please help
From the code it looks like your js function call is bound to click event of the button and not submit event of the form. The click event does not do any form validations.
Try this: http://jsfiddle.net/Dde4U/
$(function() {
$(".btn").click(function(){
if($('.form-horizontal').valid()){ //Checks if form is valid
$(this).button('loading').delay(1000).queue(function() {
$(this).button('reset');
$(this).dequeue();
});
}
});
});
I applied your code and i fill up the textboxes. the page is submitting but what i need is to change the "Login" to "Loading" while the page is submitting or loading.
There is negligible time between click and submit to observe that.
I applied it to my other form data entry in my resume' form. Because it takes time to save all info from CV thats why i want to put loading in the button. i just create username and password as a sample.
|
In My Africa
Summary
Plot
[Title Card]
The scene then goes to Cheikh and his mother leaving Senegal to move to America, in Elwood City.
Major
* D.W. Read
* Cheikh
* The Brain
Minor
* Arthur Read
* Cheikh's mother
* Cheikh's father
* Ms. Morgan
* Timmy and Tommy Tibble
Cameo
* Emily
* James MacDonald
* Keith
* Edwin
* Maryann
* Liam
* Amanda Hulser
* Elias Howe
Songs
* "In My Africa"
Trivia
* is located in the United States in Michigan.
Cultural references
* D.W., Cheikh and the Brain walk across a zebra crossing, a reference to ''The Beatles album '.
* This entire episode is about the countries in Africa.
Episode connections
Errors
* D.W. pronounces as Le-so-tho, not Lee-soo-too, as it is supposed to be pronounced.
Production notes
Gallery
{{CollapsingGallery|
Screenshots
}}
|
Methods, reagents and cells for biosynthesizing glutarate methyl ester
ABSTRACT
This document describes biochemical pathways for producing 2,4-pentadienoyl-CoA by forming one or two terminal functional groups, comprised of carboxyl or hydroxyl group, in a C5 backbone substrate such as glutaryl-CoA, glutaryl-[acp] or glutarate methyl ester. 2,4-pentadienoyl-CoA can be enzymatically converted to 1,3-butadiene.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 62/012,722, filed Jun. 16, 2014, and U.S. Provisional Application No. 62/012,586, filed Jun. 16, 2014, the disclosures of which are incorporated by reference herein in their entireties.
TECHNICAL FIELD
This invention relates to a method of increasing the activity of a polypeptide having carboxylate reductase activity on a dicarboxylic acid by enzymatically converting the dicarboxylic acid to a methyl ester using a polypeptide having malonyl-CoA methyltransferase activity. This invention also relates to methods for biosynthesizing 2,4-pentadienoyl-CoA (e.g., as a precursor to the biosynthesis of 1,3-butadiene), and more particularly to synthesizing 2,4-pentadienoyl-CoA using one or more isolated enzymes such as one or more of a malonyl-CoA O-methyltransferase, methyl ester esterase, a carboxylate reductase, or a 5-hydroxyvaleryl-CoA dehydratase, or using recombinant host cells expressing one or more of such enzymes.
BACKGROUND
1,3-butadiene (hereinafter butadiene) is an important monomer for the production of synthetic rubbers including styrene-butadiene-rubber (SBR), polybutadiene (PB), styrene-butadiene latex (SBL), acrylonitrile-butadiene-styrene resins (ABS), nitrile rubber, and adiponitrile, which is used in the manufacture of Nylon-66 (White, Chem ico-Biological Interactions, 2007, 166, 10-14).
Butadiene is typically produced as a co-product from the steam cracking process, distilled to a crude butadiene stream, and purified via extractive distillation (White, Chem ico-Biological Interactions, 2007, 166, 10-14).
On-purpose butadiene has been prepared among other methods by dehydrogenation of n-butane and n-butene (Hou dry process); and oxidative dehydrogenation of n-butene (Oxo-D or O-X-D process) (White, Chem ico-Biological Interactions, 2007, 166, 10-14).
Industrially, 95% of global butadiene production is undertaken via the steam cracking process using petrochemical-based feedstocks such as naphtha. Production of on-purpose butadiene is not significant, given the high cost of production and low process yield (White, Chem ico-Biological Interactions, 2007, 166, 10-14).
Given a reliance on petrochemical feedstocks and, for on-purpose butadiene, energy intensive catalytic steps; biotechnology offers an alternative approach via biocatalysis. Biocatalysis is the use of biological catalysts, such as enzymes, to perform biochemical transformations of organic compounds.
Accordingly, against this background, it is clear that there is a need for sustainable methods for producing intermediates wherein the methods are bio catalyst based (Jang et al., Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).
SUMMARY
This disclosure is based at least in part on the development of enzymatic systems and recombinant hosts for biosynthesizing 2,4-pentadienoyl-CoA or precursors thereof, which are useful for producing, for example, 1,3-butadiene and polymers or copolymers of 1,3-butadiene. In particular, as described herein, 2,4-pentadienoyl-CoA can be biosynthetically produced from renewable feedstocks without the need for any chemical catalysts such as metal oxides. For example, in the pathways described herein, 2,4-pentadienoyl-CoA can be produced from malonyl-CoA or malonyl-[acp] via various methyl-ester shielded routes. Such methyl-ester shielded routes include using a methyl ester esterase such as a pimelyl-[acp] methyl ester esterase or esterase to hydrolyze the methyl ester of glutaryl-[acp] methyl ester, glutaryl-CoA methyl ester, glutarate methyl ester, glutarate semialdehyde methyl ester, or 5-hydroxypentanoate methyl ester, and using a 5-hydroxyvaleryl-CoA dehydratase to introduce the first terminal vinyl group of 1,3-butadiene. For example, 1,3-butadiene can be produced from precursors stemming from 2,4-pentadienoyl-CoA as outlined in FIG. 7.
In some embodiments, the C5 aliphatic backbone for conversion to 1,3-butadiene can be formed from malonyl-[acp] or malonyl-CoA via conversion to glutaryl-[acp] methyl ester or glutaryl-CoA methyl ester, followed by (i) de-esterification of glutaryl-[acp] methyl ester or glutaryl-CoA methyl ester to glutaryl-[acp] or glutaryl-CoA respectively, or (ii) hydrolysis of glutaryl-[acp] methyl ester or glutaryl-CoA methyl ester to glutarate methyl ester. See FIG. 1-3.
In some embodiments, an enzyme in the pathway generating the C5 aliphatic backbone purposefully contains irreversible enzymatic steps.
In some embodiments, the terminal carboxyl groups can be enzymatically formed using an esterase, a thioesterase, a reversible CoA-ligase, a CoA-transferase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase or a 5-oxopentanoate dehydrogenase. See FIG. 4.
In some embodiments, the terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase and a 6-hydroxyhexanoate dehydrogenase. See FIG. 5.
In some embodiments, the terminal vinyl group can be enzymatically formed using a 5-hydroxyvaleryl-CoA dehydratase. See FIG. 6.
In one aspect this document features a method of biosynthesizing glutarate methyl ester in a recombinant host. The method includes enzymatically converting at least one of malonyl-[acp] and malonyl-CoA to glutarate methyl ester in the host using at least one polypeptide having malonyl-CoA O-methyltransferase activity and at least one polypeptide having thioesterase activity.
In some embodiments, the malonyl-[acp] is enzymatically converted to malonyl-[acp] methyl ester using the at least one polypeptide having malonyl-CoA O-methyltransferase activity. The malonyl-[acp] methyl ester can be enzymatically converted to glutaryl-[acp] methyl ester using at least one polypeptide having an activity selected from the group consisting of synthase activity, dehydrogenase activity, dehydratase activity, and reductase activity. The glutaryl-[acp] methyl ester can be enzymatically converted to glutarate methyl ester using the at least one polypeptide having thioesterase activity.
In some embodiments, malonyl-CoA is enzymatically converted to malonyl-CoA methyl ester using the at least one polypeptide having malonyl-CoA O-methyltransferase activity. The malonyl-CoA methyl ester can be enzymatically converted to glutaryl-CoA methyl ester using at least one polypeptide having an activity selected from the group consisting of synthase activity, β-ketothiolase activity, dehydrogenase activity, hydratase activity, and reductase activity. The glutaryl-CoA methyl ester can be enzymatically converted to glutarate methyl ester using the at least one polypeptide having thioesterase activity.
The polypeptide having malonyl-CoA O-methyltransferase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO:13.
The polypeptide having reductase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 11 or 12.
In some embodiments, the method further includes enzymatically converting glutarate methyl ester to glutarate semialdehyde methyl ester in the host using at least one polypeptide having carboxylate reductase activity. The polypeptide having carboxylate reductase activity can be used in combination with a polypeptide having phosphopantetheine transferase enhancer activity.
In some embodiments, the method further includes enzymatically converting glutarate methyl ester to 5-oxopentanoic acid using at least one polypeptide having an activity selected from the group consisting of carboxylate reductase activity and esterase activity. The polypeptide having carboxylate reductase activity can be used in combination with a polypeptide having phosphopantetheine transferase enhancer activity.
In some embodiments, the method further includes enzymatically converting glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid using at least one polypeptide having esterase activity. In some embodiments, the method further includes using at least one polypeptide having dehydrogenase activity to enzymatically convert glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid.
The polypeptide having esterase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 8.
In some embodiments, the method further includes enzymatically converting glutarate methyl ester to glutaric acid using at least one polypeptide having esterase activity. The method can further include enzymatically converting glutaric acid to 5-hydroxypentanoic acid using at least one polypeptide having carboxylate reductase activity and at least one polypeptide having dehydrogenase activity classified under EC 1.1.1.—. The polypeptide having carboxylate reductase activity can be used in combination with a polypeptide having phosphopantetheine transferase enhancer activity.
The polypeptide having carboxylate reductase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 2-7.
The polypeptide having thioesterase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino acid sequences set forth in SEQ ID NO: 9, 10, 14 or 15.
In some embodiments, the method further includes enzymatically converting 5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA using at least one polypeptide having an activity selected from the group consisting of CoA-transferase activity, a synthase activity, and dehydratase activity. A polypeptide having a CoA-transferase activity or a synthase activity and a polypeptide having dehydratase activity can enzymatically convert 5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA. The method can further include enzymatically converting 2,4-pentadienoyl-CoA into 1,3 butadiene using at least one polypeptide having an activity selected from the group consisting of hydratase activity, thioesterase activity, decarboxylase activity, dehydrogenase activity, CoA-transferase activity, and dehydratase activity.
The polypeptide having thioesterase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino acid sequences set forth in SEQ ID NOs: 14-15.
In another aspect, this document features a method of making glutarate. The method includes (i) enzymatically converting glutaryl-[acp] methyl ester to glutaryl-[acp] or glutaryl-CoA methyl ester to glutaryl-CoA using a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity, and (ii) enzymatically converting glutaryl-[acp] or glutaryl-CoA to glutarate using at least one polypeptide having thioesterase activity, reversible CoA-ligase activity, a CoA-transferase activity, an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, or a succinate-semialdehyde dehydrogenase activity.
The polypeptide having pimeloyl-[acp] methyl ester methylesterase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 1.
In some embodiments, glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having thioesterase activity. In some embodiments, glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having reversible CoA-ligase activity or a CoA-transferase activity. In some embodiments, glutaryl-[acp] or glutaryl-CoA is enzymatically converted to glutaric acid using a polypeptide having an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, or a succinate-semialdehyde dehydrogenase activity.
In another aspect, this document features a recombinant host cell. The recombinant host cell includes at least one exogenous nucleic acid encoding a polypeptide having malonyl-CoA O-methyltransferase activity and a polypeptide having thioesterase activity, the host producing glutarate methyl ester.
The host can further include an exogenous polypeptide having carboxylate reductase activity, the host further producing glutarate semialdehyde methyl ester. In some embodiments, the host furthers include one or more exogenous polypeptides having an activity selected from the group consisting of synthase activity, dehydrogenase activity, dehydratase activity, and reductase activity. In some embodiments, the host further includes one or more exogenous polypeptides having an activity selected from the group consisting of synthase activity, β-ketothiolase activity, dehydrogenase activity, hydratase activity, and reductase activity.
The polypeptide having malonyl-CoA O-methyltransferase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 13.
The polypeptide having thioesterase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 14-15.
The polypeptide having reductase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino acid sequences set forth in SEQ ID NOs: 11 or 12.
The host can further include an exogenous polypeptide having esterase activity, the host further producing glutaric acid or 5-oxopentanoic acid.
In some embodiments, the host includes one or more exogenous polypeptides having an activity selected from the group consisting of esterase activity, 6-hydroxyhexanoate dehydrogenase activity, 4-hydroxybutyrate dehydrogenase activity, 5-hydroxypentanoate dehydrogenase activity, and alcohol dehydrogenase activity, the host producing 5-hydroxypentanoic acid. The host can further include one or more exogenous polypeptides having an activity selected from the group consisting of CoA-transferase activity, a synthase activity, and dehydratase activity, the host producing 2,4-pentadienoyl-CoA from 5-hydroxypentanoic acid. The host can further include one or more exogenous polypeptides having an activity selected from the group consisting of hydratase activity, thioesterase activity, decarboxylase activity, dehydrogenase activity, CoA-transferase activity, and dehydratase activity, the host producing 1,3-butadiene from 2,4-pentadienoyl-CoA.
The polypeptide having thioesterase activity can at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15
The polypeptide having carboxylate reductase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 2-7.
In some embodiments, when the host includes an exogenous polypeptide having carboxylate reductase activity it is used in combination with an exogenous polypeptide having phosphopantetheine transferase enhancer activity.
In another aspect, this document features a recombinant host including at least one exogenous nucleic acid encoding a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity, and at least one polypeptide having an activity selected from the group consisting of thioesterase activity, reversible CoA-ligase activity, a CoA-transferase activity, an acylating dehydrogenase activity, an aldehyde dehydrogenase activity, a glutarate semialdehyde dehydrogenase activity, and a succinate-semialdehyde dehydrogenase activity.
The polypeptide having pimeloyl-[acp] methyl ester methylesterase activity can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 1.
In some embodiments, when the host includes an exogenous polypeptide having carboxylate reductase activity it is used in combination with an exogenous polypeptide having phosphopantetheine transferase enhancer activity.
In another aspect, this document features a bio-derived product, bio-based product or fermentation-derived product, wherein the product includes (i.) a composition including at least one bio-derived, bio-based or fermentation-derived compound as described herein, or any combination thereof; (ii.) a bio-derived, bio-based or fermentation-derived polymer including the bio-derived, bio-based or fermentation-derived composition or compound of (i.), or any combination thereof; (iii.) a bio-derived, bio-based or fermentation-derived resin including the bio-derived, bio-based or fermentation-derived compound or bio-derived, bio-based or fermentation-derived composition of (i.) or any combination thereof or the bio-derived, bio-based or fermentation-derived polymer of ii. or any combination thereof; (iv.) a molded substance obtained by molding the bio-derived, bio-based or fermentation-derived polymer of (ii.) or the bio-derived, bio-based or fermentation-derived resin of (iii.), or any combination thereof; (v.) a bio-derived, bio-based or fermentation-derived formulation including the bio-derived, bio-based or fermentation-derived composition of (i.), bio-derived, bio-based or fermentation-derived compound of (i.), bio-derived, bio-based or fermentation-derived polymer of (ii.), bio-derived, bio-based or fermentation-derived resin of (iii.), or bio-derived, bio-based or fermentation-derived molded substance of (iv.), or any combination thereof; or (vi.) a bio-derived, bio-based or fermentation-derived semi-solid or a non-semi-solid stream, including the bio-derived, bio-based or fermentation-derived composition of (i.), bio-derived, bio-based or fermentation-derived compound of (i.), bio-derived, bio-based or fermentation-derived polymer of (ii.), bio-derived, bio-based or fermentation-derived resin of (iii.), bio-derived, bio-based or fermentation-derived formulation of (v.), or bio-derived, bio-based or fermentation-derived molded substance of (iv.), or any combination thereof.
This document also features a method of increasing the activity of a polypeptide having carboxylate reductase activity on a substituted or unsubstituted C₄-C₈ dicarboxylic acid such as glutaric acid or adipic acid. The method includes enzymatically converting the C₄-C₈ dicarboxylic acid to a HOC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ ester using a polypeptide having malonyl-CoA methyltransferase activity before enzymatically converting the HOC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ ester to a HC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ using a polypeptide having carboxylate reductase activity. The method further can include enzymatically converting the HC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ to HOCH₂(C₂-C₆ alkyl)-C(═O)OCH₃ using a polypeptide having dehydrogenase activity. In some embodiments, the method further includes enzymatically converting the HOCH₂(C₂-C₆ alkyl)-C(═O)OCH₃ product to a HOCH₂(C₂-C₆ alkyl)-C(═O)OH product using a polypeptide having the activity of an esterase.
This document also features a recombinant host that includes at least one exogenous nucleic acid encoding a (i) malonyl-[acp] O-methyltransferase, (ii) a pimeloyl-[acp] methyl ester methylesterase and (iii) a thioesterase, and produce glutarate methyl ester, glutaryl-[acp] or glutaryl-CoA.
Such a recombinant host producing glutarate methyl ester further can include an esterase, and further produce glutaric acid.
Such a recombinant host producing glutaryl-[acp] further can include a thioesterase and produce glutaric acid.
Such a recombinant host producing glutaryl-CoA further can include one or more of (i) a thioesterase, (ii) a reversible CoA-ligase, (iii) a CoA-transferase, or (iv) an acylating dehydrogenase, and (v) an aldehyde dehydrogenase such as such as 7-oxoheptanoate dehydrogenase, 6-oxohexanoate dehydrogenase or 5-oxopentanoate dehydrogenase and further produce glutaric acid or 5-oxopentanoate.
A recombinant host producing 5-oxopentanoate or glutaric acid further can include one or more of (i) an alcohol dehydrogenase or (ii) a carboxylate reductase and further produce 5-hydroxypentanoate.
A recombinant host producing glutarate methyl ester further can include one or more of (i) an alcohol dehydrogenase, (ii) an esterase or (iii) a carboxylate reductase and further produce 5-hydroxypentanoate.
In another aspect, this document features a method for producing a bioderived four or five carbon compound. The method includes culturing or growing a host as described herein under conditions and for a sufficient period of time to produce the bioderived four or five carbon compound, wherein, optionally, the bioderived four or five carbon compound can be selected from the group consisting of 2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester, 5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene and combinations thereof.
In another aspect, this document features a composition including a bioderived for or five carbon compound and a compound other than the bioderived four or five carbon compound, wherein the bioderived four or five carbon compound is selected from the group consisting of 2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester, 5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene and combinations thereof. For example, the bioderived 4-carbon compound can be a cellular portion of a host cell or an organism.
This document also features a biobased polymer including the bioderived four or five carbon compound including 2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester, 5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene and combinations thereof.
This document also features biobased resin including the bioderived four or five carbon compound including 2,4-pentadienoyl-CoA, glutaryl-[acp] methyl ester, 5-hydroxypentanoic acid, 3-buten-2-one, 3-buten-2-ol, 1,3-butadiene and combinations thereof, as well as a molded product obtained by molding a biobased resin.
In another aspect this document also features a process for producing a biobased polymer including chemically reacting the bioderived four or five carbon compound with itself or another compound in a polymer-producing reaction.
In another aspect this document features a process for producing a biobased resin as described herein including chemically reacting the bioderived four or five carbon compound with itself or another compound in a resin producing reaction.
This document also features a biochemical network including a malonyl-CoA O-methyltransferase, wherein the malonyl-CoA O-methyltransferase enzymatically converts malonyl-[acp] to malonyl-[acp] methyl ester. The biochemical network can further include a synthase, a dehydrogenase, a dehydratase, a reductase, and a thioesterase, wherein the synthase, the dehydrogenase, the dehydratase, the reductase, and the thioesterase, enzymatically convert the malonyl-[acp] methyl ester to glutarate methyl ester.
In some embodiments the biochemical network further includes a carboxylate reductase, wherein the carboxylate reductase enzymatically converts glutarate methyl ester to glutarate semialdehyde methyl ester. The biochemical network can further include an esterase and a dehydrogenase, wherein the esterase and dehydrogenase enzymatically convert glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid.
In some embodiments, the biochemical network can further include a CoA-transferase and dehydratase, wherein the CoA-transferase and dehydratase enzymatically convert 5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA. The biochemical network can further include a hydratase, a thioesterase, a decarboxylase, a dehydrogenase, a CoA-transferase, and a dehydratase, wherein the hydratase, the thioesterase, the decarboxylase, the dehydrogenase, the CoA-transferase, and the dehydratase enzymatically convert the 2,4-pentadienoyl-CoA to 1,3-butadiene.
This document also features a biochemical network including a malonyl-CoA O-methyltransferase, wherein the malonyl-CoA O-methyltransferase enzymatically converts malonyl-CoA to malonyl-CoA methyl ester. The biochemical network can further include a synthase, a β-ketothiolase, a dehydrogenase, a hydratase, a reductase, and a thioesterase, wherein the synthase, the β-ketothiolase, the dehydrogenase, the hydratase, the reductase, and the thioesterase, enzymatically convert the malonyl-CoA methyl ester to glutarate methyl ester.
In some embodiments the biochemical network further includes a carboxylate reductase, wherein the carboxylate reductase enzymatically converts glutarate methyl ester to glutarate semialdehyde methyl ester. The biochemical network can further include an esterase and a dehydrogenase, wherein the esterase and dehydrogenase enzymatically convert glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid.
In some embodiments, the biochemical network can further include a CoA-transferase and dehydratase, wherein the CoA-transferase and dehydratase enzymatically convert 5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA. The biochemical network can further include a hydratase, a thioesterase, a decarboxylase, a dehydrogenase, a CoA-transferase, and a dehydratase, wherein the hydratase, the thioesterase, the decarboxylase, the dehydrogenase, the CoA-transferase, and the dehydratase enzymatically convert the 2,4-pentadienoyl-CoA to 1,3-butadiene.
This document also features a method of increasing the activity of a polypeptide having carboxylate reductase activity on a substituted or unsubstituted C4-C8 dicarboxylic acid such as glutaric acid or adipic acid. The method includes enzymatically converting the C₄-C₈ dicarboxylic acid to a HOC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ ester using a polypeptide having malonyl-CoA methyltransferase activity before enzymatically converting the HOC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ ester to a HC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ using a polypeptide having carboxylate reductase activity. The method further can include enzymatically converting the HC(═O)(C₂-C₆ alkyl)-C(═O)OCH₃ to HOCH₂(C₂-C₆ alkyl)-C(═O)OCH₃ using a polypeptide having dehydrogenase activity. In some embodiments, the method further includes enzymatically converting the HOCH₂(C₂-C₆ alkyl)-C(═O)OCH₃ product to a HOCH₂(C₂-C₆ alkyl)-C(═O)OH product using a polypeptide having the activity of an esterase.
Any of the methods can be performed in a recombinant host by fermentation. The host can be subjected to a cultivation strategy under aerobic, anaerobic, or micro-aerobic cultivation conditions. The host can be cultured under conditions of nutrient limitation such as phosphate, oxygen or nitrogen limitation. The host can be retained using a ceramic membrane to maintain a high cell density during fermentation.
In some embodiments, the host is subjected to a cultivation strategy under aerobic or micro-aerobic cultivation conditions.
In some embodiments, a biological feedstock can be used as the principal carbon source for the fermentation. For example, the biological feedstock can be, or can derive from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.
In some embodiments, a non-biological feedstock can be used as the principal carbon source for the fermentation. The non-biological feedstock can be, or can be derived from, natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
In any of the embodiments described herein, the host can be a prokaryote. The prokaryote can be selected from the group consisting of Escherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delft ia; Bacillus s; Lactobacillus; Lactococcus; and Rhodococcus. For example, the prokaryote can be selected from the group consisting of Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delft ia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, Lactococcus lactis, and Rhodococcus equi.
In any of the embodiments described herein, the host can be a eukaryote. The eukaryote can selected from the group consisting of Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces. For example, the eukaryote can be selected from the group consisting of Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, and Kluyveromyces lactis.
In some embodiments, the host exhibits tolerance to high concentrations of a C5 building block, and wherein the tolerance to high concentrations of a C5 building block is improved through continuous cultivation in a selective environment.
In some embodiments, the host's endogenous biochemical network is attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA, (2) create a cofactor, i.e. NADH or NADPH, imbalance that may be balanced via the formation of glutarate methyl ester, 2,4-pentadienoyl-CoA, or 1,3-butadiene, (3) prevent degradation of central metabolites, central precursors leading to and including glutarate methyl ester, 2,4-pentadienoyl-CoA, or 1,3-butadiene and (4) ensure efficient efflux from the cell.
In some embodiments, the host includes one or more of the following: the intracellular concentration of oxaloacetate for biosynthesis of a C5 building block is increased in the host by overexpressing recombinant genes forming oxaloacetate; wherein an imbalance in NADPH is generated that can be balanced via the formation of a C5 building block; wherein an exogenous lysine biosynthesis pathway synthesizing lysine from 2-oxoglutarate via 2-oxoadipate is introduced in a host using the meso 2,6 diaminopimelate pathway for lysine synthesis; wherein an exogenous lysine biosynthesis pathway synthesizing lysine from oxaloacetate to meso 2,6 diaminopimelate is introduced in a host using the 2-oxoadipate pathway for lysine synthesis; wherein endogenous degradation pathways of central metabolites and central precursors leading to and including C5 building blocks are attenuated in the host; or wherein the efflux of a C5 building block across the cell membrane to the extracellular media is enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C5 building block.
Any of the recombinant hosts described herein further can include one or more of the following attenuated polypeptides having attenuated activity of a: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an acetyl-CoA specific β-ketothiolase, an acetoacetyl-CoA reductase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxo acid decarboxylase producing isobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, a transhydrogenase dissipating the cofactor imbalance, a glutamate dehydrogenase specific for the co-factor for which an imbalance is created, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C5 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.
Any of the recombinant hosts described herein further can overexpress one or more genes encoding a polypeptide having: an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a feedback resistant threonine deaminase; a puri dine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6 di phosphatase; a propionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a L-glutamine synthetase; a lysine transporter; a dicarboxylate transporter; and/or a multidrug transporter activity.
The reactions of the pathways described herein can be performed in one or more cell (e.g., host cell) strains (a) naturally expressing one or more relevant enzymes, (b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes. Alternatively, relevant enzymes can be extracted from of the above types of host cells and used in a purified or semi-purified form. Extracted enzymes can optionally be immobilized to the floors and/or walls of appropriate reaction vessels. Moreover, such extracts include lysates (e.g. cell lysates) that can be used as sources of relevant enzymes. In the methods provided by the document, all the steps can be performed in cells (e.g., host cells), all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein including GenBank and NCBI submissions with accession numbers are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
One of skill in the art understands that compounds containing carboxylic acid groups (including, but not limited to, organic mono acids, hydroxy acids, aminoacids, and dicarboxylic acids) are formed or converted to their ionic salt form when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include, but are not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt of the present invention is isolated as a salt or converted to the free acid by reducing the pH to below the pKa, through addition of acid or treatment with an acidic ion exchange resin.
One of skill in the art understands that compounds containing amine groups (including, but not limited to, organic amines, aminoacids, and dia mines) are formed or converted to their ionic salt form, for example, by addition of an acidic proton to the amine to form the ammonium salt, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids including, but not limited to, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandel ic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butyl acetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt of the present invention is isolated as a salt or converted to the free amine by raising the pH to above the pKb through addition of base or treatment with a basic ion exchange resin.
