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39057269_p26
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39057269
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sec[2]/sec[2]/p[1]
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3.3. Habitat Preferences and Environmental Influences on Blowfly Species Distribution
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biomedical
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Study
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Further analysis identified specific habitat preferences among the blowfly species ( Table 3 ). Five species demonstrated a preference towards urban habitats, with C. calliphoroides and L. sericata showing a significantly higher preference than that of other species ( p < 0.001). Additionally, Calliphora vicina favored urban settings, albeit without statistical significance. Conversely, three species exhibited a preference for forest habitats, with L. porphyrina and Ch. pinguis displaying a marked inclination towards these environments ( p < 0.001). Calliphora nigribarbis did not exhibit a clear preference for either habitat type.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
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en
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39057269_p27
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39057269
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sec[2]/sec[2]/p[2]
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3.3. Habitat Preferences and Environmental Influences on Blowfly Species Distribution
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biomedical
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The distribution of blowflies is clearly reflected in the PCA, which is related to environmental influences such as season and habitat . The PCA (PC1 and PC2 accounting for 77.1% of the variation) showed that spring and autumn contrasted clearly with summer. Species belonging to the genus Calliphora were found to be associated with spring and autumn, while Ch. pinguis , Lucilia caesar , and L. illustris were generally associated with summer. The vectors for Gimhae and Tongyeong were found to have no significant explanatory power compared to the other vectors.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
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en
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39057269
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4. Discussion
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This research presents the first comprehensive analysis of blowfly communities within the Gyeongsangnam-do region. This study successfully identified 14 distinct blowfly species, with L. porphyrina (37.2%), Ch. pinguis (27.6%), L. sericata (7.6%), and L. illustris (7.1%) emerging as the predominant species. Notably, the composition of the blowfly community did not exhibit significant variations between different sites within the region. However, seasonal distinctions were apparent, with community composition differing between every season apart from winter. This seasonal variation in community composition underscores the influence of climatic factors on blowfly activity and diversity. Upon analyzing climatic factors, we found no significant differences in the annual average daily temperatures among the four study regions. This suggests that the cause affecting the distribution of blowflies in the region is not the difference in daily average temperature. However, significant differences in average temperature between seasons were confirmed. This supports the findings of this study that the seasonal division of the four regions was appropriately differentiated according to daily average temperature and that the distribution of blowflies is more influenced by seasonal factors governing different environmental conditions.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
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en
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4. Discussion
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We analyzed the distribution of blowflies by considering both species diversity and species richness to better understand their response to ecological and environmental changes in the study area. In all regions, species diversity decreased during the summer, which may be due to the dominance of L. porphyrina and Ch. pinguis , which accounted for a significant proportion of the total individuals collected during this period. Such a high prevalence of these two species in the summer suggests they have a possible competitive advantage or preference for the environmental conditions present during this season.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
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39057269_p30
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39057269
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4.1. Ecological Niches and Seasonal Distribution of Forensic Blowflies
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Our findings reveal a distinct season-dependent prevalence of the genus Calliphora , which was more abundant in spring and autumn than in summer. Specifically, C. calliphoroides (also known as Triceratopyga calliphoroides ) was absent during summer and winter, and its highest relative abundance occurred in autumn. This pattern underscores its pronounced seasonal preference. The genus Calliphora is typically found colonizing remains in significant numbers during the cool seasons, such as autumn, winter, and spring, with the exact timing varying by geographic location . In Korea, Calliphora species have been frequently observed on carcasses in spring and autumn . Forensic autopsy data obtained in Korea corroborated our findings, with species such as C. calliphoroides , C. grahami , C. nigribarbis , and C. vicina predominantly identified in these seasons , highlighting their forensic significance in determining the mPMI.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
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en
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39057269_p31
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39057269
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4.1. Ecological Niches and Seasonal Distribution of Forensic Blowflies
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The genus Lucilia was present in spring, summer, and autumn, with the most abundant during the summer. Lucilia porphyrina , the dominant species throughout Gyeongsangnam-do, exhibited limited abundance in metropolitan autopsies . Notably, L. porphyrina thrives in Southeast Asia and tropical and subtropical mountainous regions with higher altitudes , suggesting that geographical and latitudinal differences may influence its distribution. Other Lucilia species mirrored the seasonal pattern of L. porphyrina , with L. sericata being particularly prevalent in domestic autopsies, underscoring its forensic importance in these cases .