One of skill in the art understands that compounds containing both amine groups and carboxylic acid groups (including, but not limited to, aminoacids) are formed or converted to their ionic salt form by either 1) acid addition salts, formed with inorganic acids including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids including, but not limited to, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandel ic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butyl acetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like, or 2) when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Acceptable organic bases include, but are not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A salt can of the present invention is isolated as a salt or converted to the free acid by reducing the pH to below the pKa through addition of acid or treatment with an acidic ion exchange resin.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. The word “comprising” in the claims may be replaced by “consisting essentially of” or with “consisting of,” according to standard practice in patent law.
DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic of exemplary biochemical pathways leading to glutarate methyl ester or glutaryl-[acp] from malonyl-[acp].
FIG. 2 is a schematic of exemplary biochemical pathways leading to glutarate methyl ester or glutaryl-CoA from malonyl-CoA using NADPH as reducing equivalent.
FIG. 3 is a schematic of exemplary biochemical pathways leading to glutarate methyl ester or glutaryl-CoA from malonyl-CoA using NADH as reducing equivalent.
FIG. 4 is a schematic of exemplary biochemical pathways leading to glutarate using glutarate methyl ester, glutaryl-[acp] or glutaryl-CoA as central precursor.
FIG. 5 is a schematic of an exemplary biochemical pathway leading to 5-hydroxypentanoate using glutarate methyl ester or glutarate as a central precursor.
FIG. 6 is a schematic of an exemplary biochemical pathway leading to 2,4-pentadienoyl-CoA using 5-hydroxypentanoate as a central precursor.
FIG. 7 is a schematic of an exemplary biochemical pathway leading to 1,3-butadiene using 2,4-pentadienoyl-CoA as central precursor.
FIG. 8 contains the amino acid sequences of an Escherichia coli pimeloyl-[acp] methyl ester methylesterase (see Genbank Accession No. AAC76437.1, SEQ ID NO: 1), a Mycobacterium marinum carboxylate reductase (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Segniliparus rugosus carboxylate reductase (see Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis carboxylate reductase (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense carboxylate reductase (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), a Segniliparus rotundus carboxylate reductase (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), an Pseudomonas fluorescens esterase (see Genbank Accession No. AAC60471.2, SEQ ID NO: 8), a Lactobacillus brevis acyl-[acp] thioesterase (see Genbank Accession NO: ABJ63754.1, SEQ ID NO:9), a Lactobacillus plantarum acyl-[acp] thioesterase (see Genbank Accession Nos. CCC78182.1, SEQ ID NO: 10), a Treponema denticola enoyl-CoA reductase (see, e.g., Genbank Accession No. AAS11092.1, SEQ ID NO: 11), an Euglena gracilis enoyl-CoA reductase (see, e.g., Genbank Accession No. AAW66853.1, SEQ ID NO: 12), a Bacillus cereus malonyl-[acp] O-methyltransferase (see, e.g., Genbank Accession No. AAP11034.1, SEQ ID NO: 13), an Escherichia coli thioesterase (see, e.g., Genbank Accession No. AAB59067.1, SEQ ID NO: 14), and an Escherichia coli thioesterase (see, e.g., Genbank Accession No. AAA24665.1, SEQ ID NO: 15), a Bacillus subtilis phosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1, SEQ ID NO:16), a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1, SEQ ID NO:17)
FIG. 9 is a bar graph summarizing the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and activity of five carboxylate reductase preparations in enzyme only controls (no substrate).
FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20 minutes, which is a measure of the consumption of NADPH and activity of a carboxylate reductase preparation for converting glutarate methyl ester to glutarate semialdehyde methyl ester relative to the empty vector control.
FIG. 11 is a table of conversion after 1 hour of glutaryl-CoA methyl ester to glutaryl-CoA by pimeloyl-[acp] methyl ester methylesterase.
DETAILED DESCRIPTION
This document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuation s to the host's biochemical network, which can be used to synthesize 2,4-pentadienoyl-CoA and, optionally, 1,3-butadiene (also known as buta-1,3-diene, bi ethylene, or vinyl ethylene) from central precursors or central metabolites. Production of butadiene thus can proceed through a common intermediate, 2,4-pentadienoyl-CoA, even though there are a number of different feedstocks and different pathways that can be used to produce 2,4-pentadienoyl-CoA. For example, malonyl-CoA or malonyl-[acp] can be used to produce 2,4-pentadienoyl-CoA via different methyl-ester shielded routes. As used herein, the term “central precursor” is used to denote any metabolite in any metabolic pathway shown herein leading to the synthesis of 5-hydroxypentanoate, 2,4-pentadienoyl-CoA or butadiene. The term “central metabolite” is used herein to denote a metabolite that is produced in all microorganisms to support growth.
As such, host microorganisms described herein can include endogenous pathways that can be manipulated such that 2,4-pentadienoyl-CoA can be produced. In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A host microorganism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the host.
The term “exogenous” as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.
In contrast, the term “endogenous” as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell “endogenously expressing” a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host “endogenously producing” or that “endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.
For example, depending on the host and the compounds produced by the host, one or more of the following enzymes may be expressed in the host including a malonyl-[acp] O-methyltransferase, a pimeloyl-[acp] methyl ester methylesterase, an esterase, a reversible CoA-ligase, CoA-transferase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, an alcohol dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an aldehyde dehydrogenase, or a carboxylate reductase. In recombinant hosts expressing a carboxylate reductase, a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.
This document also features a recombinant host that includes at least one exogenous nucleic acid encoding (i) a polypeptide having malonyl-[acp] O-methyltransferase activity, (ii) a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity and (iii) a polypeptide having thioesterase activity, and produce glutarate methyl ester, glutaryl-[acp] or glutaryl-CoA.
Such a recombinant host producing glutarate methyl ester further can include a polypeptide having esterase activity, and further produce glutaric acid.
Such a recombinant host producing glutaryl-[acp] further can include a polypeptide having thioesterase activity and produce glutaric acid.
Such a recombinant host producing glutaryl-CoA further can include one or more of (i) a polypeptide having thioesterase activity, (ii) a polypeptide having reversible CoA-ligase activity, (iii) a polypeptide having CoA-transferase activity, or (iv) a polypeptide having acylating dehydrogenase activity, and (v) a polypeptide having aldehyde dehydrogenase activity such as a 7-oxoheptanoate dehydrogenase, 6-oxohexanoate dehydrogenase or 5-oxopentanoate dehydrogenase activity and further produce glutaric acid or 5-oxopentanoate.
A recombinant host producing 5-oxopentanoate or glutaric acid further can include one or more of (i) a polypeptide having alcohol dehydrogenase activity or (ii) a polypeptide having carboxylate reductase activity and further produce 5-hydroxypentanoate.
A recombinant host producing glutarate methyl ester further can include one or more of (i) a polypeptide having alcohol dehydrogenase activity, (ii) a polypeptide having esterase activity or (iii) a polypeptide having carboxylate reductase activity and further produce 5-hydroxypentanoate.
Within an engineered pathway, the enzymes can be from a single source, i.e., from one species or genus, or can be from multiple sources, i.e., different species or genera. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL.
Any of the enzymes described herein that can be used for production of one or more C5 building blocks can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the corresponding wild-type enzyme. It will be appreciated that the sequence identity can be determined on the basis of the mature enzyme (e.g., with any signal sequence removed) or on the basis of the immature enzyme (e.g., with any signal sequence included). It also will be appreciated that the initial methionine residue may or may not be present on any of the enzyme sequences described herein.
For example, a polypeptide having pimeloyl-[acp] methyl ester methylesterase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli (see Genbank Accession Nos. AAC76437.1, SEQ ID NO: 1) pimeloyl-[acp] methyl ester methylesterase. See FIG. 1-3.
For example, a polypeptide having carboxylate reductase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. See, FIG. 5.
For example, a polypeptide having phosphopantetheinyl transferase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus subtilis phosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1, SEQ ID NO: 16) or a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1, SEQ ID NO: 17). See FIG. 5.
For example, a polypeptide having esterase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas fluorescens esterase (see Genbank Accession Nos. AAC60471.2, SEQ ID NO: 8). See FIG. 4, 5.
For example, a polypeptide having thioesterase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Lactobacillus brevis acyl-[acp] thioesterase (see Genbank Accession Nos. ABJ63754.1, SEQ ID NO: 9), a Lactobacillus plantarum acyl-[acp] thioesterase (see Genbank Accession Nos. CCC78182.1, SEQ ID NO: 10), or a Escherichia coli thioesterase (see Genbank Accession Nos. AAB59067.1 or AAA24665.1, SEQ ID NO: 14 or 15). See FIG. 4.
For example, a polypeptide having malonyl-[acp] O-methyltransferase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus cereus (see Genbank Accession Nos. AAP11034.1, SEQ ID NO: 13) malonyl-[acp] O-methyltransferase. See FIG. 1-3.
For example, a polypeptide having enoyl-CoA reductase activity described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Treponema denticola (see Genbank Accession Nos. AAS11092.1, SEQ ID NO:11), or a Euglena gracilis (see Genbank Accession Nos. AAW66853.1, SEQ ID NO:12) enoyl-CoA reductase. See FIGS. 1-3.
The percent identity (homology) between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (e.g., www.fr.com/blast/) or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of Bl2seq are set as follows: —i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); —j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); —p is set to blastp; —o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq-i c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.
Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity (homology) is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity (homology) value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.
It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.
Functional fragments of any of the enzymes described herein can also be used in the methods of the document. The term “functional fragment” as used herein refers to a peptide fragment of a protein that has at least 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater than 100%) of the activity of the corresponding mature, full-length, wild-type protein. The functional fragment can generally, but not always, be comprised of a continuous region of the protein, wherein the region has functional activity.
This document also provides (i) functional variants of the enzymes used in the methods of the document and (ii) functional variants of the functional fragments described above. Functional variants of the enzymes and functional fragments can contain additions, deletions, or substitutions relative to the corresponding wild-type sequences. Enzymes with substitutions will generally have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservative substitutions). This applies to any of the enzymes described herein and functional fragments. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a nonconservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Additions (addition variants) include fusion proteins containing: (a) any of the enzymes described herein or a fragment thereof; and (b) internal or terminal (C or N) irrelevant or heterologous amino acid sequences. In the context of such fusion proteins, the term “heterologous amino acid sequences” refers to an amino acid sequence other than (a). A heterologous sequence can be, for example a sequence used for purification of the recombinant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine), hemagglutinin (HA), glutathione-S-transferase (GST), or maltosebinding protein (MBP)). Heterologous sequences also can be proteins useful as detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments, the fusion protein contains a signal sequence from another protein. In certain host cells (e.g., yeast host cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g., KLH) useful, e.g., in eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.
Engineered hosts can naturally express none or some (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathways described herein. Thus, a pathway within an engineered host can include all exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered hosts can be referred to as recombinant hosts or recombinant host cells. As described herein recombinant hosts can include nucleic acids encoding one or more polypeptide having the activity of a reductase, deacetylase, N-acetyltransferase, malonyl-[acp] O-methyltransferase, esterase, thioesterase, hydratase, dehydrogenase, CoA-ligase, and/or CoA-transferase as described herein.
In addition, the production of one or more C5 building blocks can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a host microorganism as a source of the enzymes, or using a plurality of lysates from different host microorganisms as the source of the enzymes.
Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of 2,4-Pentadienoyl-CoA
As depicted in FIG. 4, a terminal carboxyl group can be enzymatically formed using (i) a polypeptide having thioesterase activity, (ii) a polypeptide having reversible CoA-ligase activity, (iii) a polypeptide having CoA-transferase activity, (iv) a polypeptide having acylating dehydrogenase activity, or (v) a polypeptide having aldehyde dehydrogenase activity such as a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 5-oxopentanoate dehydrogenase activity, or (vi) a polypeptide having esterase activity.
In some embodiments, a terminal carboxyl group leading to the synthesis of glutarate is enzymatically formed by a thioesterase classified under EC 3.1.2.—, such as the gene product of YciA (SEQ ID NO: 14), tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 15) or Acot13 (see, for example, Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9), 2789-2796; or Naggert et al., J. Biol. Chem., 1991, 266(17), 11044-11050).
In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by a CoA-transferase such as a glutaconate CoA-transferase classified, for example, under EC <IP_ADDRESS> such as from Acidaminococcus fermentans. See, for example, Buckel et al., 1981, Eur. J. Biochem., 118:315-321. FIG. 4.
In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by a reversible CoA-ligase such as a succinate-CoA ligase classified, for example, under EC <IP_ADDRESS> such as from Thermococcus kodakaraensis. See, for example, Shi kata et al., 2007, J. Biol. Chem., 282(37):26963-26970.
In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by an acyl-[acp] thioesterase classified under EC 3.1.2.—, such as the acyl-[acp] thioesterase from Lactobacillus brevis (GenBank Accession No. ABJ63754.1, SEQ ID NO: 9) or from Lactobacillus plantarum (GenBank Accession No. CCC78182.1, SEQ ID NO: 10). Such acyl-[acp] thioesterases have C6-C8 chain length specificity (see, for example, Jing et al., 2011, BMC Biochemistry, 12(44)). See, e.g., FIG. 4.
In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by an aldehyde dehydrogenase classified, for example, under EC <IP_ADDRESS> (see, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192). See, FIG. 4.
In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by an aldehyde dehydrogenase classified under EC 1.2.1.—such as a glutarate semialdehyde dehydrogenase classified, for example, under EC <IP_ADDRESS>, a succinate-semialdehyde dehydrogenase classified, for example, under EC <IP_ADDRESS> or EC <IP_ADDRESS>, or an aldehyde dehydrogenase classified under EC <IP_ADDRESS>. For example, an aldehyde dehydrogenase classified under EC 1.2.1.—can be a 5-oxopentanoate dehydrogenase such as the gene product of Cp nE, a 6-oxohexanoate dehydrogenase (e.g., the gene product of Ch nE from Acinetobacter sp.), or a 7-oxoheptanoate dehydrogenase (e.g., the gene product of ThnG from Sphingomonas macrogolitabida) (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11), 5158-5162; Lopez-Sanchez et al., Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, a 6-oxohexanoate dehydrogenase can be classified under EC <IP_ADDRESS> such as the gene product of Ch nE. For example, a 7-oxoheptanoate dehydrogenase can be classified under EC 1.2.1.—.
In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by a polypeptide having esterase activity such as an esterase classified under EC 3.1.1.—such as EC <IP_ADDRESS> or EC <IP_ADDRESS>.
Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of a 2,4-Pentadienoyl-CoA
As depicted in FIG. 5, a terminal hydroxyl group can be enzymatically formed using an alcohol dehydrogenase such as a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase.
For example, a terminal hydroxyl group leading to the synthesis of 5-hydroxypentanoate can be enzymatically formed by a dehydrogenase classified, for example, under EC 1.1.1.—such as a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC <IP_ADDRESS>8 (e.g., the gene from of Ch nD), a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.—such as the gene product of Cp nD (see, for example, Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684), a 5-hydroxypentanoate dehydrogenase from Clostridium viride, or a 4-hydroxybutyrate dehydrogenase such as ga bD (see, for example, Lütke-Eversloh & Steinbüchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See, FIG. 5.
Enzymes Generating the Terminal Vinyl Group in the Biosynthesis of a 2,4-Pentadienoyl-CoA
As depicted in FIG. 6, a terminal vinyl group can be enzymatically formed using a dehydratase such as 5-hydroxypentanoyl-CoA dehydratase from Clostridium viride (Eikmanns and Buckel, 1991, Eur. J. Biochem., 197, 661-668).
Biochemical Pathways
Pathway to Glutarate Methyl Ester, Glutaryl-CoA or Glutaryl-[Acp] from Malonyl-[Acp] or Malonyl-CoA
As shown in FIG. 1, glutarate methyl ester can be synthesized from malonyl-[acp] by conversion of malonyl-[acp] to malonyl-[acp] methyl ester by a malonyl-CoA O-methyltransferase classified, for example, under EC <IP_ADDRESS> such as the gene product of bioC; followed by conversion to 3-oxoglutaryl-[acp] methyl ester by condensation with malonyl-[acp] and a β-ketoacyl-[acp] synthase classified, for example, under EC 2.3.1.—such as EC <IP_ADDRESS>, EC <IP_ADDRESS> or EC <IP_ADDRESS> (e.g., the gene product of fabB, fa bF or fa bH); followed by conversion to 3-hydroxy-glutaryl-[acp] methyl ester by a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.—such as EC <IP_ADDRESS> (e.g., the gene product of fa bG); followed by conversion to 2,3-dehydroglutaryl-[acp] methyl ester by a 3-hydroxyacyl-[acp] dehydratase classified, for example, under EC <IP_ADDRESS> such as the gene product of fab Z; followed by conversion to glutaryl-[acp] methyl ester by a trans-2-enoyl-CoA reductase classified, for example, EC 1.3.1.—such as EC <IP_ADDRESS> such as the gene product of fabI; followed by (i) conversion to glutarate methyl ester by a thioesterase classified, for example, under EC 3.1.2.—such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No. AAO77182) or a Lactobacillus plantarum acyl-CoA thioesterase (GenBank Accession No. CCC78182.1) or (ii) conversion to glutaryl-[acp] by a pimeloyl-[acp] methyl ester methylesterase classified, for example, under EC <IP_ADDRESS> such as bio H (SEQ ID NO: 1).
As shown in FIG. 2, glutarate methyl ester can be synthesized from malonyl-CoA by conversion of malonyl-CoA to malonyl-CoA methyl ester by a malonyl-CoA O-methyltransferase classified, for example, under EC <IP_ADDRESS> such as the gene product of bioC; followed by conversion to 3-oxoglutaryl-CoA methyl ester by condensation with acetyl-CoA by a β-ketothiolase classified, for example, under EC <IP_ADDRESS> such as the gene product of bk tB or by condensation with malonyl-CoA by a β-ketoacyl-[acp] synthase classified, for example, under EC <IP_ADDRESS> such as the gene product of fa bH; followed by conversion to 3-hydroxy-glutaryl-CoA methyl ester by a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.—such as EC <IP_ADDRESS> (e.g., the gene product of fa bG) or EC <IP_ADDRESS> (e.g., the gene product of ph aB); followed by conversion to 2,3-dehydroglutaryl-CoA methyl ester by an enoyl-CoA hydratase classified, for example, under EC <IP_ADDRESS> such as the gene product of ph aJ (Shen et al., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915; Fukui et al., Journal of Bacteriology, 1998, 180(3), 667-673); followed by conversion to glutaryl-CoA methyl ester by a trans-2-enoyl-CoA reductase classified, for example, EC 1.3.1.—such as EC <IP_ADDRESS>, EC <IP_ADDRESS>, EC <IP_ADDRESS> or EC <IP_ADDRESS>; followed by (i) conversion to glutarate methyl ester by a thioesterase classified, for example, under EC 3.1.2.—such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No. AAO77182) or a Lactobacillus plantarum acyl-ACP thioesterase (GenBank Accession No. CCC78182.1) or (ii) conversion to glutaryl-CoA by a pimeloyl-[acp] methyl ester methylesterase classified, for example, under EC <IP_ADDRESS> such as bio H (SEQ ID NO: 1).
As shown in FIG. 3, glutarate methyl ester can be synthesized from malonyl-CoA by conversion of malonyl-CoA to malonyl-CoA methyl ester by a malonyl-CoA O-methyltransferase classified, for example, under EC <IP_ADDRESS> such as the gene product of bioC; followed by conversion to 3-oxoglutaryl-CoA methyl ester by condensation with acetyl-CoA by a β-ketothiolase classified, for example, under EC <IP_ADDRESS> such as the gene product of bk tB or by condensation with malonyl-CoA by a β-ketoacyl-[acp] synthase classified, for example, under EC <IP_ADDRESS> such as the gene product of fa bH; followed by conversion to 3-hydroxy-glutaryl-CoA methyl ester by a 3-hydroxyacyl-CoA dehydrogenase classified, for example, under EC 1.1.1.—such as EC <IP_ADDRESS> or EC <IP_ADDRESS> (e.g., the gene product of fa dB or hbd); followed by conversion to 2,3-dehydroglutaryl-CoA methyl ester by an enoyl-CoA hydratase classified, for example, under EC <IP_ADDRESS> such as the gene product of crt; followed by conversion to glutaryl-CoA methyl ester by a trans-2-enoyl-CoA reductase classified, for example, under EC <IP_ADDRESS> such as the gene product of ter or td ter; followed by (i) conversion to glutarate methyl ester by a thioesterase classified, for example, under EC 3.1.2.—such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No. AAO77182) or a Lactobacillus plantarum acyl-CoA thioesterase (GenBank Accession No. CCC78182.1) or (ii) conversion to glutaryl-CoA by a pimeloyl-[acp] methyl ester methylesterase classified, for example, under EC <IP_ADDRESS> such as bio H (SEQ ID NO: 1).
Pathway to Glutarate or 5-Oxopentanoate Using Glutarate Methyl Ester, Glutaryl-[Acp] or Glutaryl-CoA as a Central Precursor
As depicted in FIG. 4, glutarate methyl ester can be converted to glutarate by an esterase classified, for example, EC 3.1.1.—, such as EC <IP_ADDRESS> or EC <IP_ADDRESS> such as estC (SEQ ID NO: 8).
As depicted in FIG. 4, glutaryl-CoA can be converted to glutarate by a (i) a thioesterase classified, for example, EC 3.1.2.—, such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No. AAO77182) or a Lactobacillus plantarum acyl-CoA thioesterase (GenBank Accession No. CCC78182.1) (ii) a reversible CoA-ligase classified, for example, under EC <IP_ADDRESS>, (iii) a CoA-transferase classified, for example, under EC 2.8.3.—such as EC <IP_ADDRESS>, or (iv) an acylating dehydrogenase classified under, for example, EC <IP_ADDRESS> or EC <IP_ADDRESS> such as encoded by P duB or Pd uP and an aldehyde dehydrogenase classified under EC 1.2.1.—such as a glutarate semialdehyde dehydrogenase classified, for example, under EC <IP_ADDRESS>, a succinate-semialdehyde dehydrogenase classified, for example, under EC <IP_ADDRESS> or EC <IP_ADDRESS>, or an aldehyde dehydrogenase classified under EC <IP_ADDRESS>. For example, a 5-oxovalerate dehydrogenase such as the gene product of Cp nE, a 6-oxohexanoate dehydrogenase such as the gene product of Ch nE, or a 7-oxoheptanoate dehydrogenase (e.g., the gene product of ThnG from Sphingomonas macrogolitabida) can be used to convert 5-oxopentanoic acid to glutarate.
As depicted in FIG. 4, glutaryl-[acp] can be converted to glutarate by a thioesterase classified, for example, EC 3.1.2.—, such as the tesB (SEQ ID NO:15), YciA (SEQ ID NO:14) or Acot13, a Bacteroides thetaiotaomicron acyl-ACP thioesterase (GenBank Accession No. AAO77182) or a Lactobacillus plantarum acyl-CoA thioesterase (GenBank Accession No. CCC78182.1).
Pathway to 5-Hydroxypentanoate Using Glutarate Methyl Ester as a Central Precursor
As depicted in FIG. 5, 5-hydroxypentanoate can be synthesized from the central precursor glutarate methyl ester by conversion of glutarate methyl ester to glutaric acid by an esterase classified under EC 3.1.1.—(e.g., the gene product of estC) such as a carboxyl esterase classified under EC <IP_ADDRESS> or an acetylesterase classified under EC <IP_ADDRESS>; followed by conversion of glutaric acid to glutarate semialdehyde by a carboxylate reductase classified, for example, under EC <IP_ADDRESS> such as the gene product of car in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:16) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:17) gene from Nocardia), or the gene product of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion to 5-hydroxypentanoate by a dehydrogenase classified, for example, under EC 1.1.1.—such as a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC <IP_ADDRESS>8 (e.g., the gene from of Ch nD), a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.—such as the gene product of Cp nD (see, for example, Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684), or a 4-hydroxybutyrate dehydrogenase such as ga bD (see, for example, Lütke-Eversloh & Steinbüchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See, FIG. 5.
As depicted in FIG. 5, 5-hydroxypentanoate can be synthesized from the central precursor glutarate methyl ester by conversion of glutarate methyl ester to glutarate semialdehyde methyl ester by a carboxylate reductase classified, for example, under EC <IP_ADDRESS> such as from a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:16) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:17) gene from Nocardia), or the gene product of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion to glutarate semialdehyde by an esterase classified under EC 3.1.1.—(e.g., the gene product of estC) such as a carboxyl esterase classified under EC <IP_ADDRESS> or an acetylesterase classified under EC <IP_ADDRESS>; followed by conversion to 5-hydroxypentanoate by a dehydrogenase classified, for example, under EC 1.1.1.—such as a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC <IP_ADDRESS>8 (e.g., the gene from of Ch nD), a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.—such as the gene product of Cp nD, or a 4-hydroxybutyrate dehydrogenase such as ga bD.
As depicted in FIG. 5, 5-hydroxypentanoate can be synthesized from the central precursor glutarate methyl ester by conversion of glutarate methyl ester to glutarate semialdehyde methyl ester by a carboxylate reductase classified, for example, under EC <IP_ADDRESS> such as from a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:16) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:17) gene from Nocardia), or the gene product of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion to 5-hydroxypentanoate methyl ester by an alcohol dehydrogenase classified, for example, under EC 1.1.1.—(e.g., EC <IP_ADDRESS>, EC <IP_ADDRESS>, EC <IP_ADDRESS>, or EC <IP_ADDRESS>) such as the gene product of YMR318C, YqhD, or the protein having GenBank Accession No. CAA81612.1; followed by conversion to 5-hydroxypentanoate by an esterase classified under EC 3.1.1.—(e.g., the gene product of estC) such as a carboxyl esterase classified under EC <IP_ADDRESS> or an acetylesterase classified under EC <IP_ADDRESS>.
Pathway to 2,4-Pentadienoyl-CoA Using 5-Hydroxypentanoate as a Central Precursor
As depicted in FIG. 6, 2,4-pentadienoyl-CoA can be synthesized from 5-hydroxypentanoate by conversion of 5-hydroxypentanoate to 5-hydroxypentanoyl-CoA by a 5-hydroxypentanoate CoA-transferase or 4-hydroxybutryrate CoA-transferase classified, for example, under EC 2.8.3.—such as EC <IP_ADDRESS> or EC <IP_ADDRESS> or by a synthase classified, for example, under EC 6.2.1.—such as a 3-hydroxypropionyl-CoA synthase classified under EC <IP_ADDRESS>; followed by conversion to 2,4-pentadienoyl-CoA by a dehydratase such as 5-hydroxypentanoy-CoA dehydratase classified, for example, under EC 4.2.1.—obtained from Clostridium viride.
Cultivation Strategy
In some embodiments, the cultivation strategy entails achieving an aerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrification cultivation condition. Enzymes characterized in vitro as being oxygen sensitive require a micro-aerobic cultivation strategy maintaining a very low dissolved oxygen concentration (See, for example, Chayabatra & Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson and Bouwer, 1997, Journal of Industrial Microbiology and Biotechnology, 18(2-3), 116-130).
In some embodiments, a cyclical cultivation strategy entails alternating between achieving an anaerobic cultivation condition and achieving an aerobic cultivation condition.
In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate or oxygen limitation.
In some embodiments, a final electron acceptor other than oxygen such as nitrates can be utilized. In some embodiments, a cell retention strategy using, for example, ceramic membranes can be employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.
In some embodiments, the principal carbon source fed to the fermentation in the synthesis of one or more C5 building blocks can derive from biological or non-biological feedstocks.
In some embodiments, the biological feedstock can be or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.
The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol., 2011, 90:885-893).
The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms such as Cupriavidus necator and Pseudomonas putida in the synthesis of 3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin and Prather, J. Biotechnol., 2009, 139:61-67).
The efficient catabolism of lignin-derived aromatic compounds such as benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).
The efficient utilization of agricultural waste, such as olive mill waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008, 99(7):2419-2428).
The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al., J. Bioscience and Bioengineering, 1999, 87(5):647-654).
The efficient utilization of furfural, derived from a variety of agricultural lignocellulosic sources, has been demonstrated for Cupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).
In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.
The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).
The efficient catabolism of CO₂ and H₂, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al., Energy, Sustainability and Society, 2012, 2:11).
The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Köpke et al., Applied and Environmental Microbiology, 2011, 77(15):5467-5475).
The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demonstrated for numerous microorganisms, such as Delft ia acidovorans and Cupriavidus necator (Ramsay et al., Applied and Environmental Microbiology, 1986, 52(1):152-156).
Metabolic Engineering
The present document provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such steps. Where less than all the steps are included in such a method, the first, and in some embodiments the only, step can be any one of the steps listed.
Furthermore, recombinant hosts described herein can include any combination of the above enzymes such that one or more of the steps, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps, can be performed within a recombinant host. This document provides host cells of any of the genera and species listed and genetically engineered to express one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms of any of the enzymes recited in the document. Thus, for example, the host cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.
In addition, this document recognizes that where enzymes have been described as accepting CoA-activated substrates, analogous enzyme activities associated with [acp]-bound substrates exist that are not necessarily in the same enzyme class.
Also, this document recognizes that where enzymes have been described accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.
This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or co-substrate, such as acetyl-CoA, many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this document recognizes that where enzymes have high specificity for e.g., a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.
In some embodiments, the enzymes in the pathways outlined herein are the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co-factor specificity.
In some embodiments, the enzymes in the pathways outlined here can be gene dosed, i.e., overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.
In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to a 2,4-pentadienoyl-CoA.
Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.
In some embodiments, fluxomic, metabolomic and transcriptomal data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to 2,4-pentadienoyl-CoA.
In some embodiments, the host microorganism's tolerance to high concentrations of 2,4-pentadienoyl-CoA can be improved through continuous cultivation in a selective environment.
In some embodiments, the host microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA, (2) create a NADH or NADPH imbalance that may be balanced via the formation of 2,4-pentadienoyl-CoA, and/or (3) prevent degradation of central metabolites, central precursors leading to and including 2,4-pentadienoyl-CoA.
In some embodiments requiring intracellular availability of acetyl-CoA-CoA for C5 building block synthesis, endogenous enzymes catalyzing the hydrolysis of acetyl-CoA such as short-chain length thioesterases can be attenuated in the host organism.
In some embodiments requiring condensation of acetyl-CoA and malonyl-CoA for 2,4-pentadienoyl-CoA synthesis, one or more endogenous β-ketothiolases catalyzing the condensation of only acetyl-CoA to acetoacetyl-CoA such as the endogenous gene products of At oB or ph aA can be attenuated.
In some embodiments requiring the intracellular availability of acetyl-CoA for 2,4-pentadienoyl-CoA synthesis, an endogenous phosphotransacetylase generating acetate such as pta can be attenuated (Shen et al., Appl. Environ. Microbiol., 2011, 77(9):2905-2915).
In some embodiments requiring the intracellular availability of acetyl-CoA for 2,4-pentadienoyl-CoA synthesis, an endogenous gene in an acetate synthesis pathway encoding an acetate kinase, such as ack, can be attenuated.
In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for 2,4-pentadienoyl-CoA synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to lactate such as a lactate dehydrogenase encoded by ld hA can be attenuated (Shen et al., 2011, supra).
In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for 2,4-pentadienoyl-CoA synthesis, endogenous genes encoding enzymes, such as menaquinol-fumarate oxidoreductase, that catalyze the degradation of phophoenolpyruvate to succinate such as frdBC can be attenuated (see, e.g., Shen et al., 2011, supra).
In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for 2,4-pentadienoyl-CoA synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of acetyl-CoA to ethanol such as the alcohol dehydrogenase encoded by adhE can be attenuated (Shen et al., 2011, supra).
In some embodiments, where pathways require excess NADH co-factor for 2,4-pentadienoyl-CoA synthesis, a recombinant formate dehydrogenase gene can be overexpressed in the host organism (Shen et al., 2011, supra).
In some embodiments, acetyl-CoA carboxylase can be overexpressed in the host organisms.
In some embodiments, one or more of 3-phosphoglycerate dehydrogenase, 3-phosphoserine aminotransferase and phosphoserine phosphatase can be overexpressed in the host to generate serine as a methyl donor for the S-Adenosyl-L-methionine cycle.
In some embodiments, a methanol dehydrogenase or a formaldehyde dehydrogenase can be overexpressed in the host to allow methanol catabolism via formate.
In some embodiments, where pathways require excess NADH or NADPH co-factor for 2,4-pentadienoyl-CoA synthesis, a transhydrogenase dissipating the cofactor imbalance can be attenuated.
In some embodiments, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to ethanol such as pyruvate decarboxylase can be attenuated.
In some embodiments, an endogenous gene encoding an enzyme that catalyzes the generation of isobutanol such as a 2-oxo acid decarboxylase can be attenuated.
In some embodiments requiring the intracellular availability of acetyl-CoA for 2,4-pentadienoyl-CoA synthesis, a recombinant acetyl-CoA synthetase such as the gene product of acs can be overexpressed in the microorganism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
In some embodiments, carbon flux can be directed into the pentose phosphate cycle to increase the supply of NADPH by attenuating an endogenous glucose-6-phosphate isomerase (EC <IP_ADDRESS>).
In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al., 2003, Biotechnology Progress, 19(5), 1444-1449).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of 2,4-pentadienoyl-CoA, a gene such as Ud hA encoding a puri dine nucleotide transhydrogenase can be overexpressed in the host organisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant glyceraldehyde-3-phosphate-dehydrogenase gene such as Gap N can be overexpressed in the host organisms (Brigham et al., 2012, supra).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant malic enzyme gene such as ma eA or mae B can be overexpressed in the host organisms (Brigham et al., 2012, supra).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant glucose-6-phosphate dehydrogenase gene such as z wf can be overexpressed in the host organisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant fructose 1,6 di phosphatase gene such as fbp can be overexpressed in the host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 building block, endogenous triose phosphate isomerase (EC <IP_ADDRESS>) can be attenuated.
In some embodiments, where pathways require excess NADPH co-factor in the synthesis of 2,4-pentadienoyl-CoA, a recombinant glucose dehydrogenase such as the gene product of gdh can be overexpressed in the host organism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
In some embodiments, endogenous enzymes facilitating the conversion of NADPH to NADH can be attenuated, such as the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases classified under EC <IP_ADDRESS> (NADH-specific) and EC <IP_ADDRESS> (NADPH-specific).
In some embodiments, an endogenous glutamate dehydrogenase (EC <IP_ADDRESS>) that utilizes both NADH and NADPH as co-factors can be attenuated.
In some embodiments, a membrane-bound enoyl-CoA reductase can be solubilized via expression as a fusion protein to a small soluble protein such as a maltose binding protein (Glo erich et al., FEBS Letters, 2006, 580, 2092-2096).
In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the endogenous polyhydroxyalkanoate synthase enzymes can be attenuated in the host strain.
In some embodiments using hosts that naturally accumulate lipid bodies, the genes encoding enzymes involved with lipid body synthesis are attenuated.
In some embodiments, a L-glutamate dehydrogenase, a L-glutamine synthetase, or a glutamate synthase can be overexpressed in the host to regenerate L-glutamate from 2-oxoglutarate as an amino donor for w-transaminase reactions.
In some embodiments, enzymes such as pimeloyl-CoA dehydrogenase classified under, EC <IP_ADDRESS>; an acyl-CoA dehydrogenase classified, for example, under EC <IP_ADDRESS> or EC <IP_ADDRESS>; and/or a glutaryl-CoA dehydrogenase classified, for example, under EC <IP_ADDRESS> that degrade central metabolites and central precursors leading to and including C5 building blocks can be attenuated.
In some embodiments, endogenous enzymes activating C5 building blocks via Coenzyme A esterification such as CoA-ligases (e.g., a glutaryl-CoA synthetase) classified under, for example, EC <IP_ADDRESS> can be attenuated.
Producing 2,4-Pentadienoyl-CoA Using a Recombinant Host
Typically, 2,4-pentadienoyl-CoA can be produced by providing a host microorganism and culturing the provided microorganism with a culture medium containing a suitable carbon source as described above. In general, the culture media and/or culture conditions can be such that the microorganisms grow to an adequate density and produce 2,4-pentadienoyl-CoA efficiently. For large-scale production processes, any method can be used such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demain and J. E. Davies, ASM Press; and Principles of Fermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, or more tank) containing an appropriate culture medium is inoculated with a particular microorganism. After inoculation, the microorganism is incubated to allow biomass to be produced. Once a desired biomass is reached, the broth containing the microorganisms can be transferred to a second tank. This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank. In addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank.
Once transferred, the microorganisms can be incubated to allow for the production of 2,4-pentadienoyl-CoA. Once produced, any method can be used to produce 1,3-butadiene from 2,4-pentadienoyl-CoA such as depicted in FIG. 7. The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES Example 1 Enzyme Activity of Carboxylate Reductase Using Glutarate Methyl Ester as Substrate and Forming Glutarate Semialdehyde Methyl Ester
A nucleotide sequence encoding a His-tag was added to the genes from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium massiliense, and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 2-4, 6 and 7, respectively (GenBank Accession Nos. ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, and ADG98140.1, respectively) (see FIG. 8) such that N-terminal HIS tagged carboxylate reductases could be produced. Each of the modified genes was cloned into a pET Duet expression vector alongside a sfp gene encoding a His-tagged phosphopantetheine transferase from Bacillus subtilis, both under control of the T7 promoter. Each expression vector was transformed into a BL21[DE3] E. coli host along with the expression vectors from Example 2. Each resulting recombinant E. coli strain was cultivated at 37° C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 37° C. using an auto-induction media.
The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferase were purified from the supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated via ultrafiltration.
The enzyme activity assay was performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM glutarate methyl ester, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. The enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase or the empty vector control to the assay buffer containing the glutarate methyl ester and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. The enzyme only control without glutarate methyl ester demonstrated low base line consumption of NADPH. See FIG. 9.
The gene product of SEQ ID NO 2, 3, 6, and 7, enhanced by the gene product of sfp, accepted glutarate methyl ester as substrate as confirmed against the empty vector control (see FIG. 10) and synthesized glutarate semialdehyde methyl ester.
Example 2 Enzyme Activity of Pimeloyl-[Acp] Methyl Ester Methylesterase Using Glutaryl-CoA Methyl Ester as Substrate and Forming Glutaryl-CoA
A sequence encoding an C-terminal His-tag was added to the gene from Escherichia coli encoding the pimeloyl-[acp] methyl ester methylesterase of SEQ ID NO: 1 (see FIG. 8) such that C-terminal HIS tagged pimeloyl-[acp] methyl ester methylesterase could be produced. The resulting modified gene was cloned into a pET28b+ expression vector under control of the T7 promoter and the expression vector was transformed into a BL21[DE3] E. coli host. The resulting recombinant E. coli strain was cultivated at 37° C. in a 500 mL shake flask culture containing 100 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 18° C. using 0.3 mM IPTG.
The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation. The pimeloyl-[acp] methyl ester methylesterase was purified from the supernatant using Ni-affinity chromatography, buffer exchanged and concentrated into 20 mM HEPES buffer (pH=7.5) via ultrafiltration and stored at 4° C.
Enzyme activity assays converting glutaryl-CoA methyl ester to glutaryl-CoA were performed in triplicate in a buffer composed of a final concentration of 25 mM Tris.HCl buffer (pH=7.0) and 5 [mM] glutaryl-CoA methyl ester. The enzyme activity assay reaction was initiated by adding pimeloyl-[acp] methyl ester methylesterase to a final concentration of 10 [μM] to the assay buffer containing the glutaryl-CoA methyl ester and incubated at 30° C. for 1 h, with shaking at 250 rpm. The formation of glutaryl-CoA was quantified via LC-MS.
The substrate only control without enzyme showed no trace quantities of the substrate glutaryl-CoA. See FIG. 11. The pimeloyl-[acp] methyl ester methylesterase of SEQ ID NO. 1 accepted glutaryl-CoA methyl ester as substrate and synthesized glutaryl-CoA as reaction product as confirmed via LC-MS. See FIG. 11.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
What is claimed is:
1. A method of biosynthesizing glutarate methyl ester in a recombinant host comprising at least one exogenous nucleic acid encoding a polypeptide having malonyl-CoA O-methyltransferase activity, the method comprising enzymatically converting at least one of malonyl-[acp] and malonyl-CoA to glutarate methyl ester in said host using a polypeptide having malonyl-CoA O-methyltransferase activity, a polypeptide having thioesterase activity, or a combination thereof, wherein said polypeptide having malonyl-CoA O-methyltransferase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:13 and is capable of enzymatically converting malonyl-CoA to malonyl-CoA methyl ester or malonyl-[acp] to malonyl-[acp] methyl ester, and wherein said polypeptide having thioesterase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:15 and is capable of enzymatically converting glutaryl-CoA methyl ester to glutarate methyl ester, wherein malonyl-CoA is enzymatically converted to malonyl-CoA methyl ester using said at least one polypeptide having malonyl-CoA O-methyltransferase activity; or wherein malonyl-[acp] is enzymatically converted to malonyl-[acp] methyl ester using said at least one polypeptide having malonyl-CoA O-methyltransferase activity, the method optionally further comprising enzymatically converting glutarate methyl ester to glutarate semialdehyde methyl ester in said host using at least one polypeptide having carboxylate reductase activity, wherein said polypeptide having carboxylate reductase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 and is capable of enzymatically converting glutarate methyl ester to glutarate semialdehyde methyl ester, wherein said enzymatic conversion occurs in the host using said polypeptide having malonyl-CoA O-methyltransferase activity, said polypeptide having thioesterase activity, or a combination thereof.
2. The method of claim 1, wherein malonyl-[acp] methyl ester is enzymatically converted to glutaryl-[acp] methyl ester using at least one polypeptide having an activity selected from the group consisting of synthase activity, dehydrogenase activity, dehydratase activity, and reductase activity, wherein said polypeptide having reductase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 or 12 and is capable of enzymatically converting malonyl-[acp] methyl ester to glutaryl-[acp] methyl ester, and wherein glutaryl-[acp] methyl ester is enzymatically converted to glutarate methyl ester using at least one polypeptide having thioesterase activity, wherein said polypeptide having thioesterase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:15 and is capable of enzymatically converting glutaryl-CoA methyl ester to glutarate methyl ester.
3. The method of claim 1, further comprising enzymatically converting glutarate methyl ester to 5-oxopentanoic acid using at least one polypeptide having an activity selected from the group consisting of carboxylate reductase activity and esterase activity, the method optionally further comprising enzymatically converting glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid using at least one polypeptide having esterase activity, wherein the polypeptide having esterase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:8 and is capable of enzymatically converting glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid, the method optionally further comprising using at least one polypeptide having dehydrogenase activity classified in EC 1.1.1- to enzymatically convert glutarate semialdehyde methyl ester to 5-hydroxypentanoic acid.
4. The method of claim 1, said method further comprising enzymatically converting glutarate methyl ester to glutaric acid using at least one polypeptide having esterase activity, wherein the polypeptide having esterase activity has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:8, the method optionally method further comprising enzymatically converting glutaric acid to 5-hydroxypentanoic acid using at least one polypeptide having carboxylate reductase activity and at least one polypeptide having dehydrogenase activity classified under EC 1.1.1.-, wherein the polypeptide having carboxylate reductase activity has at least 90% sequence identity to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 2-7 and is capable of enzymatically converting glutarate methyl ester to glutaric acid.
5. The method of claim 4, further comprising enzymatically converting 5-hydroxypentanoic acid to 2,4-pentadienoyl-CoA using at least one polypeptide having an activity selected from the group consisting of CoA-transferase activity, a synthase activity, and dehydratase activity.
6. The method of claim 5, wherein (i) the polypeptide having a CoA-transferase activity or a synthase activity and (ii) the polypeptide having dehydratase activity enzymatically convert 5-hydroxypentoic acid to 2,4-pentadienoyl-CoA, the method optionally further comprising enzymatically converting 2,4-pentadienoyl-CoA into 1,3 butadiene using at least one polypeptide having an activity selected from the group consisting of hydratase activity, thioesterase activity, decarboxylase activity, dehydrogenase activity, CoA-transferase activity, and dehydratase activity, wherein the polypeptide having thioesterase activity has at least 90% sequence identity to any one of the amino acids set forth in SEQ ID NO:14-15 and is capable of enzymatically converting 5-hydroxypentoic acid to 2,4-pentadienoyl-CoA.
7. The method of claim 1, wherein the host is subjected to a cultivation strategy under aerobic or micro-aerobic cultivation conditions.
8. The method of claim 1, wherein the host is cultured under conditions of nutrient limitation either via nitrogen, phosphate or oxygen limitation.
9. The method of claim 1, wherein the host is retained using a ceramic membrane to maintain a high cell density during fermentation.
10. The method of claim 1, wherein a principal carbon source fed to the fermentation is derived from a biological feedstock.
11. The method of claim 10, wherein the biological feedstock is, or derives from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.
12. The method of claim 1, wherein a principal carbon source fed to the fermentation is derived from a non-biological feedstock.
13. The method of claim 12, wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid I isophthalic acid mixture waste streams.
14. The method of claim 1, wherein the host is a prokaryote selected from the group consisting of Escherichia; Clostridia; Corynebacteria; Cupriavidus; Pseudomonas; Delft ia; Bacillus; Lactobacillus; Lactococcus; and Rhodococcus, or a eukaryote selected from the group consisting of Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyyeromyces.
15. The method of claim 1, wherein the host exhibits tolerance to high concentrations of a C5 building block, and wherein the tolerance to high concentrations of a C5 building block is improved through continuous cultivation in a selective environment.
16. The method of claim 1, wherein said host expresses one or more exogenous polypeptides selected from the group consisting of having an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a feedback resistant threonine deaminase; a puri dine nucleotide transhydrogenase; a formate dehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6 di phosphatase; a propionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a L-glutamine synthetase; a lysine transporter; a dicarboxylate transporter; and a multidrug transporter activity.
17. The method of claim 1, wherein the host comprises an attenuation of one or more polypeptides having an activity selected from the group consisting of: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, an acetyl-CoA specific/J-ketothiolase, an acetoacetyl-CoA reductase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxo acid decarboxylase producing isobutanol, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, a transhydrogenase dissipating the cofactor imbalance, a glutamate dehydrogenase specific for the co-factor for which an imbalance is created, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C5 building blocks and central precursors as substrates; a glutaryl-CoA dehydrogenase; and a pimeloyl-CoA synthetase.
18. The method of claim 2 wherein the at least one polypeptide having synthase activity is classified under EC 2.3.1.-, the polypeptide having dehydrogenase activity is classified under EC 1.1.1-, and the polypeptide having dehydratase activity is classified under EC 4.2.1-.
19. The method of claim 5 wherein the polypeptide having CoA-transferase activity is classified under EC 2.8.3-, the polypeptide having synthase activity is classified under EC 2.3.1.-, and the polypeptide having dehydratase activity is classified under EC 4.2.1.-.
20. The method of claim 6 wherein the polypeptide having hydratase activity is classified under EC <IP_ADDRESS> or EC <IP_ADDRESS>, the polypeptide having dehydrogenase activity is classified under EC 1.1.1.-, the polypeptide having CoA-transferase activity is classified under 2.8.3.-, and the polypeptide having dehydratase activity is classified under EC 4.2.1.-.
|
/*
MIT License
Copyright(c) 2018
Antonio Di Nucci
Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in all
copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE
SOFTWARE.
*/
using System;
namespace LogicMine.Api.Filter
{
/// <summary>
/// A term within a filter, a term is a constraint placed on a single property
/// </summary>
public class FilterTerm : IFilterTerm
{
/// <inheritdoc />
public string PropertyName { get; }
/// <inheritdoc />
public FilterOperators Operator { get; }
/// <inheritdoc />
public object Value { get; }
/// <summary>
/// Construct a new FilterTerm
/// </summary>
/// <param name="propertyName">The name of the property the term applies to</param>
/// <param name="op">The type of filtering to perform</param>
/// <param name="value">The value to filter on</param>
public FilterTerm(string propertyName, FilterOperators op, object value)
{
if (string.IsNullOrWhiteSpace(propertyName))
throw new ArgumentException("Value cannot be null or whitespace.", nameof(propertyName));
PropertyName = propertyName;
Operator = op;
Value = value;
}
}
}
|
Fun talk:Baseball
From Conservapedia Talk:What is going on at CP? (10/31/07)
what?
Er ... What's a Red Sox? Susan You don't have to talk, but ... 15:34, 31 October 2007 (EDT)
Er ... "important"? Susan You don't have to talk, but ... 15:45, 31 October 2007 (EDT)
* Important to them and a few deranged baseball fans in Japan, PR, and DR, yes.
* That doesn't make it any easier for the poor players! How they suffer!
* Also, bonus points for using "pshaw" in a sentence. --Kels 17:53, 31 October 2007 (EDT)
Sick Freak!!! Landed!!!
20:41, 2 November 2007 (EDT)
"World series"
Apparently this was named after a long defunct newspaper.
Anyone care to develop the rounders angle? <IP_ADDRESS> (talk) 17:22, 18 April 2011 (UTC)
* Only if you can make it funny. 04:28, 19 April 2011 (UTC)
Page title.
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Add compile dependency protobuf-3
Issue: cassandra-commons:compileJava build failed for missing protobuf
JIRA: https://dcosjira.atlassian.net/browse/CASSANDRA-26
Can one of the admins verify this patch?
Can one of the admins verify this patch?
@mesosphere-ci: retest this please
|
FRANK MAXA, JR., v. THOMAS NEIDLEIN.
[No. 29,
October Term, 1932.]
Decided December 8th, 1932.
The cause was argued before Bond, C. J., Pattison, Henee, Adkins, Oeetttt, Digges, Paeke, and Sloan, JJ.
John S. Young, for the appellant.
Robert H. Archer and Aaron C. Snyder, with whom were J. Wilmer Cronin and J. Glasgow Archer on the brief, for the appellee.
Urner, J.,
delivered the opinion of the Court.
The trial of this action for assault and battery resulted in a verdict and judgment for the plaintiff, and the defendant has appealed. The chiefly contested ruling submitted to the jury, by granted prayers, the plaintiff’s claim for punitive damages. Ho doubt is created by the record as to the propriety of that ruling.
The defendant had been paying attentions to the plaintiff’s wife. In an effort to interrupt their objectionable relationship, the plaintiff accosted them as they were alighting fronr ah automobile in Baltimore and were about to enter the home of the wife’s brother. What occurred then and subsequently is thus described in the plaintiff’s testimony:
“I told him that I saw that he was still running with my wife, and he said that he had just picked her up- as she was coming out of the moving pictures. Q. Did she do that ? A. She got out and went into the house1, and he followed her in. I went to the door and he said that he would settle with me later. Then I went out, got in my car and started for home; and when I was out on the Philadelphia Boad, he pulled around in front of me. Q. You saw him on the Philadelphia Boad after you left- the house ? A. He pulled right in front- of me and stopped. * * * Q. Whereabouts was this? A. It was just outside of Baltimore City. * * * Q. You were headed towards Aberdeen? A. Yes, sir. Q. What happened then? A. He asked me if I was ready to settle with him there and I told him that I would not fight him in Baltimore City, and I said that I was going on up- the road. He said that he would follow me anywhere- — any place — to- settle with me; and I came on up the road to- White Harsh. Q. How did you come to stop the second time? A. He- asked me if I was ready to settle with him there. Q. Where did he drive ? A. In front of me. Q. Did he stop- his car a second time? A. Yes, sir. Q. What did he do- then? A. He asked me if I was ready to settle with him then, and I told him that I was. Q. What did he do then? A. He stood in front of the lights on the road, and I told him that I had caught him with my wife after I had told him to keep away from her. Then I stepped out into the road and had nothing with me at that time. Then I picked up something out of my car and started for him. Q. What did you pick up ? A. Then what happened — did he start to run? A. Started back after him, caught him and we wrestled and fell to the ground. Q. Did you hit him with the jack? A. Ho-, sir. Q. Why not — because he was holding it? A. Ho, sir. After we wrestled and fell on the ground, I started back for my car; and as I pulled the door open to get in, he hit me. Q. Where did he hit you? A. Right along side the nose. Q. Did he knock you into- the road? A. Yes, sir. Q1. What was the damage to you ? A. He broke my nose and two ribs. Q. What broke your ribs? A. I think he kicked me after I fell. Q. Were you conscious when you fell? A. Ho, sir. Q. Where was he when you came to- ? A. He was gone. Q. On which side <of the ear were you lying when you came to? A. On the left side. Q. In the- Philadelphia Road ? A. Yes, sir.”
The testimony of the plaintiff’s physician confirmed his statement as to the nature and extent of his injuries.
The defendant’s version of his meeting with the plaintiff in Baltimore and of their encounter on the Philadelphia Road is given in the following extracts from his testimony: “Q. A. Yes, sir. Q. Where did you see Mr. Xeidlein — from which way did he appear? A. I don’t know which way he came from. Q. When did he appear — how soon after you had driven up ? A. Immediately. Q. He has testified that he didn’t draw a pistol on you there — tell the jury whether or not he did? A. Yes, sir; he did. I had driven up there and was about ready to open the door for Mrs. Xeidlein to get out; and, when I turned and looked to the left, he was standing there with a gun poked into my side. Then I got out of my car and he ran around to the other side of the car and tried to hold the door shut so she could not get out. I told him to let her get out and she went on into the house there, and he said, ‘All right, I will settle wdth you later.’ * * * Q. Hid he leave there before or after you did? A. He left there before I left. Q. When you did leave and whichever way yon went, you did get to the stop-light at the crossing of the Baltimore and Ohio Railroad over the Philadelphia Road before he did ? A. Yes, sir; I did. * * * Q. How as to the altercation that yon had on the Philadelphia Road — where was it? A. At White Marsh. * * * Q. How did you come out there — who got there first ? A. Mr. Xeidlein wras driving ahead of me; and I was driving somewhere around a hundred and fifty to two hundred yards behind him; and, when we got that far — it was a right dark lonely place — he pulled off to the side of the road. There was no machine coming in the opposite direction, and I pulled in hack of him and stopped and I got out and walked towards the front of my car. When I got in the lights of my car, I saw Mr. Xeidlein walking towards me. Q. Hid he have a pistol in his hand then? A. Yes, six; he had a pistol in his band then, and then he immediately ran hack, put the pistol up and got a jack. Q. What did he do with that? A. He kit me two or threé times — tried to hit me in the face with it. I held up my left hand and he hurt that badly, and I could not stand that any more, so I tried to get back away from hiip, and he followed me and we both fell down, and I got possession of the jack. When we got up, I took the jack up along the edge of the road and threw it on the running board of my car. Mr. Neidlein had started to his car, I ran up to him; and, about the time he was ready to- get in his ear, I hit him. * * * Q. Why did you strike him ? A. I was afraid he would get something out of the car. I had only one arm that was good and I had already taken the jack away from him. * * * Q. Did you have any weapon or pistol when you struck him, or did you hit him with a club ? A. No, sir; I didn’t have anything but this pair of gloves. * * * Q. Now, when you-hit him, did he fall? A. Yes, sir. Q. What did you do after that ? A. I walked back to my car, stood there a couple of seconds until he got up, and then I jumped in my car and drove off. * * * Q. Did you kick him after he was down? A. No, sir.”