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
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https://doi.org/10.3390/insects15070536
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N/A
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https://creativecommons.org/licenses/by/4.0/
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en
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39057269_p32
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39057269
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4.1. Ecological Niches and Seasonal Distribution of Forensic Blowflies
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Within the genus Chrysomya , both Ch. pinguis and Ch. megacephala were collected, but their abundances varied significantly. Chrysomya pinguis emerged as the second most abundant species, exhibiting a strong association with summer. Although Ch. megacephala is globally recognized as forensically significant , it constituted only 0.4% of the total population in this study. Chrysomya pinguis has a notable presence across various Asian countries and is a key forensic species in Korea that is frequently found on carcasses and in autopsies across spring, summer, and autumn , which is further supported by its high abundance in this study. The differential abundances of Ch. pinguis and Ch. megacephala may result from ecological niche differentiation, with potential interspecific competition for limited resources . This competition might lead to Ch. megacephala being ecologically displaced by Ch. pinguis . Alternatively, Yang and Shiao proposed that temperature differentiation might influence the ecological niches of these species, but our study did not obtain constant abundance data to fully elucidate seasonal distribution differences only for those species.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
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N/A
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https://creativecommons.org/licenses/by/4.0/
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en
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39057269_p33
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39057269
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sec[3]/sec[1]/p[0]
|
4.2. Ecological Niches and Habitat Preferences of Forensic Blowflies
| 4.171875 |
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In this study, species within the Calliphora genus exhibited a general preference for urban habitats. Calliphora calliphoroides and C. grahami demonstrated a clear bias toward urban environments, while C. vicina was more frequently found in urban habitats, albeit without statistical significance. Despite C. vicina ’s known association with urban habitats, regional and environmental variations may influence its lifecycle dynamics . Altitude has been documented to affect blowflies’ seasonal distribution and oviposition behavior . Although no significant habitat preference was noted for C. nigribarbis in this study, previous research indicates its tendency to migrate to higher altitudes during summer for breeding, returning to lower altitudes in autumn . In addition, Kimura discussed the migration patterns of C. nigribarbis and C. grahami in the context of univoltine life cycles, distinguishing between upward and downward migration, emphasizing the possibility of diapause-associated migration of low-altitude individuals. In particular, only some of the high-altitude individuals were moving at low altitudes; therefore, whether this migration can be classified as dispersion remains controversial. The similarities between migration and dispersion complicate identifying a clear definition, underscoring the need for further research on these species’ elevation-based life cycles and migration behaviors.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
39057269_p34
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39057269
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sec[3]/sec[1]/p[1]
|
4.2. Ecological Niches and Habitat Preferences of Forensic Blowflies
| 4.074219 |
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The Lucilia genus displayed varied habitat preferences among species. Lucilia caesar and L. porphyrina showed a predilection for forested habitats, while L. sericata and L. illustris favored urban settings. Lucilia porphyrina ’s migration from the highlands to the lowlands for hibernation, followed by returning to the highlands for oviposition in summer , may explain its forest-biased habitat preference, as its lifecycle is predominantly spent in woodlands during summer. Here, L. sericata exhibited a strong preference for urban habitats and is known as an urban species with human affinities; it has been found in many autopsies in domestic indoor spaces . Lucilia caesar and L. illustris are known to be morphologically similar sister species ; the habitat distribution in this study showed conflicting results. Lucilia caesar is generally known to be abundant in shady and sparsely populated forested habitats, where its ecology is influenced by low temperature and high humidity factors . These habitat differences may be related to niche partitioning. As carcasses are a limited and transient resource, carrion ecosystems are characterized by intense resource competition . Therefore, it is possible that the two species have diverged to adapt to different habitats to minimize direct competition.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057269_p35
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39057269
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sec[3]/sec[1]/p[2]
|
4.2. Ecological Niches and Habitat Preferences of Forensic Blowflies
| 2.224609 |
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Chrysomya pinguis exhibited a preference for forested habitats, aligning with observations of its prevalence on corpses in less densely populated regions and its abundance in mountainous areas during summer . This species likely dominates high-land or forest habitats in summer, with potential spring and autumn migrations to low-land areas, possibly related to the diapause-associated migration behaviors described for C. nigribarbis and C. grahami .
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
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en
| 0.999996 |
39057269_p36
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39057269
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4.3. Ecological Insights and Forensic Dynamics of Blowflies
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This year-long investigation in Gyeongsangnam-do, South Korea, yielded fundamental forensic entomological data, laying the groundwork for enhancing forensic case studies in this region. The species cataloged in this research have been observed in both domestic animal carcasses and human autopsies, affirming their forensic relevance within South Korea . The local blowfly data obtained by this study underscores the necessity for a comprehensive nationwide blowfly distribution dataset for refining forensic insect estimation techniques in future investigations.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057269_p37
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39057269
|
sec[3]/sec[2]/p[1]
|
4.3. Ecological Insights and Forensic Dynamics of Blowflies
| 4.109375 |
biomedical
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Study
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This study was limited to blowfly colonization during the early stages of carcass decomposition because the bait used to replace the odor of the decomposing carcass was only allowed to colonize for two days after the onset of decomposition. Despite this limitation, our findings support the hypothesis that different blowfly species exhibit distinct habitat preferences, as well as specific seasonal and geographical distribution patterns. These observed patterns are instrumental for forensic entomologists to determine the geographical origin of insects found on a corpse, assess potential corpse movement, and deduce the location of the crime. Understanding the ecological niches and behavior of blowfly species can significantly enhance the utility of forensic entomology in criminal investigations. By recognizing species-specific habitat preferences and their seasonal and geographical distribution, forensic entomologists can draw inferences about the environmental conditions surrounding a crime scene . This information can help identify the likely locations where a body may have been moved from or to, providing critical evidence in crime scene reconstruction .