As between the narratives of the plaintiff and defendant, concerning incidents to which they alone testified, it was the right and duty of the jury to determine which statement was more worthy of belief. The verdict shows that the jury accepted the plaintiff’s version, which unquestionably described an attack upon him by the defendant sufficiently wanton, unprovoked, and excessive to justify an instruction that punitive as well as compensatory damages might be awarded. Zell v. Dunaway, 115 Md. 1, 80 A. 215; Stockham v. Malcolm, 111 Md. 615, 74 A. 569, 19 Ann. Cas. 759; Sloan v. Edwards, 61 Md. 89. While the first of the plaintiff’s granted prayers referred only to compensatory damages, but nevertheless permitted the pecuniary circumstances of the defendant to'be considered, -which, as said in Stockham v. Malcolm, supra, are pertinent only where punitive damages are in issue, yet, as further held in that case, the granting of a prayer in that form is not ground of reversal when, as here, the plaintiff’s testimony, if true, supports the theory that exemplary or punitive damages are allowable.
The first six evidence exceptions are abandoned. Exception number eight is immaterial, since it refers to a ruling on. an inquiry which the witness then under examination said he could not answer. The seventh, ninth, eleventh, and twelfth exceptions were taken because of the admission of evidence as to the defendant’s pecuniary circumstances. This was a proper subject of inquiry, in view7 of the issues involved.
The thirteenth and fifteenth exceptions relate to an incident at the home of the defendant’s father and mother, in regard to which the defendant had been questioned and had testified without objection on his part, as follows: “Q. Didn’t Air. A. Air. Xeidlein came up there and told my mother and father that; and when he got through, 1 opened the door and told him to get out.”
Referring to that occasion, the plaintiff was allowed to testify that, when he told the defendant’s mother and father about finding his wife and the defendant together on the previous night, and about the subsequent assault, the defendant "opened the door and told me to get out or he would black my other eye.” This testimony was admissible as tending to prove that the defendant’s attitude toward the plaintiff had been aggressive.
There was no error in the ruling which is the subject of the fourteenth exception. A witness who had testified to a conversation between the plaintiff and an officer to whom he had first made complaint after the assault was allowed to be recalled and to contradict a specific statement which the officer, testifying for the defendant, quoted the plaintiff as having made in the course of the interview.
The sixteenth exception, relating to the prayers, has already been discussed.
The ground of the remaining exception was the refusal of the court to prevent counsel for the plaintiff, in arguing the ease before the jury, from referring to the fact that the plaintiff, on the day of the assault, had found his children alone when he went home, and that he had gone to Baltimore and found Ms wife in the defendant’s company. That fact having been proved without objection or dispute, as being immediately involved in the altercation preceding the assault, it could properly be mentioned in the argument.
Judgment affirmed, with costs.
|
Some analysts prefer to heat the tube in a water bath, but this is a slow process, and as the only object of this method is to obtain a rapid result with fair accuracy, it is best to proceed as quickly as possible.
When the liquid is quite free from clots it is cooled down, and the tube filled up to the 50-c.c. mark with ether. The tube is corked up, well shaken, and allowed to stand until the ethereal layer has completely separated.
Ten c.c. of this ethereal extract is measured off into a weighed dish, evaporated to dryness, and weighed. The result, after subtracting the weight of the dish, will be one-fifth of the weight of fat in 10 c.c. of milk. Knowing the specific gravity of the milk, it is easy to calculate the percentage of fat.
309. Apparatus. — The apparatus, which is shown in fig. 46, consists of a bottle in which the solution of the fat takes place, and a tube surrounded by a water jacket and enclosing a delicate hydrometer giving specific gravities from 743 to 766. Attached to the hydrometer is a thermometer. The bottle may be connected with the water-jacketed tube by inserting an india-rubber stopper through which pass two tubes arranged as for a wash bottle, the delivery tube being attached to the apparatus whilst the blowing tube has an india-rubber blower fixed to it. This apparatus is complete for one determination. Should a large number be required in rapid succession, a large number of bottles will be required.
310. The Operation. — Measure out 200 c.c. of milk into one of the bottles; add 10 c.c. of caustic potash solution (the solution used for expelling ammonia after heating with H 2 S0 4 in the acid process of nitrogen estimation, paragraph 91, may be used) and 60 c.c. of ether. The ether must be
|
import { Schema, model, Model } from 'mongoose';
import validator from 'validator';
import bcryptjs from 'bcryptjs';
import { UserBodyProtocol, UserModelProtocol, UserProtocol } from '../interfaces/modelsProtocols';
class User implements UserProtocol {
private model: Model<UserModelProtocol>;
public constructor() {
this.model = this.createModel();
}
get db(): Model<UserModelProtocol> {
return this.model;
}
private async check(body: UserBodyProtocol): Promise<string[]> {
const errors: string[] = [];
if (!body.name) errors.push('Nome requerido');
if (!body.email) errors.push('E-mail requerido');
if (!body.password) errors.push('Senha requerido');
if (!validator.isEmail(body.email)) errors.push('E-mail inválido');
if (body.password.length < 3 || body.password.length > 50)
errors.push('A senha precisa ter entre 3 e 50 caracteres');
return errors;
}
public async register(body: UserBodyProtocol): Promise<UserModelProtocol | string[]> {
const checkResult = await this.check(body);
if (checkResult.length > 0) return checkResult;
const user = await this.model.findOne({ email: body.email });
if (user) return ['E-mail já existe'];
const salt = bcryptjs.genSaltSync();
// eslint-disable-next-line no-param-reassign
body.password = bcryptjs.hashSync(body.password, salt);
return this.model.create(body);
}
public async login(body: UserBodyProtocol): Promise<UserModelProtocol | string[]> {
const checkResult = await this.check(body);
if (checkResult.length > 0) return checkResult;
const user = await this.model.findOne({ email: body.email });
if (!user) return ['Usuario não existe'];
if (!bcryptjs.compareSync(body.password, user.password)) return ['Usuario não existe'];
return user;
}
private createModel(): Model<UserModelProtocol> {
const schema = new Schema({
name: {
type: String,
required: true,
},
email: {
type: String,
required: true,
},
password: {
type: String,
required: true,
},
});
return model<UserModelProtocol>('user', schema);
}
}
export default new User();
|
Filter device and fume extraction device comprising filter device
ABSTRACT
A filter device for a fume extraction device includes at least two filter elements arranged at an angle relative to one another, and a holding frame for holding the at least two filter elements. An edge of one of the at least two filter elements lies adjacent to an edge of the other one of the at least two filter element. Each of the at least two filter elements has a surface with a variable distance between opposite outer faces of the filter element.
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is the U.S. National Stage of International Application No. PCT/EP2019/000114, filed Apr. 8, 2019, which designated the UnitedStates and has been published as International Publication No. WO2019/197055 A1 and which claims the priority of European Patent Application, Serial No. 18290031.6, filed Apr. 10, 2018, pursuant to 35U.S.C. 119(a)-(d).
BACKGROUND OF THE INVENTION
The present invention relates to a filter device for a fume extractiondevice and to a fume extraction device with such a filter device.
In the case of fume extraction devices, which are used in particular in kitchens, it is known to use filter devices by means of which grease andother contaminants are filtered out of the fumes and vapors sucked intothe fume extraction device. The filter devices represent, for example,so-called filter cartridges, which consist of multiple filter layers arranged one above the other in parallel, in particular expanded metal filter layers, which are held in a frame. Such a filter cartridge is described, for example, in DE 10 2013 212 921 A1.
In the case of fume extraction hoods, which are usually mounted on a room wall or ceiling and where the contaminated air is hence sucked up into the fume extraction device, the filter cartridge is built into the suction opening such that it covers the area of the suction opening.Multiple filter cartridges can also be inserted next to one another inthe suction opening. To offer an adequate filter area that enables reliable cleaning of air, a large suction opening is therefore necessary for fume extraction hoods. With a large suction opening, however, thefume extraction hood must be operated at high power in order to ensure a reliable intake of air.
In the case of fume extraction devices which are installed in or next toa hob and which thus suck the contaminated air down into the fumeextraction device, the area available for the suction opening is however small. With this type of fume extraction device too, which can also be referred to as a downdraft fan or downdraft extractor, the use of plate-shaped filter elements is likewise known. For example, DE 20 2009008 286 U1 describes a device for extracting cooking fumes in a direction pointing below a hob level with a cooking fume intake device.According to one embodiment, in this device a plate-shaped filterelement is introduced horizontally in a duct-like insert and the duct-like insert is suspended from a mounting opening. Thus in this embodiment the available filter area is limited to the size of the suction opening and is therefore small. According to an alternative embodiment a plate-shaped filter element is introduced obliquely into an enlargement of an exhaust air duct. The filter element is here placed ona holder-like projection of a part of the exhaust air duct, inparticular a collecting channel formed in the exhaust air duct. Although this embodiment offers a larger filter area, it is unfavorable in terms of handling. In this embodiment the user has to reach into the exhaust air duct through the smaller suction opening in order to get to thefilter element located in the enlargement located below this, forexample if said filter element is to be removed for cleaning purposes.
BRIEF SUMMARY OF THE INVENTION
The object of the present invention is therefore to create a solution with which, with a simple structure of the filter device and the fumeextraction device, a reliable intake and cleaning of air is possible atthe same time as a low power output of the fume extraction device.
According to a first aspect the object is achieved by a filter device for a fume extraction device which comprises at least two filterelements and at least one holding frame for holding the at least twofilter elements. The filter device is characterized in that each filterelement has a variable distance between the opposite outer faces of thefilter element over the surface of the filter element and that at least two filter elements are arranged at an angle relative to one another,and an edge of one of the filter elements lies adjacent to an edge ofthe other filter element.
The filter device has at least two filter elements and at least one holding frame. A filter device for a fume extraction device is understood, according to the invention, as a structural unit which canbe introduced on or into a suction opening of the fume extraction device and by means of which the air can be freed of contaminants. For this purpose, the filter device has at least two filter elements. The filterelements preferably represent elongated elements. The filter element can have filter material which is held in a filter frame. Alternatively, thefilter element consists of filter material and does not have its own filter frame. The filter material can, for example, be expanded metal layers, a fleece, a knitted fabric or a warp-knitted fabric. Inparticular, the filter material can consist of wire. In addition to theat least two filter elements, the inventive filter unit has a holding frame. The holding frame is used to hold the filter elements in thefilter device.
According to the invention, each filter element has a variable distance between the opposite outer faces of the filter element over the surface of the filter element. The outer faces of the filter element can also be referred to as outer walls. The outer faces, between which the distance varies, are in particular the upper face and the lower face of thefilter body. When the filter device is introduced into a fume extractiondevice, the upper face preferably forms the inflow side of the filterelement and the lower face the outflow side, which is also referred to as the clean air side. According to the invention, the distance betweenthe outer faces preferably varies over the width of the filter element.The width is understood here as the distance between the longitudinal edges of the filter element. The distance between end faces of thefilter element is understood as the length. The dimension between the outer faces of the filter element, in particular between the upper face and lower face of the filter element, is referred to as the height or thickness of the filter element. The height can vary over the width and/or the length of the filter element. Particularly preferably,however, the height of the filter element varies only in the width direction and has a constant cross-section over the length. Particularly preferably, the height of the filter element, i.e. the distance betweenthe opposite outer faces, is greater in the central area of the width ofthe filter body than in the lateral edge areas of the width.
At least two filter elements are provided in the filter device. If morethan two filter elements are provided, their number is preferably a multiple of two. If multiple filter elements are provided, they are preferably arranged such that at least two filter elements each abut one another on their end faces. The filter elements are therefore preferably arranged adjacent to one another in their length direction. For a better understanding, the preferred embodiment of the filter device with only two filter elements is mainly discussed below.
According to the invention, at least two filter elements are arranged atan angle relative to one another. Here, one edge of one of the filterelements lies adjacent to one edge of the other filter element. This arrangement of the two filter elements can also be referred to as aV-shaped arrangement.
Because the filter elements firstly have a cross-section which changes over the width and/or length of the filter element and the filterelements are moreover present in a V-shaped arrangement in the filterdevice, a number of advantages can be achieved.
Firstly, thanks to the shape of the filter elements, in particular thanks to the variable height of the filter elements at least over the width, a targeted setting of the air permeability of the filter elements takes place with respect to an air flow which strikes an outer face ofthe filter element and exits again on the opposite outer face of thefilter element. A constant flow resistance can here be set over the width of the filter element or the flow resistance can be varied overthe width, for example be set lower in the center than at the lateral edge areas. Moreover, thanks to this three-dimensional configuration ofthe filter element, an optimal pressure drop can be achieved at thefilter device, which is so low that a sufficient flow rate of air through the filter element can be ensured and which is nevertheless large enough that a sufficient separation of contaminants at the filterelement can be ensured. Thanks to the three-dimensional configuration ofthe filter element the volume in which contaminants can be stored in thefilter element can moreover be maximized. Besides the storage volume inthe filter element, the three-dimensional configuration of the filterelements also increases the filter area. The filter area is understood here to mean the area onto which contaminated air can flow during operation of the fume extraction device. Furthermore, the geometry ofthe filter element can also be adapted to the shape of the filterdevice, in that for example the distance between the outer faces of thefilter element is small, at least at the longitudinal edges of thefilter element. As a result, the size of a holding element at these longitudinal edges is small and the blockage of the air flowing throughthe filter device is small, as a result of which the demands on the performance of the fan of the fume extraction device in which the filterdevice is used are reduced. Because the at least two filter elements are held in one common holding frame, the filter device can easily be removed from the fume extraction device. It is not necessary to remove the filter elements from the fume extraction device separately. Thanks to the inventive arrangement of the filter elements in a V-shape in the holding frame the filter area is further enlarged, in addition to the enlargement of the filter area by the three-dimensional configuration ofthe filter elements, without the size of the suction opening of the fumeextraction device having to be increased. Compared to a single filterelement which is introduced obliquely and nevertheless has the same filter area as the inventive filter elements which are arranged at an angle relative to one another, the V-shaped arrangement also reduces the space requirement needed in the height direction of the fume extractiondevice.
Thus with a simple structure of the filter device, a reliable intake and cleaning of air is enabled with, at the same time, a low power output ofthe fume extraction device. Moreover, the handling of the fumeextraction device is simplified.
According to one embodiment, the edges of the two filter elements, which lie adjacent to one another, abut one another. The edges can in this case abut one another directly or can abut opposite sides of a web witha small thickness. In this embodiment, the size of the part of the holding frame on which these edges are held is thus small. The blockageof the air flow which flows through the filter device is thus also low.Moreover, this also further minimizes the required overall height of thefilter device for a given size of the suction opening and the required size of the filter area. If the edges were to be spaced apart from one another, for example spaced apart from one another by a plate, the angle between the filter elements would have to be chosen to be smaller inorder to be able to achieve the required filter area and nevertheless beable to introduce the filter device into the suction opening.
According to a preferred embodiment, the two filter elements are at an angle of less than 90° and preferably less than 40° to one another. The angle here is preferably between the center lines which extend in the width direction of the filter elements from one longitudinal edge to the opposite longitudinal edge. Due to the variable distance between the outer faces of the filter element, the angle between the facing outer faces of the two filter elements can be smaller or larger than the angle between the center lines of the filter elements. Thanks to the small angle between the center lines of the filter elements the filter area can be maximized for a given size of the suction opening.
According to a preferred embodiment, the distance between the outer faces of a filter element from one edge of the filter element in the direction of the opposite edge increases from a minimum to a maximum andto the other edge decreases from the maximum to the minimum. The edges between which the distance increases from a minimum to a maximum and decreases again to the minimum are preferably the longitudinal edges ofthe filter element. The change in thickness is thus preferably located in the width direction of the filter element. Preferably, the maximum ofthe thickness lies in the center of the width direction. The filterelement can hence have an oval cross-section, for example. Because the thickness of the filter element is preferably small at the edges, thefilter element can easily be held at these edges in the holding frame ofthe filter device. In particular, only a part of the holding frame witha small size is required to hold the thin edges. This further reduces the blockage of the air. However, since there is a greater thickness over the width of the filter element, the storage volume of the filterelement is still large.
According to a preferred embodiment, the filter element has a diamond-shaped cross-section. In addition to the advantages mentioned ofthe thickness of the filter element increasing to a maximum, this embodiment has the particular advantage that the filter element can easily be produced and can be reliably held at the longitudinal edges inthe holding frame by simple means, for example rails.
According to one embodiment, the holding frame has two end walls whichabut end faces of the at least two filter elements, and rails of the holding frame run between the end walls. Parts of the holding frame which have an elongated channel shape are referred to here as rails. The rails hence each serve to accommodate an edge, in particular a longitudinal edge of the filter elements. The end walls and rails are preferably made of plastic or metal. The end walls delimit the space which is formed between the filter elements arranged at an angle relative to one another, at the longitudinal ends of the filterelements. The end walls hence preferably form a triangular surface or atrapezoidal surface. The filter elements can be fastened to the end walls. However, it is also within the scope of the invention for thefilter elements only to be fastened to the rails, for example to be accommodated in the rails, and for the rails to be fastened to the end walls. In addition to the rails, struts can be provided between the end walls, which extend perpendicular to the rails and connect them to one another. Because the holding frame is formed at least by end walls and rails, it has stability. The filter elements which are held in this holding frame can therefore be of simple construction. For example, thefilter elements can consist exclusively of filter material and inparticular not have a separate filter frame. For example, the filterelements can be formed from filter layers which are held together at the edges by the rails of the holding frame.
According to a preferred embodiment, a handle is formed on each of the end walls. The handle can be formed on an edge of the end face. Forexample, in the case of a trapezoidal end wall, the handle can be fastened to or integrally molded onto the longer, parallel edge of thetrapezoid. In this embodiment, it must be possible to introduce thefilter device so deep into the fume extraction device that the handle does not protrude above the suction opening when it is introduced.
According to a preferred embodiment, however, a handle is formed in each case on the side of each of the end walls which faces the rails and thus the other end wall, said handle extending in the longitudinal direction of the rails. A handle which protrudes over the surface of the end wallin this direction is understood here to mean a handle extending in the longitudinal direction of the rails. The handle is hence preferably embodied as a projection. The handle can for example have the shape of a partial circle. If the end wall consists of metal, for example sheetmetal, the handle can be introduced by stamping. In the case of a front wall made of plastic, the handle can for example be produced by injection molding during manufacture. Because a handle is fastened tothe inside of each of the end walls, the filter device can easily be removed from the fume extraction device and in particular from the suction opening.
According to one embodiment, the holding frame has a first rail in whichthe edges of the at least two filter elements which lie adjacent to one another are held, and has two second rails in which the opposite,spaced-apart edges of the two filter elements are held. One advantage ofthis embodiment is that the edges of the filter elements are closed andthe filter material is thus held securely. Thus, for example, expanded metal filter layers, wire mesh or wire fabric can be used as filter material and can be securely held in the holding frame without a separate filter frame.
According to a further embodiment, the first rail has a web running inthe longitudinal direction, which the edges of the two filter elements,which lie adjacent to one another, abut on opposite sides. Because thefilter elements are not supported directly on one another but on the web which is formed in the first rail, the stability of the filter device is increased.
The holding frame can consist of multiple parts. In particular, the rails to the end walls can be present separately. In this embodiment,the parts of the holding frame can for example consist of metal, inparticular of sheet metal. The parts can be connected to one another,for example by screws. According to a preferred embodiment, however, the holding frame is formed in one piece. In this embodiment, the holding frame consists, for example, of plastic and can be produced by injection molding. However, in the one-piece embodiment the holding frame can also consist of metal and be produced for example by bending and stamping.The advantage of a one-piece holding frame is the simplified production and the greater stability of the filter device.
To further increase the stability of the filter device, at least one filter element can have a support element which extends in the longitudinal direction of the filter element in its interior. The support element can represent a tube, for example, and preferably extends over the entire length of the support element. At the longitudinal ends of the support element, it can be screwed to the end walls of the filter device. For this purpose, a screw can be guided through the end wall from the outside on each end wall and screwed intothe longitudinal end of the tubular support element.
According to a further aspect, the invention relates to a fumeextraction device which has a fume extraction housing with a suctionopening in the upper face of the fume extraction housing, and a fan which is arranged offset below relative to the suction opening. The fumeextraction device is characterized in that the fume extraction device has at least one inventive filter device.
Advantages and features which are described in respect of the inventive filter device also apply—where applicable—for the inventive fumeextraction device and vice versa.
A fume extraction device is understood as a device by means of which fumes and vapors, in particular in a kitchen, can be sucked in and cleaned. In particular, the fume extraction device represents a device in which steam and/or vapors sucked into the suction opening of the fumeextraction device are directed downward and cleaned there by at least one filter device.
The fume extraction device can be a so-called downdraft fan. A downdraftfan is understood as a fume extraction device in which the suctionopening is located horizontally adjacent to the hob or in a recess inthe hob. A housing of the fume extraction device is downwardlycontiguous to this suction opening, in which the at least one filterdevice is provided via which the fumes and vapors are cleaned. The interior of the housing is accessible from above via the suctionopening. An exhaust air opening is provided on the housing of the fumeextraction device, and is connected to the fan of the fume extractiondevice via further duct elements. The air cleaned by the filter device is discharged from the housing via the exhaust air opening. The exhaust air opening can hence also be referred to as a clean air opening.
In the case of a downdraft fan, the entire interior of the housing,which is contiguous with the suction opening, is available to accommodate a filter device. Moreover, in the case of a downdraft fan,the suction opening is provided at the level of the hob or slightly offset upward and the filter device is offset downward relative to the suction opening. Thus particles that fall out of the filter device andin particular out of the filter elements cannot fall onto the hob, as isthe case with a fume extraction hood arranged above the hob. Thus, forexample, knitted metal or warp-knitted metal can also be used as filter material for the filter element, although with this filter material metal fibers can partially detach. Furthermore, in the case of adowndraft fan, the holding frame can easily be used to collect contaminants. Finally, in the case of a downdraft fan, the interior ofthe housing of the fume extraction device is easily accessible, suchthat the filter device accommodated in the housing can be easily removed and reinserted after cleaning.
In the case of the inventive fume extraction device, this has a fumeextraction housing with a suction opening in the upper face of the fumeextraction housing. The suction opening preferably has a rectangular cross-section which is slightly larger than the width and length of thefume extraction device.
Directional indicators such as above and below refer to the fumeextraction device and the parts thereof in an assembled state, in whichthe suction opening lies horizontally.
The fan of the fume extraction device is arranged offset downward relative to the suction opening. The fan is in particular fluidicallyconnected to the fan via the fume extraction housing and any duct elements connected thereto. Thus as a result of the negative pressure generated by the fan, air is sucked in downward through the suctionopening and flows from above into the fume extraction housing. The fumeextraction device has at least one inventive filter device. The filterdevice is preferably located in the vicinity of the suction opening inthe fume extraction housing. As a result, the filter device is easily accessible to the user from above through the suction opening. The filter device is particularly preferably held in the fume extraction housing by a holding geometry which is provided on two walls of the fumeextraction housing. The holding geometry can be formed by one or more projections, for example one or more strips of material, which extend inward from the walls into the fume extraction housing.
Particularly preferably, the filter device is introduced into the fumeextraction housing such that the edges of the filter elements which lie adjacent to one another face away from the suction opening. The V-shaped filter device is thus introduced into the fume extraction housing suchthat the tip of the V-shape points downward. Thanks to this arrangement of the filter device, the drawn-in air is sucked into the space which is formed between the filter elements at an angle relative to one another and the end walls which are preferably provided. The rail, in which the edges of the two filter elements lie adjacent to one another, also serves here to collect separated contaminants such as grease. The air drawn in flows completely against the filter area of the two filterelements.
According to a preferred embodiment, the fume extraction housingenlarges downward from the suction opening at least in some areas. A duct section of the fume extraction housing with a constant cross-section can here firstly be contiguous to the suction opening over its height. The first duct section preferably has a rectangular cross-section. An area of the fume extraction housing with an increasing cross-section over the height of the area can then be contiguous to this duct section. After this enlargement area, a further duct section with a constant cross-section over its height can follow. The cross-section ofthe second duct section located further down is in this case larger thanthe cross-section of the first duct section adjoining the suctionopening. The second duct section also preferably has a rectangular cross-section. This can be closed on the lower face of the second lower duct section, i.e. can have a bottom. An exhaust air opening of the fumeextraction housing is preferably introduced on one side of the second duct section, via which air can reach the fan via further ducts.Preferably the exhaust air opening is introduced in the rear side of thefume extraction housing. The rear side is understood as the side facing away from the user of the fume extraction device. In this embodiment,the fume extraction device can for example be arranged between one or both side edges of a hob. Alternatively, the fume extraction device can also be located for example between two separate hobs in the depth direction of the hobs or be introduced into a recess in the hob which extends in the depth direction in the center of the width of the hob.
The filter device is preferably located at least partially in the enlarged area. The filter device is particularly preferably arranged such that the edges of the two filter elements which are spaced apart from one another, i.e. the upper edges, are located in the first upper duct section and there abut the inner faces of two opposite walls of thefume extraction housing. The end walls of the filter device abut the two further walls of the preferably rectangular first duct section. Since the filter elements are at an angle in respect of one another and the lower longitudinal edges of the filter elements abut one another, the distance between the filter element and the wall of the duct sectionabutting it increases. In addition, the area of the enlargement of thefume extraction housing is at a height at which some of the filterelements of the filter device are also located. In particular, the cross-section of the fume extraction housing enlarges at the walls whichthe filter elements in the first duct section abut. As a result, the distance between the filter element and the wall of the fume extraction housing also increases, in addition to the increase due to the inclined arrangement of the filter elements, due to the enlargement of the fumeextraction housing. This ensures that a uniform flow of air in the fumeextraction housing and a reliable flow through the filter elements canbe achieved. In particular, an accumulation of air, which could occur due to insufficient distance between the filter element and the wall ofthe fume extraction housing, cannot occur in the preferred embodiment ofthe fume extraction housing. Moreover, the development of noise in thefume extraction housing is reduced by the enlargement of the fumeextraction housing.
According to a particularly preferred embodiment, the enlargement area between the first and the second duct section and also the transitions from the first duct section to the enlargement area and from the enlargement area to the second duct section are formed by curved surfaces. The transition from the second duct section to the bottom ofthe fume extraction housing is particularly preferably rounded. As a result, no edges or shoulders arise between the areas of the fumeextraction housing. This means the build-up of contaminants is prevented and the fume extraction housing can easily be cleaned.
According to a preferred embodiment, the suction opening has a rectangular, elongated cross-section and a width of 150 mm. With conventional downdraft fans, the width of the suction opening is usually limited to 100 mm. By preferably providing a suction opening with a larger width, in particular 150 mm, according to the invention, on theone hand the filter area of the filter device can be maximized at a given height of the fume extraction housing. The height of the fumeextraction housing is limited in particular by further built-in components which are provided below the fume extraction device in a kitchen arrangement, for example drawers of a kitchen cabinet. Moreover,due to the wider suction opening, its cross-section is also larger and thus the development of noise is further minimized.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is explained again below with reference to the accompanying figures, in which:
FIG. 1 : shows a schematic, perspective sectional view of an embodiment of the inventive fume extraction device with a first embodiment of the inventive filter device;
FIG. 2 : shows a further schematic, perspective sectional view of the embodiment of the inventive fume extraction device according to FIG. 1 ;
FIG. 3 : shows a schematic perspective view of the first embodiment ofthe filter device according to FIG. 1 ;
FIG. 4 : shows a schematic perspective view of a second embodiment ofthe inventive filter device; and
FIG. 5 : shows a schematic sectional view of the second embodiment ofthe filter device according to FIG. 4 .
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE PRESENT INVENTION
FIG. 1 shows a schematic, perspective sectional view of an embodiment ofthe inventive fume extraction device 1 with a first embodiment of the inventive filter device 2. The fume extraction device 1 comprises a fumeextraction housing 10 and a filter device 2. In addition, the fumeextraction device 1 comprises a fan, which is not shown in FIG. 1 , but is preferably offset downward relative to the fume extraction housing10.
The fume extraction housing 10 has an elongated shape. In the assembled state, the fume extraction housing 10 lies parallel to an edge of a hob(not shown). The fume extraction housing 10 is particularly preferably arranged between two hobs (not shown) and extends in the depth direction of the hobs. Alternatively, the fume extraction housing 10 can also be introduced into a recess made in the depth direction of the hob.
The fume extraction housing 10 has a suction opening 100 in the upper face. The suction opening 100 has a rectangular cross-section. A first duct section 103 is contiguous with the suction opening 100 at the bottom, and has a rectangular cross-section corresponding to the cross-section of the suction opening 100 and whose cross-section is constant over the height of the first duct section 103. At the bottom,an enlargement 101 of the fume extraction housing 10 is contiguous withthe first duct section 103. The width of the fume extraction housing 10increases over the height of said enlargement 101. A second duct section104 is contiguous with the enlargement 101. The second duct section 104has a cross-section which is constant over its height and is closed below by a bottom 105. A duct 106 is contiguous with the rear wall ofthe fume extraction housing 10, air being able to reach the fan of thefume extraction device 1 via said duct 106. The duct 106 is curved and hence directs the air downward.
In the assembled state of the fume extraction device 1, the suctionopening 100 lies in the plane of the hob(s) (not shown). In the embodiment shown, the suction opening 100 is covered by a grille 3. Itis however also possible to close the suction opening 100, in the state in which the fume extraction device 1 is not in operation, by another cover element, for example a plate or flap.
A filter device 2 is introduced into the fume extraction housing 10.
The filter device 2, which is also shown in more detail in FIGS. 2 and 3, consists of two filter elements 20 and a holding frame 21.
The two filter elements 20 are held in the holding frame 21. Each of thefilter elements 20 has a variable distance A between the opposite outer faces 200, 201 of the filter element 20 over its surface. In particular,the filter elements 20 in the embodiment shown have a diamond-shaped cross-section. The two filter elements 20 are arranged at an angle relative to one another and an edge 203, which can also be referred to as the lower longitudinal edge, of one of the filter elements 20 lies adjacent to a further edge 203 of the other filter element 20. The twofilter elements 20 thus form a V-shape. In the embodiment shown, a support element 202 is provided in each filter element 20, which extend sin the center of the filter elements 20 in their longitudinal direction.The support elements 202 can be rods or tubes.
The holding frame 21 consists of a first rail 210 and two second rails211, 212. In the embodiment shown, the lower longitudinal edges 203 ofthe two filter elements 20 are held in a first rail 210. The rail 210moreover has a web 2100 which extends in the longitudinal direction ofthe rail 210 and lies in the center thereof. The two filter elements 20thus rest with their lower edges 203 on opposite sides of the web 2100in the first rail 210. The second rails 211, 212 run on the upper longitudinal edges 204 of the filter elements 20, i.e. the upper edges204 are each accommodated in one of the second rails 211, 212 of the holding frame 21. At the longitudinal ends, the holding frame has end walls 213, 214, each of which has a trapezoidal shape. The rails 210,211, 212 rest with their longitudinal ends on the end walls 213, 214 andare preferably fastened thereto or configured therewith in one piece.
As can be seen from FIGS. 2 and 3 , a handle 216 is provided on the inner face of the end wall 213, which in the embodiment shown has a partial circular shape. A handle, which is not visible in the figures,is also preferably provided on the inner face of the end wall 214.Moreover, in the embodiment shown, the filter device 2 has support walls217 which extend inward from the end walls 213, 214 and at the end-face ends of the filter elements 20 over the width of the filter elements 20abut the outer faces 200, 201 of the filter elements 20. The support walls thus run perpendicular to the rails 210, 211, 212.
Moreover, the holding frame 21 has struts 215 running between the second rails 211, 212 and the first rail 210. These extend perpendicular to the rails 211, 212, 210 and have a small width. The struts 215 serve to additionally hold the filter elements 20 and in particular the filter material.
The filter device 2 is introduced into the fume extraction housing 10such that the tip of the V-shape is directed downward. In particular,the second rails 211, 212 of the holding frame 21 lie in the vicinity ofthe suction opening 100 in the first duct section 103 of the fumeextraction housing 10. The first rail 210 is offset downward relative tothe second rails 211, 212. In the embodiment shown, the first rail 210and thus the lower face of the filter device 2 lies in the region of the second duct section 104 of the fume extraction housing 10.
As can be seen from FIG. 2 , in the embodiment shown a holding geometry102 is formed on the end walls of the fume extraction housing 10, which extends inward from the end wall of the fume extraction housing 10 and supports the filter device 2 at its longitudinal ends from below. Inparticular, the holding geometry 102 therefore represents a V-shaped strip of material.
FIGS. 4 and 5 show a second embodiment of the inventive filter device 2.This embodiment differs from the embodiment shown in FIGS. 1 to 3 only in that the rails 210, 211 and 212 to the end walls 213, 214 represent separate components. In this embodiment, the rails 210, 211, 212 are configured such that inwardly directed folded edges are provided on their open side which engage in the filter material of the filterelements 20 and are thus fastened to the filter material. Moreover, in contrast to the first embodiment, no web is provided in the case of thefirst rail 210. The folded edges of the lower rail 210, which are provided on the upper edges thereof, each engage onto an outer face 200,which forms the clean air side of the filter elements 20. In the second embodiment of the filter device 2, the end walls 213, 214 are connected to the filter elements 20 by screws (see FIG. 4 ). The screw protrudesthrough the end wall 213 or 214 and engages in the longitudinal end ofthe support element 202 of the filter element 20, which represents a tube. As a result, the filter device 2 forms a structural unit and canbe introduced and removed as a whole into the fume extraction housing 10of a fume extraction device 1.
The present invention has a number of advantages. In particular, by combining a V-shaped arrangement of filter elements and the three-dimensional configuration of the filter elements, in particular a diamond shape, the filter area through which fumes and vapors can be filtered can be maximized.
The invention claimed is:
1. A filter device for a fume extractiondevice, said filter device comprising: at least two filter elements arranged at an angle relative to one another, with an edge of one of theat least two filter elements lying adjacent to an edge of the other oneof the at least two filter element, each of the at least two filterelements having a surface with a variable distance between opposite outer faces of the filter element; and a holding frame for holding theat least two filter elements, wherein the distance between the opposite outer faces of each of the at least two filter elements from one edge ofthe filter element in a direction of an opposite edge increases from a minimum to a maximum and from the maximum to the opposite edge decreases to the minimum, and wherein the filter element has a diamond-shaped cross-section.
2. The filter device of claim 1, wherein the adjacent edges of the at least two filter elements abut one another.
3. The filter device of claim 1, wherein the at least two filter elements are at an angle of less than 90°.
4. The filter device of claim 1, wherein the holding frame includes two end walls which abut end faces of the atleast two filter elements, and rails running between the end walls. 5.The filter device of claim 4, wherein each of the end walls has a side which faces the rails, said holding frame including a handle formed onthe side and extending in a longitudinal direction of the rails.
6. The filter device of claim 4, wherein a first one of the rails is configured to hold the adjacent edges of the at least two filter elements, and wherein a second one of the rails is configured to hold one of the spaced-apart edges of the at least two filter elements.
7. The filterdevice of claim 6, wherein the first one of the rails includes a web running in a longitudinal direction, said adjacent edges of the at least two filter elements abutting opposite sides of the web.
8. The filterdevice of claim 1, wherein the holding frame is formed in one piece. 9.The filter device of claim 1, wherein at least one of the at least twofilter elements includes a support element which extends in a longitudinal direction of the filter element in its interior.
10. A fumeextraction device, comprising: a fume extraction housing having an upper face with a suction opening; a fan arranged offset downward relative tothe suction opening; and a filter device comprising at least two filterelements arranged at an angle relative to one another, with an edge of one of the at least two filter elements lying adjacent to an edge of theother one of the at least two filter element, each of the at least twofilter elements having a surface with a variable distance between opposite outer faces of the filter element, and a holding frame for holding the at least two filter elements, wherein the distance betweenthe opposite outer faces of each of the at least two filter elements from one edge of the filter element in a direction of an opposite edge increases from a minimum to a maximum and from the maximum to the opposite edge decreases to the minimum, and wherein the filter element has a diamond-shaped cross-section.
11. The fume extraction device of claim 10, wherein the adjacent edges of the at least two filter elementsabut one another.
12. The fume extraction device of claim 10, wherein the at least two filter elements are at an angle of less than 90°. 13.The fume extraction device of claim 10, wherein the holding frame includes two end walls which abut end faces of the at least two filterelements, and rails running between the end walls.
14. The fumeextraction device of claim 13, wherein each of the end walls has a side which faces the rails, said holding frame including a handle formed onthe side and extending in a longitudinal direction of the rails.
15. The fume extraction device of claim 13, wherein a first one of the rails is configured to hold the adjacent edges of the at least two filterelements, and wherein a second one of the rails is configured to hold one of the spaced-apart edges of the at least two filter elements. 16.The fume extraction device of claim 15, wherein the first one of the rails includes a web running in a longitudinal direction, said adjacent edges of the at least two filter elements abutting opposite sides of the web.
17. The fume extraction device of claim 10, wherein the holding frame is formed in one piece.
18. The fume extraction device of claim10, wherein at least one of the at least two filter elements includes a support element which extends in a longitudinal direction of the filterelement in its interior.
19. The fume extraction device of claim 10,wherein the filter device is arranged in the fume extraction housing such that the adjacent edges of the at least two filter elements face away from the suction opening.
20. The fume extraction device of claim10, wherein the fume extraction housing has at least one area configured to enlarge downward from the suction opening, said filter device having at least one area located in the enlarged area.
21. A fume extractiondevice, comprising: a fume extraction housing having an upper face witha suction opening; a fan arranged offset downward relative to the suction opening; and a filter device comprising at least two filterelements arranged at an angle relative to one another, with an edge of one of the at least two filter elements lying adjacent to an edge of theother one of the at least two filter element, each of the at least twofilter elements having a surface with a variable distance between opposite outer faces of the filter element, and a holding frame for holding the at least two filter elements, wherein the suction opening has a rectangular, elongated cross-section and preferably has a width of150 mm.
22. The filter device of claim 3, wherein the at least twofilter elements are at an angle of less than 40° relative to one another.
23. The fume extraction device of claim 12, wherein the atleast two filter elements are at an angle of less than 40° relative toone another.
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User talk:TheZebra
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Welcome to the 141
I hope you enjoy editing on the wiki recruit! This is Shepherd, out.
* -- --DARTH SIDIOUS 2 (Contact) 18:23, June 29, 2010 (UTC)
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Deleting duplicates from a list of lists if some duplicates do not have the same order
My problem is very similar to the one linked below except that if there was a [2,1] element I would need that to be deleted too.
Removing duplicates from a list of lists
I've tried all sorts of things but just can't make it work. Any help would be much appreciated!
Thanks.
Couldn't you just sort all your sublists and then apply the solution you linked to?
doh! Don't know why I didn't think of that.
Oh I remember why I didn't think of it, I need the sublists to remain in order!
Maybe what you really want is a set of sets
unique = set(map(set, list_of_lists))
Edit: well, but that doesn't work. alas, sets cannot contain sets because sets are unhashable. frozenset is, though:
unique = set(map(frozenset, list_of_lists))
This works, but it doesn't preserve the ordering of the sublists:
def bygroup(k):
k = sorted(sorted(x) for x in k)
return [k for k,_ in itertools.groupby(k)]
>>> k = [[1, 2], [4], [5, 6, 2], [1, 2], [3], [4], [2, 1]]
>>> bygroup(k)
[[1, 2], [2, 5, 6], [3], [4]]
In Python 2.7 or 3.2, you could use an OrderedDict if you need to preserve the order within sublists and also the general order of the list (except for duplicates), but it's much slower:
def bydict(k):
s = collections.OrderedDict()
for i in k:
s[tuple(sorted(i))] = i
return s.values()
>>> bydict(k)
[[2, 1], [4], [5, 6, 2], [3]]
I tested with 100,000 iterations using timeit. The bydict function took about 4 times longer in Python 2.7.2 and about 3 times longer in Python 3.2.
Thanks, but alas I need the order of the sublists to be preserved.
As Tim Pietzcker suggested at the moment I am sorting each sublist as it is created and then am using the fastest method discussed in the question that I linked to. Do you know off the top of your head if your method would be faster than this? (when I have got time I can check for myself).
|
Lens mounting
ABSTRACT
A cam sleeve for axially moving a lens as it rotates about an optical axis is provided with a pair of camming grooves that depict the same locus. And, a holding member for the lens to be guided by a guide member is provided with a pair of pins corresponding to the pair of camming grooves. These pins engage in the pair of camming grooves to achieve good stability of movement of the lens when the cam sleeve rotates.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the structure of a lens mounting of relatively large size capable of attaching to a television camera, single lens reflex camera or video camera, and more particularly to improvements of the cam mechanism for controlling movement of lenses along an optical axis of the photographic optical system.
2. Description of the Related Art
In the zoom lenses of the mechanical compensation type, it has been common in the art that the movement of the movable lens units is controlled by the camming slits of the cam sleeve. In other words, the guide pins planted on the holding rings of the movable lens units engage in the linear camming slits provided in the fixed tube in parallel to the optical axis and the non-linear camming slits of the cam sleeve so that as the cam sleeve rotates about the optical axis, the guide pins move in the camming slits.
Particularly in the lens mounting of relatively small size such as the interchangeable one for 8 mm cine cameras or single lens reflex cameras, the cam sleeve in many cases is provided with a plurality of camming slits.
On the other hand, a sectional view of the conventional lens mounting of relatively large size for television cameras is shown in FIGS. 5 and 6. For note, FIG. 6 is a cross-sectional view taken along line C--C of FIG. 5. 201 is a magnification varying lens system, and 202 is a compensating lens system. 101 is a focusing sleeve; 102 is a fixed barrel; and 103 is a cam sleeve connected with a zoom actuating ring 110 by a connection pin 109 to rotate, and provided with camming grooves, one for each of holding members 105 and 108, to axially move the magnification varying lens system (variator) 201 and the compensating lens system (compensator) 202 through the holding members 105 and 108. Also, in order to guide the axial movement of the holding members 105 and 108 while restraining them from rotation in accompaniment with rotation of the cam sleeve 103, there are provided a round bar 104 and a round bar 106, both of which are fixed to the fixed barrel 102. The round bar 104 and the holding member 105 engage each other along a certain length, l, in the axial direction. Meanwhile, the round bar 106 is positioned in almost 180° spaced relation to the round bar 104. This round bar 106 generally has a function that restrains it from rotation about the optical axis and makes it engage the holding member 105 with a radial looseness to achieve smoothness of movement of the latter.
However, the zoom lens of this type had a drawback that when in use at an inclined angle, or with the front lens unit pointed either upward or downward, an image shift in the longitudinal or lateral direction was caused to occur, because the holding member 105 holding the variator 201 and the holding member 108 holding the compensator 202 were tilted.
The reason why the holding members tilt is that there is need to provide a minute gap (Δx) between the round bar 104 and a part 105a of the holding member 105 which engage each other, while the lower engagement of the round bar 106 and the holding member 105 is free in the radial directions. Therefore, when the lens is tilted with the front member downward or upward, the variator 201 and compensator 202 incline to an angle θ=tan⁻¹ (Δx/l), because it is fulcrumed mainly about a forked portion 105b or so-called one-sided ly held. In this case, to reduce this angle, there is a method of lessening the gap ΔX between the round bar 104 and the holding member 105. But, if it is too much done, the required driving power becomes very large. So, the zooming becomes very difficult to perform. Another method is to increase the length, l, of engagement of the round bar 104 and the holding member 105. But, this leads to increase the size of the lens barrel. Also, because there is a limit on the size of the round bar 104 in the longitudinal direction, too much an increase of the length is not desirable.
SUMMARY OF THE INVENTION
A first object of the invention is to achieve a good stability of lens movement.
A second object of the invention is to reduce the variation of the image shift even when the lens is tilted.
To achieve this object, the present invention has a feature that the cam sleeve for axially moving the lens as it rotates is provided with a pair of camming grooves that depict the same locus, and the holding member of the aforesaid lens is provided with a pair of pins corresponding to the aforesaid one pair of camming grooves, the pair of pins being made to insert into the aforesaid one pair of camming grooves.
Further objects of the invention will become apparent from the following drawings and description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a longitudinal section view of a lens mounting of the invention.
FIG. 2 is a cross-sectional view taken along line A--A of FIG. 1.
FIG. 3 is a cross-sectional view taken along line B--B of FIG. 1.
FIG. 4 is an expanded view of the cam sleeve of FIGS. 1 to 3.
FIG. 5 is a longitudinal view of the conventional lens mounting.
FIG. 6 is a cross-sectional view taken along line C--C of FIG. 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention is next described by reference to the drawings.
FIG. 1 is a sectional view of the lens mounting according to the present invention. 1 is a focusing barrel, and 2 is a fixed barrel. A cam sleeve 3 is rotatably fitted in the inner diameter of the fixed barrel 2, and, as will be described later, provided with two kinds of cam for a magnification varying system 20 and a compensating system 21, each kind of cam having two grooves. An expanded view of this cam sleeve 3 is shown in FIG. 4.
A zoom ring 10 is rotatably fitted on the outer diameter of the fixed barrel 2, and connected as a unit to the cam sleeve 3 by a connection pin 9. A round guide bar 4 is fixedly mounted to the fixed barrel 2. A holding member 5 contains the magnification varying optical system 20, and another holding member 8 contains the compensating optical system 21. These holding members 5 and 8 are axially movably carried on the round bar 4. A round guide bar 6 is fixedly mounted to the fixed barrel 2 to restrain the first holding member 5 from rotation when its forked portion 5a contacts the bar 6 through a length L, as shown in FIG. 2.
A round guide bar 7 also mounted to the barrel 2 adjacent bar 6 restrains the second holding member 8 from rotation when its forked portion contacts the bar 7 (FIG. 3) through a certain length (not shown).
FIGS. 2 and 3 are cross-sectional views taken along lines A--A and B--B of FIG. 1 respectively. Two pins 51 and 52 are planted on the holding member 5 in almost 180° spaced relation and are fitted in a pair of camming grooves 31 and 32 respectively machined in the inner surface of the cam sleeve 3. The second holding member 8 also has two pins 81 and 82 positioned in almost 108° spaced relation and slidably fitted in a pair of camming grooves 33 and 34 respectively machined in the inner surface of the cam sleeve 3.
And, as shown in FIG. 4, the first and second pairs of camming grooves 31 and 32, and 33 and 34 are 180° opposed to each other in the cam sleeve 3 and have similar non-linear forms to each other, but the two grooves in each pair are so configured that when the cam sleeve 3 rotates, the magnification varying optical system 20 and the compensating optical system 21 axially move in desired differential relation. For note, the camming grooves 31 to 34 are not penetrated through the entire wall, that is, formed so as to have a bottom.
Now, the axis along which the holding members 5 and 8 move during the rotation of the cam sleeve 3 must be maintained in coincidence with the optical axis. For this purpose, the holding member 5 is made to contact the guide bar 4 and round bar 6, which is disposed almost 180° opposite bar 4, which restrain rotation and permit movement of member parallel to the optical axis through a length, l. The holding member 5 and the round bar 6 engage each other through a length L, leaving some radial gap therebetween.
For note, 22 is a front lens unit for focusing. and 23 is an image forming lens unit.
The operation of the lens mounting of such construction is described below. Concerning the tilt in the horizontal plane as viewed in FIG. 2, since the contact area of the holding member 5 on the round bar 6 for restraining rotation is the sufficiently long length L, the tilting is limited by this to a negligible amount. As for the tilt in the vertical plane in FIG. 2, since the pins 51 and 52 are spaced 180° from each other and extend in opposite direction to rest on the abutments of the camming grooves 31 and 32 for the magnification varying system machined in the cam sleeve 3, no tilting occurs.
Hence the holding member 5 for the magnification varying system is maintained stable throughout the entire range of axial movement. In this state, when the photographer rotates the zoom ring 10, the cam sleeve 3 is rotated in unison through the connection pin 9. Since the pins 51 and 52 are fitted in the camming grooves 31 and 32 for the magnification varying system, the holding member 5 for the magnification varying system is caused to move axially. Here suppose the lens as a whole is pointed downward (upward likewise), a force is applied on the holding member 5 for the magnification varying system to tilt in the vertical plane in FIG. 2. But, since the pins 51 and 52 which also serve to prevent tilting lie on opposite side of the optical axis (both sided), no tilting will occur.
Exactly the same holds for the second holding member 8 for the compensating system.
As has been described above, the use of the features of the invention produces the following advantages:
(1) It is possible to reduce the deviation of an image plane of sharp focus from the film plane when the entire lens is tilted.
(2) Thanks to the both-sided support of the holding members in the vertical plane, the required driving force of the zoom ring is reduced. Also, even a smoother zooming operation than was heretofore can be carried out.
Though, in the illustrated embodiment, two camming grooves are provided for each movable lens unit, it is needless to say that its number may be increased to 3 or 4.
What is claimed is:
1. A zoom lens comprising:an optical system having first and second movable lenses simultaneously moving when zooming and having an optical axis; a fixed barrel accommodating said optical system; a cam sleeve supported on said fixed barrel rotatably about the optical axis; a first pair of camming grooves for controlling the movement of said first movable lens provided in said cam sleeve; a second pair of camming grooves for controlling the movement of the second movable lens provided in said cam sleeve; a first holding ring holding said first movable lens and having projected portions in engagement with said first pair of camming grooves; a second holding ring holding said second movable lens and having projected portions in engagement with said second pair of camming grooves; a first guide bar for guiding the axial movements of said first and said second holding rings provided on said fixed barrel so as to lie above said optical axis; a second guide bar for guiding the axial movement of said first holding ring fixed to said fixed barrel; and a third guide bar for guiding the axial movement of said second holding ring provided on said fixed barrel.
2. A zoom lens according to claim 1, wherein the projected portions of each of said first and said second holding rings are provided almost symmetrical with respect to said optical axis.
3. A lens mounting according to claim 1, wherein the length in the direction of the optical axis of engaging portion of said first holding ring and said first guide bar is longer than the length in the direction of the optical axis of engaging portion between said first holding ring and said second guide bar.
|
#!/usr/bin/env python3
"""A text scroller for panels or terminals"""
import argparse
import sys
from distutils.util import strtobool
from re import search
from shlex import split
from subprocess import CalledProcessError, check_output
from time import sleep, time
from unicodedata import east_asian_width
def str_to_bool(string):
"""Convert a string to a boolean.
:param string: the string to convert
:type string: str
:raise ValueError: raised when the string cannot be converted
:return: the boolean interpretation of the string
:rtype: bool
"""
return bool(strtobool(string.lower()))
# pylint: disable-msg=C0103
# pylint: disable-msg=W0603
parser = argparse.ArgumentParser()
# allow for a positional argument or piping into
parser.add_argument(
'scroll_text',
nargs='?',
default=sys.stdin.read() if not sys.stdin.isatty() else None,
help="""
text to scroll; will print in place if not longer than -l scroll
length; can be read from stdin, e.g. echo text | zscroll
""",
)
parser.add_argument(
'-l',
'--length',
type=int,
default=40,
help="length of scrolling text excluding padding (default: 40)",
)
parser.add_argument(
'-r',
'--reverse',
type=str_to_bool,
default=False,
help="whether to scroll the text from left to right (default: false)",
)
parser.add_argument(
'-b',
'--before-text',
default='',
help="""
stationary padding text to display to the left of the scroll-text
(default: "")
""",
)
parser.add_argument(
'-a',
'--after-text',
default='',
help="""
stationary padding text to display to the right of the scroll-text
(default: "")
""",
)
parser.add_argument(
'-p',
'--scroll-padding',
default=' - ',
help="""
padding text to display between the start and end of the scroll-text
only when it is scrolling (default: " - ")
""",
)
parser.add_argument(
'-d',
'--delay',
type=float,
default=0.4,
help="""
delay (in seconds) for scrolling update; lower this for faster
scrolling (default: 0.4)
""",
)
parser.add_argument(
'-m',
'--match-text',
nargs=2,
action='append',
default=[],
help="""
takes 2 arguments: the regexp to search for in match-command output to
determine whether to change settings and the new settings themselves;
see the man page for more info (default: none)
""",
)
parser.add_argument(
'-M',
'--match-command',
action='append',
default=[],
help="command(s) to search output of (default: none)",
)
parser.add_argument(
'-t',
'--timeout',
type=float,
default=0,
help="""
time in seconds to wait before exiting; 0 means don't exit (default:
0)
""",
)
# no args
parser.add_argument(
'-s',
'--scroll',
type=str_to_bool,
default=True,
help="whether to scroll; meant to be used with -m (default: true)",
)
parser.add_argument(
'-u',
'--update-check',
type=str_to_bool,
default=False,
help="""
specifies that the positional argument is a command that should be
checked to determine if the scroll-text should be updated; when the
output changes, the scroll-text is updated to the new output (default:
false)
""",
)
parser.add_argument(
'-U',
'--update-interval',
type=float,
default=0,
help="""
time in seconds to wait in between running update checking commands
(i.e. the command specified by the positional argument when
-u/--update-interval is specified and commands specified with
-M/--match-command) (default: 0)
""",
)
parser.add_argument(
'-n',
'--newline',
type=str_to_bool,
default=True,
help="""
print a newline after each update; printing a newline may be
necessary for panels (default: true)
""",
)
initial_args = None
args = None
scroll_text = None
last_text_len = None
last_update_check_time = None
def visual_len(text):
"""Determine the "visual" length of text.
Halfwidth characters are counted as length 1 and fullwidth characters are
counted as length 2.
:param text: the text to examine
:type text: str
:return: the visual length of the text
:rtype: int
"""
visual_length = 0
for char in text:
width = east_asian_width(char)
if width == 'W' or width == 'F':
visual_length += 2
else:
visual_length += 1
return visual_length
def make_visual_len(desired_visual_length, text):
"""Coerce the text to the given "visual" length.
The text will be altered by removing characters and padding with spaces as
necessary to reach the exact desired length.
:param desired_visual_length: the desired visual length
:type desired_visual_length: int
:param text: the text to modify
:type text: str
:return: the altered text
:rtype: str
"""
visual_length = 0
altered_text = ''
for char in text:
if visual_length < desired_visual_length:
width = east_asian_width(char)
if width == 'W' or width == 'F':
visual_length += 2
else:
visual_length += 1
altered_text += char
else:
break
if visual_length == desired_visual_length + 1:
# remove final wide character and add a single space as padding
altered_text = altered_text[:-1] + ' '
elif visual_length < desired_visual_length:
altered_text += ' ' * (desired_visual_length - visual_length)
return altered_text
def shell_output(command):
"""Get the output of a shell command as a string.
If the command fails, return "". This is useful in certain cases where a
command may not work initially but will work later so that zscroll does not
need to be restarted (e.g. mpc command fails because mpd is not yet
running).
:param command: the command to run
:type command: str
:return: the command output
:rtype: str
"""
try:
return (
check_output(split(command)).decode(encoding='UTF-8').rstrip('\n')
)
except CalledProcessError:
return ''
def update_check():
"""Update the scroll-text if the command output has changed.
:return: whether the scroll-text has changed
:rtype: bool
"""
global scroll_text
compare_text = shell_output(args.scroll_text)
if scroll_text != compare_text:
scroll_text = compare_text
return True
else:
return False
def set_args(new_args):
"""Update args without altering the match text and command lists.
:param new_args: the new arguments to set args to
:type new_args: argparse.Namespace
"""
global args
args = new_args
# -m and -M should never be altered
args.match_command = initial_args.match_command
args.match_text = initial_args.match_text
def match_update_args():
"""Check match-command(s) for match-text and update args if necessary.
:return: whether the scroll-text was changed
:rtype: bool
"""
match_args = None
search_text = shell_output(args.match_command[0])
text_was_updated = False
for i in range(len(args.match_text)):
if len(args.match_command) > 1 and i > 0:
search_text = shell_output(args.match_command[i])
if search(args.match_text[i][0], search_text):
match_args = parser.parse_args(split(args.match_text[i][1]))
text_was_updated = (
match_args.before_text != args.before_text
or match_args.after_text != args.after_text
or match_args.scroll_padding != args.scroll_padding
or match_args.scroll_text != args.scroll_text
)
if match_args:
set_args(match_args)
return text_was_updated
def build_display_text(text, needs_scrolling, pad_with_space):
"""Shorten text if necessary and add static left and right padding to it.
:param text: the text to add the left and right padding to
:type text: str
:param needs_scrolling: whether the text is long enough to be scrolled
:type needs_scrolling: bool
:param pad_with_space: whether to pad the beginning with a space (used when
phasing in or out a fullwidth character)
:type pad_with_space: bool
:return: the altered text
:rtype: str
"""
if needs_scrolling:
if pad_with_space:
text = ' ' + text
text = make_visual_len(args.length, text)
return args.before_text + text + args.after_text
def print_text(text):
"""Print text in place or with newlines depending on the user's settings.
:param text: the text to print
:type text: str
"""
# flush is necessary for lemonbar/panels (but not in terminal)
global last_text_len
text_len = visual_len(text)
if args.newline:
print(text, flush=True)
else:
if last_text_len and text_len < last_text_len:
# need to overwrite previous characters with spaces
text = make_visual_len(last_text_len, text)
print(text, end='\r', flush=True)
last_text_len = text_len
def maybe_update_text_and_settings():
"""Update the scroll-text and/or settings if necessary.
:return: whether the text and/or settings were updated
:rtype: bool
"""
global last_update_check_time
updated = False
if (
not args.update_interval
or last_update_check_time is None
or last_update_check_time + args.update_interval < time()
):
if len(args.match_command) >= 1 and match_update_args():
updated = True
# want to always check, especially if new match (because -u setting
# could have changed)
if args.update_check and update_check():
updated = True
last_update_check_time = time()
return updated
def zscroll(lines=0):
"""Update settings as needed and continuously prints scroll-text.
:param lines: the number of lines to print (used for testing/debugging
purposes)
:type lines: int
"""
# don't modify global var when shifting; keep for comparison
shift_text = scroll_text
should_restart_printing = True
needs_scrolling = False
last_hidden_was_wide = False
next_hidden_is_wide = True
end_time = time() + args.timeout
num_lines = 0
while True:
if (args.timeout and time() > end_time) or (
lines > 0 and num_lines == lines
):
break
else:
num_lines += 1
if maybe_update_text_and_settings():
should_restart_printing = True
if should_restart_printing:
shift_text = scroll_text
last_hidden_was_wide = False
next_hidden_is_wide = False
needs_scrolling = visual_len(scroll_text) > args.length
if needs_scrolling:
shift_text += args.scroll_padding
display_text = build_display_text(
shift_text,
needs_scrolling,
(last_hidden_was_wide or next_hidden_is_wide),
)
if args.scroll and needs_scrolling or should_restart_printing:
print_text(display_text)
should_restart_printing = False
if args.scroll and needs_scrolling:
if last_hidden_was_wide:
# don't shift the text for one update
last_hidden_was_wide = False
elif next_hidden_is_wide:
next_hidden_is_wide = False
shift_text = shift_text[-1] + shift_text[0:-1]
else:
if args.reverse:
if visual_len(shift_text[-1]) == 2:
# phase in in two steps
next_hidden_is_wide = True
else:
shift_text = shift_text[-1] + shift_text[0:-1]
else:
last_hidden_was_wide = visual_len(shift_text[0]) == 2
shift_text = shift_text[1:] + shift_text[0]
sleep(args.delay)
def pre_parse_argv(argv):
"""Ensure that argv can be properly parsed by argparse.
This is a workaround for the fact that argparse treats everything that
starts with a hyphen as an option even after options that take a fixed
number of arguments (https://bugs.python.org/issue9334).
:param argv: the argument list as obtained from sys.argv
:type argv: list
:return: the pre-parsed argv list
:rtype: list
"""
for i in range(1, len(argv)):
# note: don't need to check for --opt= because these work correctly
if argv[i] in {
'-b',
'--before-text',
'-a',
'--after-text',
'-p',
'--scroll-padding',
# unlikely to be an issue
'-M',
'--match-command',
}:
argv[i + 1] = ' ' + argv[i + 1]
elif argv[i] in {'-m', '--match-text'}:
argv[i + 1] = ' ' + argv[i + 1]
argv[i + 2] = ' ' + argv[i + 2]
return argv
def parse_argv(argv):
"""Parse command line arguments and update default values.
Also remove leading spaces that wee added to arguments to prevent argparse
from incorrectly parsing them.
:param argv: the argument list as obtained from sys.argv
:type argv: list
"""
global initial_args, args, scroll_text
args = parser.parse_args(argv[1:])
args.before_text = args.before_text.replace(' ', '', 1)
args.after_text = args.after_text.replace(' ', '', 1)
if args.scroll_padding != ' - ':
args.scroll_padding = args.scroll_padding.replace(' ', '', 1)
args.match_text = [
[j.replace(' ', '', 1) for j in i] for i in args.match_text
]
args.match_command = [i.replace(' ', '', 1) for i in args.match_command]
initial_args = args
# if -u was specified, this will be correctly updated later
scroll_text = args.scroll_text
# update defaults to those specified by the users
parser.set_defaults(**vars(args))
def validate_args(check_args):
"""Ensure that user-given arguments are valid.
Exit with an error message if the arguments are not valid.
:param check_args: the args to check
:type check_args: argparse.Namespace
"""
if not check_args.scroll_text:
parser.print_help()
sys.exit("Text to scroll must be specified.")
if len(check_args.match_text) > 0 and len(check_args.match_command) == 0:
parser.print_help()
sys.exit("At least one -M is necessary for -m.")
if len(check_args.match_command) > 1 and len(
check_args.match_command
) != len(check_args.match_text):
parser.print_help()
sys.exit(
"If there is more than one '-M', the number must match the"
+ " number of '-m's."
)
def main():
"""Scroll text, handling invalid opts and keyboard interrupts."""
argv = pre_parse_argv(sys.argv)
parse_argv(argv)
validate_args(args)
for arg_string in args.match_text:
arg_string = arg_string[1]
match_args = parser.parse_args(split(arg_string))
# -m and -M should never be altered
# ignore them if the user tries to alter them even if they are wrong
match_args.match_command = args.match_command
match_args.match_text = args.match_text
validate_args(match_args)
try:
zscroll()
except KeyboardInterrupt:
sys.exit(0)
if __name__ == '__main__':
main()
|
Extjs4.1 - Get Selected in treepanel fails
I have a treepanel and i try to get node when i selected like http://jsfiddle.net/kTedM/
Ext.create('Ext.tree.Panel', {
title: 'Simple Tree',
width: 200,
height: 200,
store: store,
rootVisible: false,
dockedItems: [{
xtype: 'toolbar',
items: {
text: 'Get Selected nodes',
handler: function(){
var s = this.up('panel').getSelectionModel().getSelection();
if (s[0])
alert(s[0].data.text + ' was selected');
else alert('no selected');
}
}
}],
renderTo: Ext.getBody()
});
But If u follow below step u will see bug.
step1: run code and click get selected nodes u will get correct alert is no selected
step2: Double click in homework node and click get selected nodes u will see
But i see that node's not selected? How to fix that thanks
+1 for the good complete running stand-alone code and the accurate description of the steps needed to reproduce.
Actually, the 'homework' node is and should be selected. There's no reason that double-clicking it should deselect it.
The bug is that the fact that this node is selected is not correctly represented visually. Clearly a bug.
It has been fixed by Sencha in Ext4.2. See this update of your fiddle; I've just changed the version to 4.2.0 and there's nothing surprising going on...
So, to answer your very question, I'd say that in order to fix it, you just have to upgrade to the last version. I advice against the very last 4.2.1 that introduces several new bugs, but rather for the 4.2.0.x.
Now, here's some code, because I am forced by SO to post some in order to be allowed to link to the fiddle:
// Same code as you
Ext.onReady(function () {
var store = Ext.create('Ext.data.TreeStore', {
fields: [
{name: 'id', type: 'string'},
{name: 'text', type: 'string'},
{name: 'selected', type: 'string'}
],
root: {
expanded: true,
id: '0',
children: [{
text: "detention",
id: '1',
leaf: true
}, {
text: "homework",
id: '2',
expanded: true,
children: [{
id: '3',
text: "book report",
leaf: true
}, {
id: '4',
text: "alegrbra",
leaf: true,
selected: 'true'
}]
}, {
id: '5',
text: "buy lottery tickets",
leaf: true
}]
}
});
Ext.create('Ext.tree.Panel', {
title: 'Simple Tree',
width: 200,
height: 200,
store: store,
rootVisible: false,
dockedItems: [{
xtype: 'toolbar',
items: {
text: 'Get Selected nodes',
handler: function(){
var s = this.up('panel').getSelectionModel().getSelection();
if (s[0])
alert(s[0].data.text + ' was selected');
else alert('no selected');
}
}
}],
renderTo: Ext.getBody()
});
});
|
Talk:Dennis Connoly/@comment-4504913-20130409015926
fixed it. Ofcorse their ruining my characters page. I'm this close to ditching thoe guys
|
/**
* author: Jun
* date: 2014/3/30
**/
(function(window, document) {
function AccordionSlider(config) {
this.id = config.id;
this.folder = config.folder;
this.images = config.images;
this.speed = config.speed;
this.boxWidth = config.boxWidth;
this.imgWidth = config.imgWidth;
}
AccordionSlider.prototype = {
constructor: AccordionSlider,
init: function() {
var li_node, temp_node;
var self = this;
var parent_node = document.getElementById(this.id);
var container_node = document.createElement("div");
container_node.setAttribute("class", "ac-slider-container");
var pic_list_node = document.createElement("ul");
pic_list_node.setAttribute("class", "ac-slider-list");
for(var i = 0; i < this.images.length; i++) {
li_node = document.createElement("li");
li_node.setAttribute("class", "ac-slider-item");
temp_node = document.createElement("img");
temp_node.setAttribute("src", this.folder + "/" + this.images[i]);
li_node.appendChild(temp_node);
pic_list_node.appendChild(li_node);
}
container_node.appendChild(pic_list_node);
parent_node.appendChild(container_node);
this.run();
},
run: function() {
var parent_id = "#" + this.id;
var img_width = this.width;
var current_pic = 0;
var self = this;
if(this.images.length > 1) {
var move_pixel = this.imgWidth - (this.boxWidth - this.imgWidth) / (this.images.length - 1);
}
else {
var move_pixel = 0;
}
$(parent_id).find(".ac-slider-container").css({"width": this.boxWidth + "px", "overflow": "hidden"});
$(parent_id).find(".ac-slider-list").css({"width": this.imgWidth * this.images.length + "px"});
$(parent_id).find(".ac-slider-list .ac-slider-item").css({"position": "relative", "float": "left"});
$(parent_id).find(".ac-slider-list .ac-slider-item img").css({"width": this.imgWidth + "px", "cursor": "pointer"});
for(var i = 2; i < this.images.length; i++ ) {
$(parent_id).find(".ac-slider-container .ac-slider-list .ac-slider-item").eq(i).css({"left": -move_pixel * (i - 1) + "px"});
}
var slider_interval = setInterval(sliderPic, this.speed);
$(parent_id).find(".ac-slider-list .ac-slider-item").mouseover(function() {
window.clearInterval(slider_interval);
if(current_pic <= $(this).index()) {
for(var i = current_pic; i < $(this).index(); i++) {
sliderPic();
}
}
else {
for(var i = current_pic; i > $(this).index(); i--) {
if(i == 0) {
continue;
}
$(parent_id).find(".ac-slider-container .ac-slider-list .ac-slider-item").eq(i).animate({left: -move_pixel * (i - 1) + "px"});
current_pic--;
}
current_pic = $(this).index();
}
});
$(parent_id).find(".ac-slider-list").mouseout(function() {
window.clearInterval(slider_interval);
slider_interval = setInterval(sliderPic, self.speed);
});
function sliderPic() {
if(current_pic < self.images.length - 1) {
current_pic++;
$(parent_id).find(".ac-slider-container .ac-slider-list .ac-slider-item").eq(current_pic).animate({left: -move_pixel * current_pic + "px"});
}
else {
for(var i = 1; i < self.images.length; i++) {
$(parent_id).find(".ac-slider-container .ac-slider-list .ac-slider-item").eq(i).animate({left: -move_pixel * (i - 1) + "px"});
}
current_pic = 0;
}
}
}
}
function createSlider(config) {
if(!config.folder) {
config.folder == "";
}
if((config.speed == undefined) || isNaN(config.speed)) {
config.speed = 3000;
}
if((config.boxWidth == undefined) || isNaN(config.boxWidth)) {
config.boxWidth = $("#" + config.id).width();
}
if((config.imgWidth == undefined) || isNaN(config.imgWidth)) {
if(config.images.length != 1) {
config.imgWidth = 0.8 * config.boxWidth;
}
else {
config.imgWidth = boxWidth;
}
}
var slider_temp = new AccordionSlider(config);
slider_temp.init();
}
$.as = createSlider;
})(window, document);
|
<?php
namespace App;
use Illuminate\Database\Eloquent\Model;
class Item extends Model
{
protected $table = 'items';
protected $fillable = ['name', 'unit', 'supplier_id'];
protected $primaryKey = 'id';
}
|
Arktika-class icebreaker
History
Arktika was the first surface ship to reach the North Pole, on August 17, 1977.
Design and Construction
OK-900A Reactors
Propulsion
|
import VectorTileWorkerSource from '../source/vector_tile_worker_source';
import Actor from './actor';
// 统一管理workerSource 实例化
export default class Worker {
constructor(self) {
this.self = self;
this.actor = new Actor(self, this);
this.workerSourceTypes = {
vector: VectorTileWorkerSource
};
this.workerSources = {};
this.self.registerWorkerSource = (name, WorkerSource) => {
if (this.workerSourceTypes[name]) {
throw new Error(`Worker source with name "${name}" already registered.`);
}
this.workerSourceTypes[name] = WorkerSource;
};
this.layerStyles = {};
}
loadTile(mapId, params, callback) {
this.getWorkerSource(mapId, params.type, params.sourceID).loadTile(params, callback);
}
abortTile(mapId, params, callback) {
this.getWorkerSource(mapId, params.type, params.sourceID).abortTile(params, callback);
}
removeTile(mapId, params, callback) {
this.getWorkerSource(mapId, params.type, params.sourceID).removeTile(params, callback);
}
setLayers(mapId, layercfgs, callback) {
this.layerStyles[mapId] = layercfgs; // mapid layerID
if (this.workerSources[mapId]) {
for (const sourceId in this.workerSources[mapId].vector) {
this.workerSources[mapId].vector[sourceId].layerStyle = layercfgs;
}
}
callback();
}
// updateLayers(id, params, callback) {
// }
/**
* 获取workerSource
* @param {string} mapId WorkerPool Id
* @param {string} type 瓦片类型 目前支持Vector
* @param {string} source souce ID
* @return {*} WorkerSource
*/
getWorkerSource(mapId, type, source) {
if (!this.workerSources[mapId]) {
this.workerSources[mapId] = {};
}
if (!this.workerSources[mapId][type]) {
this.workerSources[mapId][type] = {};
}
if (!this.workerSources[mapId][type][source]) {
// use a wrapped actor so that we can attach a target mapId param
// to any messages invoked by the WorkerSource
const actor = {
send: (type, data, callback) => {
this.actor.send(type, data, callback, mapId);
}
};
this.workerSources[mapId][type][source] = new this.workerSourceTypes[type](actor, this.layerStyles[mapId]);
}
return this.workerSources[mapId][type][source];
}
}
self.worker = new Worker(self);
|
Share Data between two pages/tabs angular2
i have two different pages sender and receiver and are on open in two different tabs
i.e one tab of browser has http://localhost:4200/sender
i.e 2nd tab of borwser has http://localhost:4200/receiver
receiver.component.ts
this component will detect/sync the change that will be applied by the second page/tab i.e It will change the content based on the boolean attribute OnMain
import {Component,bind,CORE_DIRECTIVES,OnInit} from 'angular2/core';
import {MainComponent} from 'src/MainComponent';
import {SharedService} from 'src/shared.service';
@Component({
selector: 'my-app',
directives:[MainComponent],
template: `<h1>AppComponent {{onMain}}</h1>
<div *ngIf="onMain == false">
Hello
<br>Show this content if false<br>
</div>
})
export class AppComponent implements OnInit {
onMain: Boolean;
constructor(ss: SharedService) {
this.onMain = false;
this.ss = ss;
}
ngOnInit() {
this.subscription = this.ss.getMessage()
.subscribe(item => this.onMain=item);
}
}
sender.component.ts
the page/tab having sender component wants to alter the content of first page/tab
import {Component,bind,CORE_DIRECTIVES} from 'angular2/core';
import {SharedService} from 'src/shared.service';
@Component({
selector: 'main-app',
template: `<h1> Sencond Page Component</h1>
<button (click)="changeFirstPageMenu()">Hide Menu</button>
`
})
export class MainComponent {
constructor(ss: SharedService) {
this.ss = ss;
}
changeFirstPageMenu() {
this.ss.sendMessage(true);
}
}
Data Service.ts
this is a shared service having the data that has to be altered by one page and the change will be synced to the other page(i.e receiver)
import { Subject } from 'rxjs/Subject';
@Injectable()
export class LandingService {
private subject = new Subject<any>();
sendMessage(message: any) {
this.subject.next({ text: message });
}
clearMessage() {
this.subject.next();
}
getMessage(): Observable<any> {
return this.subject.asObservable();
}
}
now the problem is the components are loaded on two different tabs/pages of a browser and these two tabs/pages have no relation they have their own instances declared and so any service(subject,behaviour),observers are not able to share the data between these two tabs so how can i share the data between two pages , and the change must be sync or reflected on the receiver page
I guess this question is similar to http://stackoverflow.com/questions/43737772/how-do-you-sync-data-between-two-browser-tabs-in-angular2
You can ping to your back end service at any time to get the most recent updated data.
Assume that you update data in first tab and you want it in another tab. When you open second tab (i.e focus second tab) you give a back end request (call a service) once so that it will fetch you the latest data on second tab which was updated from first tab.
You will get many posts to observe the tab focused event.
http://www.thefutureoftheweb.com/blog/detect-browser-window-focus
|
Korosechev
Etymology
Pre-Server History
Settling Down
The Formation Of Korosechev
Calm Before The Storm
The "War" of Independence
Lost To The Silence
T-Minus: 1 Minute Till Midnight
Two Governments, One Island, And a lot of Instability
The Red People's Reforms
* Allowing civilians to make their own money via selling
* Writing a constitution
* Diversification of crops grown
General Expansion
Renaming into Ball Ball Ball 2
Peace and Quiet
Two 2nds
The Koro Renaissance & Sledgehammer Doctrine
Uniting With Cognia
War of Independence
War for the Commune
First Tree War
Government Structure
Government Locations
Politics
National Focuses
Religion
Ethnicity
Korosechev Island
"Main Article: Korosechev Island"
Natural Geography
Human Geography
Natural Geography
Human Geography
|
import React, { Component } from 'react';
import { random } from 'lodash';
import './Ad.css';
class Ad extends Component {
state = {
outlined: false
}
componentDidMount() {
const blinkOutline = () => this.setState({ outlined: !this.state.outlined });
const startBlinkingOutline = () => {
this.blinker = setInterval(blinkOutline, random(100, 1250))
};
setTimeout(startBlinkingOutline, random(500))
}
componentWillUnmount() {
clearInterval(this.blinker);
}
render() {
const { href, description } = this.props;
const { outlined } = this.state;
return (
<a href={href} target="_blank" className="Ad">
<div className={`AdContent ${outlined ? 'outlined' : ''}`}>
{description}
</div>
</a>
);
}
}
export default Ad;
|
Kevin Gravel
Kevin Gravel (born March 6, 1992) is an American professional ice hockey defenseman who is currently playing for the Milwaukee Admirals in the American Hockey League (AHL) while under contract to the Nashville Predators of the National Hockey League (NHL). He was selected by the Los Angeles Kings in the fifth-round (148th overall) of the 2010 NHL Entry Draft.
Amateur
While playing for the Marquette Rangers in the North American Hockey League, Gravel committed to play for his fathers Alma mater St. Cloud State University. Gravel then played with the Sioux City Musketeers in the United States Hockey League for the 2009–10 season. In his first season, he was selected for the USHL All-Star Game and the World Junior A Challenge.
Gravel played four season for the St. Cloud State University Huskies, where he was named to the All-WCHA Academic Team for the 2012 and 2013 season. He also helped the Huskies qualify for the 2013 Frozen Four and win an NCAA regional title game in 2014.
Professional
After his senior year, Gravel continued his 2013–14 season by signing an amateur try-out contract with the Manchester Monarchs of the AHL. On August 4, 2014, Gravel continued within the Los Angeles Kings organization, agreeing to a one-year AHL contract with the Monarchs, the Kings' affiliate. In his first full professional season in 2014–15, Gravel established a position on the blueline with the Monarchs, contributing to the club's first Calder Cup Championship in their last season in the AHL.
On July 2, 2015, the Los Angeles Kings signed Gravel to a two-year entry-level contract. To begin the 2015–16 season, Gravel was reassigned to new AHL affiliate, the Ontario Reign. After 42 games with the Reign, Gravel received his first NHL recall to the Kings on February 11, 2016. Gravel recorded his first NHL goal on February 23, 2017 in a game against the Boston Bruins.
During the summer before the 2017–18 season, Gravel was diagnosed with crohn’s disease. He lost around 40 pounds, was hospitalized, and missed more than a month of training. On July 1, 2018, Gravel signed as a free agent to a one-year, two-way contract with the Edmonton Oilers. After attending the Oilers 2018 training camp, Gravel began the 2018–19 season with AHL affiliate, the Bakersfield Condors. Contributing with 1 assist in 5 games, Gravel was soon recalled by the Oilers and remained with the club for the duration of the year, recording 3 assists in 36 games.
As a free agent from the Oilers, Gravel agreed to a one-year, $700,000 contract with the Toronto Maple Leafs on July 24, 2019. He made 3 appearances with the Maple Leafs during an injury plagued 2019–20 season, featuring more prominently with AHL affiliate, the Toronto Marlies, with 3 points in 23 regular season games.
Un-signed leading into the pandemic delayed 2020–21 season, Gravel returned to former AHL club in the Bakersfield Condors after agreeing to a one-year contract on January 26, 2021.
After successful stint with the Condors, Gravel was signed as a free agent in returning to the NHL by securing a one-year, two-way contract with the Calgary Flames on July 28, 2021.
Following the conclusion of his contract with the Flames, Gravel as a free agent was signed to a two-year, two-way contract with the Nashville Predators on July 13, 2022.
|
#include <stdio.h>
#include <stdlib.h>
#include <inttypes.h>
#include <stdint.h>
#include <string.h>
#include <sys/stat.h>
#include "lz4.h"
#if __MINGW32__
#define __MSVCRT_VERSION__ 0x0601
#endif
#define MOZLZ4_MAGIC "mozLz40"
uint64_t fsize(const char *filename)
{
#if __MINGW32__
struct __stat64 st;
if (_stat64(filename, &st) == 0) return st.st_size;
#else
struct stat st;
if (stat(filename, &st) == 0) return st.st_size;
#endif
return 0;
}
int main (int argc, char *argv[])
{
FILE *in;
uint8_t *input;
uint32_t isize = 0;
uint32_t ipos = 0;
FILE *out;
uint32_t osize = 0;
uint8_t *output;
int32_t decompressed;
uint32_t n;
if (argc != 3) {
fprintf(stderr, "Mozilla LZ4 4a version decompressor.\n"
"Usage: unmoz inputfile outputfile\n"
);
return 1;
}
in = fopen(argv[1], "rb");
if (!in) { perror("Error opening input file"); return 1; }
isize = fsize(argv[1]);
if (isize < 12 || isize > 134217728) {
fprintf(stderr, "Size is %" PRIu32 " bytes, must be >12 bytes and <128MB\n", isize);
return 1;
}
input = malloc(isize * sizeof(uint8_t));
if (input == NULL) { perror("Cannot allocate memory"); return 1; }
memset(input, 0, isize*sizeof(uint8_t));
n = fread(input, sizeof(uint8_t), isize, in);
if (n != isize) {
fprintf(stderr, "Only got %" PRIu32 " input bytes. Quitting.\n", n);
perror("Read error");
return 1;
}
/* check magic number */
if (strncmp((char *)(input+ipos), MOZLZ4_MAGIC, strlen(MOZLZ4_MAGIC)+1) != 0) {
fprintf(stderr, "Magic number mismatch.\n"
"Expected:\n\t"
);
for (int i=0; i<strlen(MOZLZ4_MAGIC)+1; i++) { fprintf(stderr, "%4c", MOZLZ4_MAGIC[i]); }
fprintf(stderr, "\n\t");
for (int i=0; i<strlen(MOZLZ4_MAGIC)+1; i++) { fprintf(stderr, "%4u", MOZLZ4_MAGIC[i]); }
fprintf(stderr, "\nGot:\n\t");
for (int i=0; i<strlen(MOZLZ4_MAGIC)+1; i++) { fprintf(stderr, "%4c", input[i+ipos]); }
fprintf(stderr, "\n\t");
for (int i=0; i<strlen(MOZLZ4_MAGIC)+1; i++) { fprintf(stderr, "%4u", input[i+ipos]); }
fprintf(stderr, "\n");
return 1;
}
printf("Magic number ok!\n");
ipos += strlen(MOZLZ4_MAGIC)+1;
/* read output size - big endian */
osize = input[ipos] + (input[ipos + 1] << 8) + (input[ipos + 2] << 16) + (input[ipos + 3] << 24);
printf("Output size: %" PRIu32 "\n", osize);
ipos += sizeof(uint32_t);
output = malloc(osize * sizeof(uint8_t));
if (input == NULL) { perror("Cannot allocate memory"); return 1; }
memset(output, 0, osize*sizeof(uint8_t));
/* decompress */
decompressed = LZ4_decompress_fast((char *)(input+ipos), (char *)output, osize);
if (decompressed < 0) {
perror("Decompression problem");
return 1;
}
printf("Decompressed %" PRIu32 "/%" PRIu32 " input bytes.\n", decompressed, isize-ipos);
/* write */
out = fopen(argv[2], "wb");
if (!out) { perror("Error opening output file"); return 1; }
n = fwrite(output, sizeof(uint8_t), osize, out);
if (n != osize) {
fprintf(stderr, "Wrote %" PRIu32 "/%" PRIu32 " output bytes.\n", n, osize);
perror("Cannot write output file");
return 1;
}
fclose(in);
fclose(out);
printf("Done.\n");
return 0;
}
/* vim: set tabstop=4 softtabstop=4 noexpandtab: */
|
Give me the complete political debate on the topic of Ethics that ends with: ...tion to the Prime Minister of Canada immediately?.
|
Place:St. Cosmus and St. Damian in the Blean, Kent, England
Watchers
NameSt. Cosmus and St. Damian in the Blean
Alt namesBleansource: shortened, more modern name
TypeParish (ancient), Civil parish
Coordinates51.317°N 1.033°E
Located inKent, England
See alsoWhitstable Hundred, Kent, Englandancient county division in which it was located
Blean Rural, Kent, Englandrural district in which it was located 1894-1934
Bridge Blean Rural, Kent, Englandrural district in which it was located 1934-1974
Canterbury District, Kent, Englanddistrict municipality in which it has been located since 1974
source: Getty Thesaurus of Geographic Names
source: Family History Library Catalog
St. Cosmus and St. Damian in the Blean is a Church of England parish with one church serving two villages, Blean and Tyler Hill, which lie in ancient Kent woodland between the cathedral city of Canterbury and the seaside town of Whitstable. (Source: The history page of the parish church website which provides information from the formation of the church onward.)
St. Cosmus and St. Damian in the Blean was the original and formal name of the parish Over time the name of the parish was shortened to Blean. Tyler Hill, mentioned above, is in the parish of Hackington.
Between 1894 and 1934 St. Cosmus and St. Damian in the Blean, or simply Blean, was part of the Blean Rural District. In 1934 the Blean Rural District was abolished and its parishes became part of the larger Bridge Blean Rural District. Since 1974 the area has been part of the non-metropolitan City of Canterbury District. The parish was originally in the Whitstable Hundred.
A Vision of Britain through Time provides the following description of Blean from John Marius Wilson's Imperial Gazetteer of England and Wales of 1870-72:
"BLEAN, a parish, an ancient forest, and a [registration] district, in Kent. The parish is called also Blean-Church, St. Cosmus, and St. Damian-in-the-Blean: lies on the Whitstable railway, 2 miles NW by N of Canterbury; and has a post office, of the name of Blean, under Canterbury. Acres: 2,260. Real property: £4,414. Population: 626. Houses: 130. The property is subdivided. Much of the land in the north is under coppice. The living is a vicarage in the diocese of Canterbury. Value: £523. Patron: Eastbridge Hospital. The church is small.
"The forest belonged anciently to the Crown; extended from the vicinity of Herne to the vicinity of Chatham; was given away piecemeal, both before and after the Conquest, till nearly all was alienated; and lost gradually the character of a forest, till it became known simply as the Blean. Wild boars abounded in portions of it so late as the Reformation; and the yellow pine marten is still occasionally found."
The Gazetteer information on the registration district has been omitted.
the following text is based on an article in Wikipedia
Blean is in the Canterbury district of Kent, England. It is the name of the large civil parish and the suburban developed village within it: the latter is scattered along the road between Canterbury and Whitstable, in the middle of the Forest of Blean. The parish is mostly woodland, and much is ancient woodland.
For more information, see the EN Wikipedia article Blean.
Research Tips
• Kent County Council Archive, Local Studies and Museums Service. James Whatman Way, Maidstone, Kent ME14 1LQ. This incorporates the Centre for Kentish Studies in Maidstone and the East Kent Archives Centre near Dover.
• Canterbury Cathedral Archives see the Archives web pages on the Canterbury Catherdral site.
• For information on the area around the Medway Towns, have a look at Medway Council's CityArk site.
• Ordnance Survey Maps of England and Wales - Revised: Kent illustrates the parish boundaries of Kent when rural districts were still in existence and before Greater London came into being. The map publication year is 1931. An earlier map of 1900 may also be useful. The maps blow up to show all the parishes and many of the small villages and hamlets. Maps in this series are now downloadable for personal use.
• Census records for Kent are available on FamilySearch, Ancestry and FindMyPast. The first site is free; the other two are pay sites but have access to microfilmed images. Steve Archer produced a very useful round-up of the available sources, but this information may not be up to date.
• Registration Districts in Kent for the period 1837 to the present. By drilling down through the links you can follow any parish through the registration districts to which it was attached.
• England, Kent, Parish Registers, 1538-1911 The full database from Kent Archives Office, Maidstone, has been available online from FamilySearch since June 2016.
• Kent had five family history societies (now only four):
• Volume 2 of the Victoria County History of Kent (published 1926) is available online through the auspices of British History Online. It includes accounts of the early history of Canterbury and Rochester cathedrals, and of several sites now within the conurbation of London.
• Volume 3 of the Victoria County History of Kent (published 1932) This includes the text of, and the index to, the Kent Domesday survey. It has been provided by the Kent Archaeological Society.
• In place of the other volumes of the Victoria County History, British History Online has transcriptions of the numerous volumes of The History and Topographical Survey of the County of Kent by Edward Hasted (originally published 1797)
• English Jurisdictions 1851, a parish finding aid provided by FamilySearch, is particularly helpful in locating parishes in large ancient towns and cities like Canterbury.
• Kent Probate Records Numerous links provided by Maureen Rawson
• GENUKI lists other possible sources, however, it does not serve Kent so well as it does some other counties.
|
Talk:Michael Krohn-Dehli
External links modified
Hello fellow Wikipedians,
I have just modified 2 external links on Michael Krohn-Dehli. Please take a moment to review my edit. If you have any questions, or need the bot to ignore the links, or the page altogether, please visit this simple FaQ for additional information. I made the following changes:
* Added archive https://web.archive.org/web/20080831011533/http://www.ajax.nl/web/show/id%3D154417/contentid%3D67237 to http://www.ajax.nl/web/show/id%3D154417/contentid%3D67237
* Added archive https://web.archive.org/web/20150627145648/http://sevillafc.es/nuevaweb/actualidad/noticias/37765/el-sevilla-fc-firma-a-michael-krohn-dehli-para-las-dos-proximas-temporadas to http://www.sevillafc.es/nuevaweb/actualidad/noticias/37765/el-sevilla-fc-firma-a-michael-krohn-dehli-para-las-dos-proximas-temporadas
Cheers.— InternetArchiveBot (Report bug) 22:27, 9 June 2017 (UTC)
|
Barry "Reazar" Richards
Barry Richards (born November 23, 1946) is an American radio/television personality, concert promoter and music producer from Washington, D.C. He made an impact during the late 1960s to early 1970s by introducing progressive rock to radio on the East coast.
Personal life
Richards was born in Washington, D.C., on November 23. He lives in Beverly Hills with his wife Debra and has three sons: Stevie "Rocker" Richards (Deceased), Gary Richards (music executive) and Paul Richards. Gary married Anne Varnishung and has 2 children (Riley and Stevie). Paul Richards married Sivan Ayla Vardi and has two children (Capri and Walker).
On air
Barry first made it on the radio thanks to Don Dillard by reading on-air dedications on WDON Washington, D.C. He carried records to Don's record-hops where he played the records while Don MC’d. e also danced on The Milt Grant Show and convinced the artists who appeared on the TV show to do Don's record hops. He stayed at WDON while he was at school, until the station went Country/Western.
By 1965 he found opportunities in radio stations such as:
* WDON(AM) Washington, D.C.
* WMID(AM) station in Atlantic City
* WYRE(AM) in Annapolis
* WITH/Tiger radio(AM) in Baltimore
* WUST(AM) in Washington, D.C.
* WINX(AM) in Washington, D.C.
* WHMC(AM) Washington, D.C. – Program director and afternoon drive making WHMC the first progressive Rock station on the east coast. It wasn't until radio station owner Nick Chaconas gave Barry the creative freedom to program album cuts and become the first underground radio station, which later morphed into free-form radio. He became known as your heavy Head Leader. He would play Led Zeppelin, Janis Joplin, Jimi Hendrix, Jefferson Airplane.
* WKTK(FM) in Baltimore – Program director and afternoon drive
* WEEL(AM) Washington, D.C. – Nighttime air personality
* WKYS(FM) in Washington, D.C. – Station known as Disco 92, making it the first all disco station in the country
* WMOD(FM) in Washington, D.C. – Known now as WMZQ he was again nighttime air personality
* WEAM(AM) in Washington, D.C. – Program director and afternoon drive – In 1976–1978, a time where African-American and white music didn't mix, radio stations such as WEAM broke the mold by playing songs with no ethnic political or stylistic boundaries. Known as Urban Contemporary.
* WAIL 105.3 FM located in New Orleans, it's a "Cinderella story" according to owner Ed Muniz, The station faced stylistic changes and needed to be successful in 1980 when he team up with experienced Barry Richards the station jump to number one.
* KGFJ(AM)/KUTE(FM) in Los Angeles – Program director and afternoon drive
* KBOS-FM/B95 in Fresno – Program director and afternoon drive
* National program director for Bresson-Hafler Media Group with stations in Philadelphia, Youngstown, Ohio, Columbia and Myrtle Beach, South Carolina.
* WJLQ(FM)/WCOA(AM) Pensacola, Florida – Co-owner, program director and afternoon drive
* KQQB/KAZZ Spokane, Washington – Co-owner
* KVPW (FM)Fresno with Jerry Clifton
* On air Sirius XM Radio Strobe Channel- Last on-air job
Promoting
Richards became a music consultant to many record labels and was part of a team to help Black Sabbath, Alice Cooper, Led Zeppelin, Emerson Lake & Palmer, J Geils, Jethro Tull, Edgar Winter and Johnny Winter.
He later started HARD Events with son Gary Richards, now known as Destructo.
Free-form TV
Richards has promoted many artists through several popular television shows including Turn-On, Barry Richards Rock and Soul at WDCA-TV 20 Washington, D.C., Video Disco, Studio 78, WJLA/Ch.7/Washington, D.C.,WMAR/Ch2/Baltimore, Video Trax on WWL/ch.4 and WDSU/Ch.6 New Orleans", Video Zoo on KDOC TV Los Angeles, BTV(Fresno Ch. 57) and Fox Breakfast Club Movie WPMI-TV 15 mobile Alabama FOX NETWORK. Some times local comedians as Uncle Dirty a/k/a Robert Altman, Robert Klein, Richard Pryor, Cheech and Chong will be on his shows. Richards also ran the Rhythm section of HitMakers magazine called Reazar's Records.
Turn On
On UHF Channel 20 he broadcast a rambling, chaotic blend of rock and camp mixing live acid-rock acts such as Steppenwolf, Zephyr and Dr. John with Flash Gordon serials, campy movies from the forties and fifties, Allan Freed rock musicals, interviews with Playboy bunnies, Buster Crabbe (The original Flash) and movie stars such as Charlton Heston, Robert Mitchum and Cornel Wilde. In one memorable set from 1973, Fats Domino was accompanied by Roger McGuinn and The Byrds and taught them how to perform his famous songs.
Richards called the format 'free-form television', emphasizing progressive rock acts playing live on TV for the first time. The show started at 11 o'clock on Saturday night and continued until the material was done, ending with the national anthem.
Rock'n Soul
Recording at WDCA-TV, 5202 River Road, Bethesda took place Friday nights at 1:00 am and Saturday mornings at 10:00 am; later the show moved to 8:00 pm Saturday night. This live show included young people dancing to the hits of the day and in-person performances by such artists as The Commodores, Eddie Kendricks, Kool & the Gang, The O'Jays, War, Billy Preston, Earth, Wind & Fire, Joe Simon, B. T. Express and James Brown.
Video disco
This 1970s TV show, featured disco music, disco fashion and disco dancing. The show had a budget of $150,000, backed by Indian-born entrepreneur Surinder Dhillon.
Studio 78
On July 27, 1978, WJLA began airing "Studio 78" featuring disco dancing and celebrity interviews. Sponsors were HECHT Co./ May Company, Channel 2 and Channel 7. Acts included Gloria Gaynor, Village People, Donna Summer, Andy Gibb, Evelyn King and BT Express.
Video Trax
In the 1980s Richards became known as Reazar. He joined efforts with Rod Carter a program director at channel 6 to create Video Trax, a dance show. It ran from September 1982 to May 1983, before gaining the financial and promotional support of Pepsi Cola and moving the show to WWL channel 4 in New Orleans.
Concert promoter
As a concert promoter Richards brought such progressive rock acts as Black Sabbath, Alice Cooper, Led Zeppelin, Emerson Lake & Palmer, J Geils, Jethro Tull, Edgar Winter, Johnny Winter, Joe Cocker and Steppenwolf to a roller rink in Alexandria, Virginia.
Other concerts included most of the 1970s rock and R&B shows in Washington, D.C., and Baltimore. He did several discos with superstar DJ Wolfman Jack from the Midnight Special NBC TV Show. He promoted the 1977 Brute Music Festival, a three-day weekend that headlined Earth, Wind & Fire, The Commodores, Kool & the Gang, The Emotions, The Sylvers, Bohannon, Tyrone Davis, Michael Henderson, Johnnie Taylor, Gil Scott-Heron, Jimmy Castor, Shotgun, Walter Jackson, Les McCann, Juju, Brute, The Brothers Johnson and Slave. Almost every R&B Soul artist performed, it was live acts from 12 noon to 3 am, after that was continuous disco.
Carefree Sugarfree Gum
In 1980 Richards hosted a yearlong tour as promotion director for Carefree Sugarfree Gum owned by the Squib Company in New York, they were tied in with a radio station in every city with the local big DJ. Part of the promotion was to increase awareness of Carefree Sugarless gum, which had just come out, through a series of shows taking place in preselected high schools visiting 30 cities. One school in each city that collected the most gum wrappers won a free concert with Hall & Oates, a visit from the local DJ, prizes and $1000 cash.
In the 1990s, he became editor of the Rhythm section in Hitmaker's Magazine from January 1995 to January 2004. In 2000 he started his own promotion company, Reazar Record Promotions.
Shows
He hosted California Championship Wrestling Live at the Olympic in Los Angeles as well as the San Bernardino Arena in San Bernardino. He also hosted a Latin Top 40 show, produced and hosted live music videos shows such as: a dance show called Wing Ding WDCA Washington, D.C. 1967, Grove In WTTG Washington, D.C. 1969 Barry Richards Turn On (1970–1972) WDCA TV Washington, D.C., Barry Richards Rock Show WMAR TV Baltimore, Maryland (1972–1973), Barry Richards presents Rock Movies WTOP in Washington, D.C. (1973–1974), Barry Richards Rock and Soul WDCA Washington, D.C. (1975–1977), Video Disc WDCA Washington, D.C. TV 1977, Studio 78 WJLA TV Washington, D.C. and WMAR TV Baltimore, Maryland 1978, Live at the Famous with Barry Richards WDSU New Orleans 1980, Video Tracks WDSU New Orleans (1981–1983), Video Tracks WWL TV New Orleans, Louisiana (1983–1985), Video ZOO KDOC Los Angeles, CATV there is a clip Los Angeles times magazine (1986–1988).
Press
He has been featured in magazines as the Rolling Stone, Billboard, Radio & Records, and newspapers such as The Washington Post, The Merriweather Post Magazine, The Washington Star, The Arlington News and Time Herald.
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Int. J. Mol. Sci. 2015, 16(5), 11550-11573; https://doi.org/10.3390/ijms160511550
Article
De Novo Assembly and Characterization of the Transcriptome of the Chinese Medicinal Herb, Gentiana rigescens
1
College of Resources and Environment, Yuxi Normal University, Yuxi 653100, China
2
Plant and Food Research, Mt Albert Research Centre, Private Bag, Auckland 92169, New Zealand
3
Institute of Medicinal Plants, Yunnan Academy of Agricultural Sciences, Kunming 650223, China
4
University of Chinese Academy of Sciences, Beijing 100039, China
*
Author to whom correspondence should be addressed.
Academic Editor: Marcello Iriti
Received: 31 March 2015 / Accepted: 14 May 2015 / Published: 20 May 2015
Abstract
:
Gentiana rigescens is an important medicinal herb in China. The main validated medicinal component gentiopicroside is synthesized in shoots, but is mainly found in the plant’s roots. The gentiopicroside biosynthetic pathway and its regulatory control remain to be elucidated. Genome resources of gentian are limited. Next-generation sequencing (NGS) technologies can aid in supplying global gene expression profiles. In this study we present sequence and transcript abundance data for the root and leaf transcriptome of G. rigescens, obtained using the Illumina Hiseq2000. Over fifty million clean reads were obtained from leaf and root libraries. This yields 76,717 unigenes with an average length of 753 bp. Among these, 33,855 unigenes were identified as putative homologs of annotated sequences in public protein and nucleotide databases. Digital abundance analysis identified 3306 unigenes differentially enriched between leaf and root. Unigenes found in both tissues were categorized according to their putative functional categories. Of the differentially expressed genes, over 130 were annotated as related to terpenoid biosynthesis. This work is the first study of global transcriptome analyses in gentian. These sequences and putative functional data comprise a resource for future investigation of terpenoid biosynthesis in Gentianaceae species and annotation of the gentiopicroside biosynthetic pathway and its regulatory mechanisms.
Keywords:
Gentiana rigescens; gentiopicroside; regulation; transcriptome
1. Introduction
Gentiana rigescens, also named Caine gentian, belongs to Gentiana, a member of the Gentianaceae family. It is a geoherb of great importance to China’s Yunnan province. G. rigescens is a perennial, growing amongst hillside grasses, bushes, and trees, at relatively high altitudes. It is usually harvested in late October, with the roots being used as bulk herbs [1] and raw materials for more than 180 different Chinese traditional medicines [2]. Recent research has shown that it possesses potential functions in liver protection and immune promotion [3,4]. The main effective component of G. rigescens is gentiopicroside [3], which is mainly found in the vacuoles of root cells, although it is synthesized in shoots [5,6]. The content of gentiopicroside in roots was far higher than that in shoots at the flower stage [7]. In addition, there are other active components including swertiamarin, sweroside, erythricine, ursolic acid, oleanolic acid, loganic acid, gentianidine, and gentiana aldin [5,8].
In recent years, the wild resources of G. rigescens have declined sharply, with shortages of gentian, as demand for its use in clinical, pharmaceutical, and veterinary areas increases [1]. It has now been classified as a protected plant in China [1]. Similarly, many other Gentiana species have become endangered species [9]. Studies have suggested that the chromosome number of Gentiana triflora, Gentiana scabra, Gentiana manshurica, and other herbs containing gentiopicroside, is 2n = 26, while that of Gentiana lutea and Gentiana punctata is 2n = 40 [10]. The former three share a similar genome size (5 × 109 bp/1C), approximately 33 times that of Arabidopsis thaliana [10,11]. Gentian genome resources are very scarce due to its large genome, genomic heterozygosity brought by distal hybridization, long growth cycle, and the lack of genetic information [10]. The Japanese gentian’s genetic linkage map was the first map of the Gentianaceae to be published, although its coverage is still low (about 1/3 genome coverage) and the phenomena of separation distortion (whereby there is unequal segregation of pairs of alleles) emerged in 30% of the molecular markers tested in progeny [10]. Therefore, the development of batches of EST-SSR (Expression Sequence Tag-Simple Sequence Repeat) molecular makers by RNA-Seq would be an improvement. In Japan, G. scabra and G. triflora are important cut and potted flowers, so research has focused on the anthocyanin biosynthesis pathway and its regulation [12,13,14]. Other studies have been on seed germination [4,15], elemental analysis [16], and active ingredient content [17,18]. However, there has been little research on the gentiopicroside biosynthesis pathway and its regulation. Recently, a seven-year breeding project of G. rigescens, whose goals are high yield, high gentiopicroside content, disease resistance, mechanized production, and wider planting, has been launched in Yunnan province [2].
To protect wild gentian resources as a source of plant material, a better understanding of the plant’s biology and growth is required. Transcriptome research provides a method of fast, high-throughput, comprehensive interpretation of the plant’s genome information, including new gene function information, the biosynthesis of the active ingredients and their regulation, and germplasm evaluation and expansion [19].
The objective of this research was to compare the transcriptomes of the leaf and root of G. rigescens, using Illumina Hiseq2000. To determine genes involved in the gentiopicroside biosynthesis pathway and its regulatory mechanism, transcripts from leaves and roots of gentian were isolated, quantified, sequenced, and annotated. The results described here will aid further functional genomic studies in gentian.
2. Results and Discussion
2.1. Sequencing and Assembly
To determine the transcriptomes of the leaves and roots of G. rigescens, two sequencing libraries were prepared and sequenced with the Illumina paired-end technique. As a result, over 50 million clean reads per library were obtained after cleaning and quality checks were performed. The sequencing data quality assessments are shown in Table 1. The error rate of both root and leaf is 0.03% (Q20 and Q30 are over 96% and 90%, separately), indicating a high quality of sequence. The sequencing raw data has been deposited into the Short Reads Archive (SRA) database under the accession number SRP027253.
Table 1. The quality assessment of the sequencing data.
Table 1. The quality assessment of the sequencing data.
ItemSampleRaw ReadsClean ReadsClean Bases (G)Error (%)Q20 (%)Q30 (%)GC (%)
LeafLeaf_157,802,91356,289,4865.630.0397.9793.0943.00
Leaf_257,802,91356,289,4865.630.0397.4892.6143.04
RootRoot_153,933,88250,596,0965.060.0397.0290.7943.23
Root_253,933,88250,596,0965.060.0396.5990.3443.31
Leaf_1, Root_1, Leaf_2, Root_2: The left and the right reads, separately; Raw reads: Statistical raw sequence data with four lines as a unit, to sum the sequence number of each file; The Clean Reads of the Leaf or Root were the sum of the left and right reads. Error rate: Bases error rate; Q20 and Q30: The percentages of the bases whose Phred values were more than 20 and 30, separately.
The clean reads were combined and assembled by using the Trinity program, which has been shown to be an excellent assembler for de novo transcriptome assembly from short-read RNA-Seq data [20]. Assembled sequences were subjected to cluster using the Trinity algorithm. As a result, 191,541 contigs clustered into 78,433 Trinity components (mean size = 743 bp, N50 = 1365 bp). Each Trinity component defines a collection of transcripts that are most likely to be derived from the same locus (except a portion from very closely related paralogs) [20,21]. This component was defined as a unigene and the longest transcript in each component was used to represent the corresponding unigene in this study. After removal of 1716 (2.2% of total) contaminant unigene sequences from non-plant species (see Materials and Methods), a transcriptome of 76,717 unigenes with a total size ~57.7 Mb was established for G. rigescens. The sequences of the unigenes (longer than 200 bp) were deposited in the NCBI (National Center for Biotechnology Information) Transcriptome Shotgun Assembly Sequence Database (TSA) according to its standard criteria (downloaded from the BioProject: PRJNA211794, Accession Number: GDAB00000000). The full list of transcript sequences is also available upon request.
2.2. Assembly Assessment
By comparing our results to Gentiana sequences downloaded from the NCBI (Available online: http://www.ncbi.nlm.nih.gov), we demonstrated that the assembly succeeded in constructing a large amount of transcripts with desirable length. Of 43,611 Gentiana sequences, 33,773 (77.4%) sequences were represented in our assembly (Megablast, E-value was 10−9), among which 23,908 (70.8%) sequences were matched with more than 80% identity and 80% coverage. RNA-Seq reads were mapped back to the assembly to calculate the proportion of reads assembled, indicating a statistic report comparable to other de novo assemblies. The total alignment rate was 92.72% (Table 2), and 78.3% of the mapped paired-reads aligned concordantly, which showed good physical evidence of sequence contiguity. Transcript length (such as N50, average length) is another broadly used parameter to overview the quality of the transcriptome assembly. As shown in Figure 1, the unigenes ranged from 201 to 16,728 bp, with a mean length of 753 bp and an N50 length of 1384 bp, which is comparable to similar RNAseq reports. Thus, we have successfully constructed a desirable assembly from Illumina paired-end sequencing.
Table 2. Summary of the transcriptome assembly of G. rigescens.
Table 2. Summary of the transcriptome assembly of G. rigescens.
ItemContigsUnigenes
Total number189,57676,717
Total length (bp)228,624,91257,734,637
Mean length (bp)1206753
N50 (bp)19961384
GC content (%)39.739.5
Number of length ≥ 500 bp120,52529,795
Number of length ≥ 1000 bp81,91916,332
Reads mapping rate (%)92.72-
Figure 1. Length distribution frequency of the unigenes in G. rigescens.
Figure 1. Length distribution frequency of the unigenes in G. rigescens.
2.3. Gene Function Annotation and Classification
All the 76,717 assembled putative unigenes were aligned using the BLAST program against the NR, NT, Swiss-Prot and COG databases with the E-value cutoff of 10−5. A total of 33,855 unigenes were annotated, accounting for 44.13% (Table 3). Among them, 26,686 unigenes (34.78%) showed high homology, with sequences in the NR database, 24,371 unigenes (31.77%) matched to protein sequences in TAIR10, and 18,627 unigenes (24.28%) showed homology with known genes in SwissProt. The detailed results are shown in Table 3 and Table S1–S3. Based on the top-hit species distribution of the homology result against NR databases, 26,361 unigenes (92.08%) showed high homology with sequences from land plants, among which the highest matches were to genes from Coffea canephora (36.08%), followed by Vitis vinifera (8.57%), and Sesamum indicum (7.36%) (Figure 2).
Table 3. Statistics result of gene annotation.
Table 3. Statistics result of gene annotation.
ItemNumber of Unigenes (n)Percentage (%)
Annotated in NR26,68634.78
Annotated in NT815810.64
Annotated in TAIR1024,37131.77
Annotated in KEGG799810.43
Annotated in SwissProt18,62724.28
Annotated in PFAM23,28730.35
Annotated in GO26,49434.53
Annotated in KOG/COG10,52413.72
Annotated in all Databases30193.94
Annotated in at least one Database33,85544.13
Total queries/unigenes76,717100
Figure 2. Species distribution of the top BLAST (Basic Local Alignment Search Tool) hits for each unigene against NR (Non-redundant) database.
Figure 2. Species distribution of the top BLAST (Basic Local Alignment Search Tool) hits for each unigene against NR (Non-redundant) database.
Putative protein sequences were obtained by translating using a standard codon table. The CDSs of unigenes that did not match the above databases were predicted with the ESTSCAN software. The gene length distribution is shown in Figure 3. The length of peptides predicted by BLASTp ranges from 60–810, while that of ESTSCAN are 30–240.
Figure 3. Length distributions of predicated peptides. (A) Predicated by BLAST; (B) Predicated by ESTScan program (Available online: http://www.ch.embnet.org/software/ESTScan.html). The abscissa represents the peptide length, while the ordinate represents thce number of the corresponding number.
Figure 3. Length distributions of predicated peptides. (A) Predicated by BLAST; (B) Predicated by ESTScan program (Available online: http://www.ch.embnet.org/software/ESTScan.html). The abscissa represents the peptide length, while the ordinate represents thce number of the corresponding number.
In this study, all unigenes were searched against the GO database. Out of 76,717 unigenes, 26,494 were successfully annotated and classified into three GO categories: biological process, cellular component, and molecular function, and assigned to 56 functional groups (Figure 4). As shown in Figure 4, assignments which fell under cellular component ranked the highest, followed by biological process, and molecular function. In the biological process category, “cellular process” (16,075, 60.67%) and “metabolic process” (15,223, 57.46%) were the two most representative subcategories. In the cellular component category, unigenes related to “cell” (10,308, 38.91%) and “cell part” (10,282, 38.81%) were dominant, while in the molecular function category, the majority of unigenes were involved in “binding” (14,903, 56.25%) and “catalytic activity” (12,326, 46.52%). These results suggested that many kinds of enzyme pathways were active in gentian.
A total of 10,524 sequences were classified into 26 KOG/COG (Clusters of Orthologous Groups of proteins) groups (Figure 5), where “General function prediction only” category accounted for the most frequent group (1948, 18.51%), with the second largest group being “Post-translational modification, protein turnover, chaperon” (1319, 12.53%), followed by “Signal transduction” (932, 8.86%) and “Translation” (654, 6.21%). These results showed that in the flower stage of gentian, the protein translation and signal transduction are active.
Figure 4. GO classification map. The abscissa represents the next level GO term of the three GO categories, while the ordinate represents the number of genes annotated into the corresponding term, and its proportion of the total number of annotated genes.
Figure 4. GO classification map. The abscissa represents the next level GO term of the three GO categories, while the ordinate represents the number of genes annotated into the corresponding term, and its proportion of the total number of annotated genes.
Figure 5. KOG/COG classification map. The abscissa represents 26 group names of KOG/COG, while the vertical axis represents the number of genes annotated into the group and its proportion of total number of annotated genes.
Figure 5. KOG/COG classification map. The abscissa represents 26 group names of KOG/COG, while the vertical axis represents the number of genes annotated into the group and its proportion of total number of annotated genes.
The KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic system is a group of metabolic maps which represents current understanding of biomolecular interaction networks. In order to determine the active pathways in flowering gentian, KEGG assignments of all unigenes were performed. Referencing the 7998 unigenes of G. rigescens through the KEGG database predicted a total of five categories (level 1, cellular processes, environmental information processing, genetic information processing, and metabolism and organismal systems), 31 sub-categories (level 2, Figure 6) and 238 pathways (level 3). Unigenes identified as related to the “Translation” (861, 10.77%), “carbohydrate metabolism” (852, 10.65%), “Folding, sorting and degradation” (699, 8.74%) and “Signal transduction” (685, 8.56%) were the top four representative pathways (Figure 6). Unigenes counts for “Terpenoid backbone biosynthesis”, “Monoterpenoid biosynthesis”, “Diterpenoid biosynthesis”, “Sesquiterpenoid and triterpenoid biosynthesis”, and “Ubiquinone and other terpenoid-quinone biosynthesis” were 55, 5, 22, 21, and 31, separately. These results indicated that the terpenoid pathways were active in flowering gentian, and the corresponding genes would be candidate genes for gentiopicroside biosynthesis.
Figure 6. KEGG classification map. The ordinate is the name of the pathway, while the abscissa is the proportion of genes belonging to this pathway. These genes were divided into five branches: (A, Cellular Processes; B, Environmental Information Processing; C, Genetic Information Processing; D, Metabolism; E, Organismal Systems.) according to the metabolic pathway they participated in.
Figure 6. KEGG classification map. The ordinate is the name of the pathway, while the abscissa is the proportion of genes belonging to this pathway. These genes were divided into five branches: (A, Cellular Processes; B, Environmental Information Processing; C, Genetic Information Processing; D, Metabolism; E, Organismal Systems.) according to the metabolic pathway they participated in.
Gene expression was calculated using the RPKM method, which takes into account both sequencing depth and gene length effects on read count [22]. On the basis of the applied criteria q-value <0.005 and log2(foldchange) >1, 3306 genes (4.31% of all genes) were identified as significantly differentially expressed genes (DEGs) between these two tissues, which comprised 2204 up-regulated genes (accounting for 67%) and 1102 down-regulated genes (33%) in leaves (Figure 7, Table S4). The log2(fold changes) ranged from one to 15. Not surprisingly, among these DEGs, most were related to photosynthesis, for example, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-a, a key enzyme of the Calvin-Benson cycle of autotrophic CO2 assimilation [23], chloroplast chlorophyll a/b-binding protein, photosystem II 22 kDa protein gene, and chloroplastic ferredoxin genes, were all up-regulated over 10-fold in leaves compared to roots. The terpenoid biosynthesis related genes, such as geranyl diphosphate synthase (GPPS), geraniol synthase (GES), geraniol 10-hydroxylase (G10H), and iridoid oxidase (IO), four key enzymes involving monoterpene biosynthesis, were all up-regulated over 10-fold in leaves compared to roots.
Figure 7. Volcano plot of Leaf ves. Root in G. rigescens. The abscissa represents changes of gene expression (Leaf vs. Root). The ordinate represents the statistical significance of change of the amount of gene expression. The less p-value, the more −log10(p value), and the more significance. The scattering dots represent genes, while the blue dots show genes without significant differences and vice versa for red dots.
Figure 7. Volcano plot of Leaf ves. Root in G. rigescens. The abscissa represents changes of gene expression (Leaf vs. Root). The ordinate represents the statistical significance of change of the amount of gene expression. The less p-value, the more −log10(p value), and the more significance. The scattering dots represent genes, while the blue dots show genes without significant differences and vice versa for red dots.
Of the down-regulated genes, a late embryogenesis abundant (LEA) protein, was 13-fold higher in roots than in leaves. Late Embryogenesis Abundant (LEA) proteins are a group of hydrophilic proteins with a high content of glycine, and are associated with stress tolerance in plants and animals through protecting enzymatic function and inhibition of aggregation in dehydration, heat, and salt stress [24,25]. In Arabidopsis thaliana, overexpression of LEA14 enhances salt stress tolerance [26]. Ectopic expression of ZmLEA5C in tobacco and yeast enhances their tolerance to osmotic and low temperature stresses [27]. A calcium-dependent protein kinase (CDPK) gene involved in plant defense responses [28] was nine-fold higher in roots than in leaves. Previous research suggests that CCaMK is an important component of the symbiosis signaling pathway [29,30,31,32,33,34]. In Zea mays, calcium/calmodulin-dependent protein kinase (ZmCCaMK) is required for abscisic acid (ABA)-induced antioxidant defense systems [35]. A high affinity nitrate transporter [36] was eight-fold higher in roots than in leaves. In higher plants, there are two nitrate uptake systems, the high and low affinity transporter systems, and the high affinity nitrate transporter functions when the nitrate concentration is low [37,38].
2.4. Putative Genes Involved in the Terpenoid Backbone Biosynthesis and Gentiopicroside Biosynthetic Pathways
Terepenoids, including monoterpenoids, diterpenoids, chlorophyls, carotenoids, abscisic acid, cytokinin gibberellins, sterols, sesquiterpenoids, and ubiquinones, are all closely related with the terpenoid backbone biosynthesis [39,40]. The terpenoid backbone is derived from the universal precursor, isopentenyl diphosphate (IPP), and its allylic isomer, dimethylallyldiphosphate (DMAPP), which are derived from the mevalonate (MVA) and/or the methylerythritol phosphate (MEP) pathways [41] (Figure S1). Transcripts encoding the enzymes involved in the MVA and MEP pathways were searched against the unigenes and transcripts present in our database (Table 4). In general, transcripts of MVA and MEP pathway genes were more abundant in leaves, as revealed by much higher numbers of reads of 3-hydroxy-3-methylglutaryl-CoA reductase (GrHMGR), 5-diphosphomevalonate decarboxylase (GrMVD), Isopentenyl diphosphate isomerase (GrIDI), 1-deoxy-Dxylulose 5-phosphate synthase (GrDXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (GrDXR), 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (GrMCS), and 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GrHDS) genes in leaves than in roots (Table 5, (Figure S2). qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction) results showed that the selected genes GrDXS1, GrHDS, and GrIDI1 were more abundant in leaves (Figure 8). These results support the observation that gentiopicroside is synthesized in shoots and allocated to the roots [6].
Table 4. Expression of putative genes in MVA and MEP biosynthesis pathways.
Table 4. Expression of putative genes in MVA and MEP biosynthesis pathways.
PathwayGene NameUnigeneRPKM in LeafRPKM in Root
MVAAACT1comp81670_c050.6552.76
AACT2comp86403_c013.1638.43
HMGScomp1196622_c00.000.49
HMGR1comp87249_c027.143.53
HMGR2comp92954_c035.5617.08
HMGR3comp4296_c00.250.57
HMGR4comp25979_c00.570.04
HMGR5comp114241_c05.720.00
MKcomp83300_c020.7416.19
PMK1comp371052_c00.550.48
PMK2comp82309_c12.211.23
PMK3comp82309_c03.942.21
PMK4comp92698_c08.0811.20
MVD1comp86107_c051.0139.76
MVD2comp73189_c00.550.58
IDI1comp81822_c0114.7989.95
IDI2comp67360_c037.6531.50
IDI3comp92050_c049.182.79
MEPDXS1 *comp87916_c045.341.45
DXS2comp89290_c010.853.64
DXS3comp93517_c048.6935.56
DXRcomp92087_c3123.96107.04
MCTcomp67067_c036.768.28
MCScomp91375_c056.6136.13
HDS *comp94424_c0151.2877.99
HDR1comp87777_c0114.0797.97
HDR2comp509208_c00.000.91
HDR3comp1116482_c00.000.36
* These genes were selected for qRT-PCR.
Table 5. Expression of putative genes in secoiridoid biosynthesis pathways.
Table 5. Expression of putative genes in secoiridoid biosynthesis pathways.
Gene NameUnigeneRPKMs in LeafRPKMs in Root
GPPS1 *comp57663_c047.610.04
GPPS2comp79818_c053.258.76
GES *comp45416_c066.710.06
G10Hcomp95013_c1304.451075.00
G10H *comp59018_c0128.370.15
G10Hcomp67411_c06.0110.68
G10Hcomp84881_c041.3389.38
G10Hcomp74631_c031.3370.85
G10Hcomp64598_c015.0442.75
G10Hcomp42518_c03.0713.76
G10Hcomp89824_c014.2628.73
G10Hcomp67522_c011.4815.46
G10Hcomp92644_c0164.26389.80
G10Hcomp67745_c013.7325.95
G10Hcomp77398_c04.417.13
G10Hcomp51247_c00.002.77
G10Hcomp67165_c016.7827.36
G10Hcomp67799_c00.000.90
G10Hcomp63189_c00.101.55
G10Hcomp76700_c01.032.28
G10Hcomp42518_c03.0713.76
8HGOcomp93669_c0161.972.80
8HGOcomp53753_c181.291.37
8HGOcomp53753_c2121.860.92
8HGOcomp76718_c065.8977.05
8HGOcomp90961_c05.522.34
8HGOcomp92998_c0219.14245.73
SLScomp94595_c0504.6220.96
SLScomp94064_c5368.1150.11
SLScomp84511_c027.9035.43
SLScomp81016_c00.590.46
SLScomp85876_c0318.25345.13
SLScomp67629_c02.533.00
SLScomp54852_c00.081.74
SLScomp55055_c00.562.47
SLScomp61732_c00.752.06
SLScomp93282_c0143.24185.59
SLScomp281520_c00.490.75
SLScomp87446_c022.8339.78
SLScomp41718_c00.094.01
SLScomp167742_c00.431.74
SLScomp94107_c0121.99222.06
SLScomp49781_c00.000.70
SLScomp90874_c014.9524.36
SLScomp212851_c01.230.83
SLScomp73409_c00.000.72
SLScomp87446_c022.8339.78
SLScomp73685_c00.952.11
SLScomp76988_c04.194.57
SLScomp103080_c04.736.98
SLScomp81659_c03.976.59
IScomp85292_c064.520.00
IOcomp84741_c0361.420.00
7-DLGTcomp82018_c065.590.00
7-DLH *comp94064_c5368.1150.11
CYP1comp84741_c0361.420.00
CYP2comp89478_c033.352.23
CYP3comp108293_c010.240.09
CYP4 *comp92783_c127.398.86
CYP5comp80146_c017.023.45
CYP6comp83496_c046.67160.59
CYP7comp94595_c0504.6220.96
CYP8 *comp97650_c029.442.77
CYP9comp90225_c016.821.16
CYP10 *comp80525_c00.0615.35
CYP11comp92026_c060.414.19
CYP12comp68870_c03.0431.70
CYP13comp85931_c059.636.58
CYP14comp79921_c075.7233.66
CYP15 *comp80492_c019.186.02
CYP16 *comp95479_c099.2914.68
CYP17comp90874_c014.9524.36
* These genes were selected for qRT-PCR.
Figure 8. The expression pattern of three selected MEP pathway genes and CYP genes in roots and leaves in G. rigescens. Means ± SE; each qRT-PCR was biologically repeated three times.
Figure 8. The expression pattern of three selected MEP pathway genes and CYP genes in roots and leaves in G. rigescens. Means ± SE; each qRT-PCR was biologically repeated three times.
Monoterpenes are mainly synthesized in the plastid using geranyl diphosphate (GPP) as a precursor [41]. Following the formation of the acyclic terpenoid structural building blocks, terpene synthases act to generate the main terpene carbon skeleton, and the cytochrome P450 (CYP450) superfamily may catalyze these reactions [42]. However, CYP450enzymes form one of the largest gene families, with over 127 plant cytochrome P450-families being described [43]. The number of CYP450s involved in gentiopicroside biosynthesis remains unclear. Most terpenoid-related CYP450s are members of the CYP71clade, a large group that comprises CYP450s involved in the metabolism of specialized compounds [44]. In the G. rigescens transcriptome data, 169 putative CYP450s transcripts were identified that belong to 60 families as dictated by the standard CYP family categories (Table S5), and the majority are CYP716B2 family members (20 unigenes).
In the differential expression analysis, several CYP450 genes were screened out. Some which had Open Reading Frames (ORFs) with a BLASTX score of E-value <10−5, were then verified by RT-PCR and sequencing. Phylogenetic analysis of the deduced protein sequences with P450s from Arabidopsis thaliana revealed that five of them (GrCYP4, GrCYP5, GrCYP11, GrCYP16, and GrCYP17) belong to the CYP71 clan (Figure 9). qRT-PCR results showed that the selected genes GrCYP4, GrCYP8, and GrCYP15 were highly expressed in leaves, however, GrCYP10 and GrCYP16 genes were more abundant in roots (Figure 8).
Figure 9. Phylogenetic analysis of CYP450s from G. rigescens. Amino acid sequences were aligned using the CLUSTALX2 program, and evolutionary distances were calculated using MEGA6 software with the Neighbor-Joining statistical method and Poisson model. The bootstrap replications were set to 1000. The GenBank accession numbers of the sequences are GrCYP1 (KP218047), GrCYP2-1 (KP218048), GrCYP2-2 (KP218049), GrCYP3 (KP218050), GrCYP4 (KP218051), GrCYP5 (KP325125), GrCYP6 (KP218052), GrCYP8 (KP325126), GrCYP9 (KP218053), GrCYP450-10 (KJ829649), GrCYP11 (KP218054), GrCYP13-1 (KP218055), GrCYP13-2 (KP218056), GrCYP15 (KJ829650), GrCYP16-1 (KP218057), GrCYP16-2 (KP218058), and GrCYP17 (KF941188). The sequences of Arabidopsis thaliana come from TAIR (Available online: https://www.arabidopsis.org).
Figure 9. Phylogenetic analysis of CYP450s from G. rigescens. Amino acid sequences were aligned using the CLUSTALX2 program, and evolutionary distances were calculated using MEGA6 software with the Neighbor-Joining statistical method and Poisson model. The bootstrap replications were set to 1000. The GenBank accession numbers of the sequences are GrCYP1 (KP218047), GrCYP2-1 (KP218048), GrCYP2-2 (KP218049), GrCYP3 (KP218050), GrCYP4 (KP218051), GrCYP5 (KP325125), GrCYP6 (KP218052), GrCYP8 (KP325126), GrCYP9 (KP218053), GrCYP450-10 (KJ829649), GrCYP11 (KP218054), GrCYP13-1 (KP218055), GrCYP13-2 (KP218056), GrCYP15 (KJ829650), GrCYP16-1 (KP218057), GrCYP16-2 (KP218058), and GrCYP17 (KF941188). The sequences of Arabidopsis thaliana come from TAIR (Available online: https://www.arabidopsis.org).
In the secoiridoid biosynthesis pathway, IPPs and DMAPPs are condensed into GPP by GPPS, which is then converted to geraniol by GES. Geraniol is catalyzed to 8-oxogeraniol by geraniol 8-oxidase (G8O, also named G10H) [42,45], and then to 8-oxogeranial by 8-hydroxygeraniol oxidoreductase (8HGO, also named 10HGO) [42,45]; 8-oxogeranial is sequentially catalyzed into loganin via several steps including iridoid synthase (IS), IO, 7-deoxyloganetic acid glucosyltransferase (7-DLGT), 7-deoxyloganic acid hydroxylase (DL7H), loganic acid O-methyltransferase (LAMT), and secologanin synthase (SLS) [45,46,47]. In Catharanthus roseus, G10H, SLS, and DL7H were three important enzymes of the monoterpenoid biosynthesis pathway [48,49,50]. In the G. rigescens transcriptome, there were annotated two GrGPPSs, one GrGES, 18 GrG10Hs, six Gr8HGOs, 24 GrSLSs, one Gr7DLH, one GrIO, one GrIS, and one Gr7-DLGT, but no sequence annotated as GrLAMT. Of interest was that GrIO, GrIS, Gr7-DLGT, and GrCYP1 were only expressed in leaves. Differential expression analysis identified five genes GrGPPS1, GrGES, GrG10H, Gr7DLH, and GrCYP1, which were upregulated 10 times more in leaves than in roots (Table 5). Meanwhile, there were three Gr8HGOs, one GrSLS, one GrIS, one Gr7-DLGT, one GrCYP3, and one GrCYP7, whose expression was five times higher in leaves than in roots (Table 5). However, the expression of one GrSLS and one GrCYP10 was downregulated more than five times in leaves compared to roots (Table 5). qRT-PCR results showed that GrGPPS1, GrGES, GrG10H, and Gr7DLH genes were more highly expressed in leaves than in roots (Figure 8), which suggested that secologanin was mainly synthesized in leaves. These results provide further evidence for gentiopicroside synthesis in shoots [6].
2.5. Candidate Transcription Factors Involved in Regulating the Terpenoid Biosynthetic Pathway
TFs play key roles in controlling gene expression [51], and the controlled transcription of biosynthetic genes is one major mechanism regulating secondary metabolite production in plant cells [52,53,54]. The floral terpenoids of snapdragon appear to be derived exclusively from the MEP pathway in plastids, and this pathway controls precursor levels for GPPS, which in turn is transcriptionally regulated [55]. In our G. rigescens unigene dataset, 7176 unigenes were annotated as transcription factors (Table S6), including bHLH (349), AP2-EREBP (172), WRKY (141), MYB (129), bZIP (115), and GRAS (94) family members. Among these, most were expressed in both root and leaf tissues, with 80 showing a significantly higher expression level in leaves than in roots (Table 6, Table S7).
Table 6. Summary of transcription factor unigenes of G. rigescens.
Table 6. Summary of transcription factor unigenes of G. rigescens.
TF FamilyNumber of Genes DetectedUp-Regulated in Leaves (log2(Fold_Change) > 2)Up-Regulated in Roots (log2(Fold_Change) > 2)
HLH349265
AP2-EREBP172204
WRKY141171
MYB12972
bZIP11534
GRAS9471
Total10008017
Members of the WRKY transcription factor family have been shown to regulate secondary metabolism pathways [56]. In Gossypium arboreum, GaWRKY1 regulates sesquiterpene biosynthesis via activation of δ-cadinene synthase (CAD1-A) [57]. In Coptis japonica, the biosynthesis of berberine is controlled by CjWRKY1 [58]. In tomato trichomes, terpene synthase are controlled by SlMYC1 and SlWRKY73 [59]. In Catharanthus roseus, CrWRKY1, a regulator in biosynthesis of terpenoid indole alkaloids, interacts with transcription factors, including ORCA3, CrMYC, and ZCTs, to play a role in determining the root-specific accumulation of serpentine [60,61]. In Nicotiana attenuata, biosynthesis of diterpene glycosides are regulated by WRKY3 and WRKY6 [62]. In leaves of Artemisia annua, AaWRKY1 activated the expression of the majority of artemisinin biosynthetic genes, including AaADS and AaHMGR [63]. In the present analysis, 141 unigenes were annotated as WRKY family transcription factors, of which 17 were more highly expressed in leaves than in roots (Table 6). qRT-PCR results showed that GrWRKY7 genes were more highly expressed in leaves than in roots, while it was the opposite for GrWRKY5 and GrWRKY6 (Figure 10). Thus, GrWRKY7 is a good candidate to study in the regulation of the biosynthesis of gentiopicroside.
Figure 10. The expression pattern of three selected WRKY genes in roots and leaves in G. rigescens. Means ± SE; each qRT-PCR was biologically repeated three times.
Figure 10. The expression pattern of three selected WRKY genes in roots and leaves in G. rigescens. Means ± SE; each qRT-PCR was biologically repeated three times.
3. Experimental Section
3.1. Plant Materials and RNA Isolation
The cultivated variety of G. rigescens was grown in pots with humus soil and yellow soil mixed in a 1:1 ratio. The fresh roots and leaves were collected from 3-year-old flowering gentian plants in October 2012 (Figure 11). To reduce biological bias, material of three individual plants was pooled to give 1 g of roots and 1 g of leaf samples. All samples were immediately frozen in liquid nitrogen and stored at −80 °C.
Total RNA of each sample (three plants mixed) was isolated by Illumina TruSeq™ RNA Sample Preparation Kit (RS-122-2001). RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, Westlake Village, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA).
3.2. Transcriptome Sample Preparation for Sequencingc
The construction of a cDNA library and the following sequencing procedures were as in Zhu et al. [64].
Figure 11. Plant materials of G. rigescens. (A) G. rigescens used in this article; (B,C) are roots and leaves used in experiments for sequencing.
Figure 11. Plant materials of G. rigescens. (A) G. rigescens used in this article; (B,C) are roots and leaves used in experiments for sequencing.
3.3. Data Filtering
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts (available upon request). In this step, clean reads were obtained by standard quality control criteria to remove all of the reads which meet any one of the following parameters: (1) The reads that aligned to adaptors with no more than two mismatches; (2) The reads with more than 10% unknown bases (N bases); (3) The reads with more than 50% of low-quality bases (quality value ≤ 5) in one read. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data was calculated. All the downstream analyses were based on clean data with a high quality of sequencing.
3.4. Transcriptome Assembly and Contamination Sequences Filtering
As there are few reference sequences available for Gentianaceae, the reads for unigenes of both root and leaf were assembled together. The left files (read1 files) from all libraries/samples were pooled into one left.fq file, and right files (read2 files) into one right.fq file, both in FastQ format. Transcriptome assembly was accomplished based on the pooled paired-end reads files (left.fq and right.fq) using Trinity software (Version 2012-10-05) [20] with min_k-mer_cov set to 2 and all other parameters settings as default. The following processes are referred to Shu et al. [65].
Contaminant sequence level was investigated according to species distribution based on protein similarity searching against NR protein databases. Coding sequences from Non-land-plant species were identified and discarded using a previously described taxonomy-based method [66]. Contaminant sequence from major plant pathogens, and human and other microorganisms (including bacteria, virus, and fungi) was investigated using the stand-alone version of DeconSeq [67].
3.5. Gene Functional Annotation
To assign putative gene function, unigenes were searched against the NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), TAIR10, PFAM (Protein family; Available online: http://pfam.sanger.ac.uk/), and Swiss-Prot (A manually annotated and reviewed protein sequence database; Available online: http://www.ebi.ac.uk/uniprot/) databases using BLAST software with an E-value cutoff of 10−5 [68]. Hmmerscan was adopted for PFAM annotation, and Blast2GO was used for GO annotation (Gene Ontology; Available online: http://www.geneontology.org/) [69] with the same E-value. To evaluate the completeness of the library and the efficacy of the annotation process, the annotated sequences were searched for the possible functions involved in KOG/COG (Available online: http://www.ncbi.nlm.nih.gov/COG/) classifications. To determine which pathways are active in leaves and roots, the annotated sequences were mapped to the reference pathways in KOG/COG, KO (KEGG Ortholog database; Available online: http://www.genome.jp/kegg/).
3.6. Differential Expression Analysis
The calculation of unigene expression used the RPKM method (Reads per kb per Million reads) [70]. Gene expression levels were estimated by RSEM [71] for each sample: (1) Clean data were mapped back onto the assembled transcriptome; (2) Readcount for each gene was obtained from the mapping results.
Differential expression analysis and GO enrichment analysis of leaves vs. roots was referred to Lv et al. [72]. To figure out the transcription factor families existing in leaves and roots, the transcript sequences were aligned against the Plant Transcription Factor Database with BLASTX and a cutoff of E-value < 10−6 [73].
3.7. KEGG Enrichment Analysis
KEGG pathway enrichment analysis of the DEGs was done using KOBAS [74].
3.8. Real-Time PCR Analysis
DNase I-treated total RNA of root and leaf was converted into first-strand cDNA by the use of PrimeScript RTase (Takara, Tokyo, Japan). qRT-PCRs were performed in an ABI7000 Fluorescence Quantitative PCR Instrument (Applied Biosystems, Foster City, CA, USA) using a SuperReal PreMix Plus Kit (Tiangen, China). The PCR condition was: 95 °C for 3 min; 95 °C for 15 s; 60 °C for 30 s. Each reaction was repeated three times. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the internal reference gene. The 2−ΔΔCt method was adopted for the relative gene expression. The primers used are listed in Table S8.
4. Conclusions
Next generation sequencing of RNA has now replaced microarrays as the preferred method for gene expression profiling. One key advantage of this method is that it enables examination of the transcriptome of non-model organisms [75]. Despite the Chinese traditional herb G. rigescens being used for thousands of years, the biosynthesis pathway and regulation of its main effective component, gentiopicroside, remains unknown. Few genetic or genomic studies have been performed. The results presented here addresses this by using the Illumina Hiseq2000 platform to identify sequences and transcript abundance levels of genes expressed in developing roots and leaves of G. rigescens. These sequences provide a starting point for further investigation of gentiopicroside biosynthesis, and include the 3306 unigenes from diverse pathways that were differentially expressed between root and leaf. The results represent a genetic resource for G. rigescens, and may serve as the foundation for further genomic research on G. rigescens and its relatives.
Supplementary Materials
Supplementary materials can be found at https://www.mdpi.com/1422-0067/16/05/11550/s1.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (81260608), Key Project of Education Ministry of Yunnan Province (2013Z075) and National Key Technology R&D Program of China (2011BAI13B02-04). Thanks to the Beijing Novogene Bioinformatics Technology Company for carrying out the sequencing of the transcriptomes.
Author Contributions
Conception and design of experiments: Xiaodong Zhang and Andrew C. Allan. Performing the experiments: Xiaodong Zhang and Caixia Li. Analysis of data: Xiaodong Zhang, Caixia Li and Qiuyang Yao. Contribution of reagents/materials/analysis tools: Yuanzhong Wang. Preparing the manuscript: Xiaodong Zhang and Andrew C. Allan.
Conflicts of Interest
The authors declare no conflict of interest.
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