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057269_p38
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39057269
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sec[3]/sec[2]/p[2]
|
4.3. Ecological Insights and Forensic Dynamics of Blowflies
| 3.75 |
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It is important to recognize that using mouse cadavers cannot perfectly replicate the condition of human remains. The differences between small mammals, which are typically hairy, and the larger human body, which has less body hair and is often clothed, complicate direct comparisons of research results to actual forensic cases. Nevertheless, insects collected from experiments using animal carcasses have also been found during human autopsies . Utilizing animal carcasses for research allows for stringent control of experimental conditions and the repetition of experiments with fewer ethical and legal constraints. Therefore, studies involving animal carcasses can sufficiently support the findings obtained from human cadaver studies.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057269_p39
|
39057269
|
sec[3]/sec[2]/p[3]
|
4.3. Ecological Insights and Forensic Dynamics of Blowflies
| 4.023438 |
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The data from this study also highlight the importance of localized research in building a robust forensic framework. The specific blowfly species identified, as well as their patterns of colonization, offer valuable insights into regional biodiversity and ecological dynamics. Detailed regional studies are crucial for developing accurate forensic models that can be applied to various scenarios, ultimately improving the precision of forensic analyses. Beyond immediate forensic applications, the ecological information gained from this study contributes to a broader understanding of blowfly ecology and their role in the decomposition process. The observed habitat preferences and distribution patterns can inform future studies on blowfly behavior and their interactions with the environment, which is vital for both forensic science and ecological research.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
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5. Conclusions
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This investigation provides essential data on the spatio-temporal distribution of blowfly species in Gyeongsangnam-do, South Korea, emphasizing the need for comprehensive regional datasets. The findings highlight the significance of species diversity, habitat preferences, and ecological niches in forensic entomology, offering valuable insights for crime scene investigations and advancing the field’s scientific foundation. Future research should focus on expanding the dataset to include various stages of decomposition and different environmental conditions to further refine forensic entomological methods and applications.
|
[
"Hyeon-Seok Oh",
"In-Seong Baek",
"Min-Gyu Kang",
"Sang-Hyun Park"
] |
https://doi.org/10.3390/insects15070536
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
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PMC11277061_p0
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PMC11277061
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1. Introduction
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Mycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis (TB), a bacterial disease that claimed about 1.5 million lives worldwide in 2022 . On infection, Mtb is phagocytosed by the sentinel cells of the host, macrophages. These cells are equipped with cell autonomous innate immune responses, which act as front-line defenders against Mtb infection . Activated macrophages upregulate various antimicrobial pathways including the expression of inducible nitric oxide synthase (INOS), phagocyte NADPH oxidase (NOX2/gp91 phox ) and xanthine oxidase system that becomes recruited to the Mtb phagosome and generates antimicrobial reactive nitrogen and oxygen species (ROS and RNS) via a respiratory burst to kill Mtb . Mice deficient in both phox and iNOS are highly susceptible to Mtb infection, which shows that RNS and ROS generation is required by the host for protection against Mtb . These molecules alter the intracellular signaling events, triggering the host antimicrobial responses that primarily aim to eliminate Mtb . The host cell ROS and RNS interacts directly with its bacterial targets and during the process, becomes converted into chemically district oxidants such as hydrogen peroxide (H 2 O 2 ), hypochlorites (HClO), peroxynitrite (ONOO − ) and hydroxy radicals . These reactive species damage Mtb DNA, oxidize lipids and proteins, including highly sensitive proteins such as iron–sulfur (4Fe-4S) cluster proteins . Thus, Mtb infection prompts macrophages to generate ROS and RNS as a mechanism to kill intracellular bacteria . The ROS and RNS production by Mtb -infected macrophages, as part of host cell-autonomous innate immune response, not only play a role in direct host defense against the bacteria, but also trigger signaling pathways that contribute to the overall antimicrobial responses of these immune cells .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
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PMC11277061_p1
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PMC11277061
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1. Introduction
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The survival of Mtb in the host not only depends on the manipulation of the host but also on its ability to encounter stresses imposed by immunologically active macrophages. Mtb employs several detoxifying antioxidant thiols and enzyme systems, such as superoxide dismutase (SOD), catalase-peroxidase, alkylhydroperoxidase (AhpC), and thioredoxin reductase . However, the gene encoding global stress regulator oxyR , commonly found in several other bacteria, is absent in Mtb . The absence of oxyR in Mtb is suggested to be compensated by the presence of several AhpC, which help the bacteria tolerate ROS and RNS induced by the infected host cells . In addition to AhpC, recent research has revealed that Mtb possesses a category of enzymes known as short-chain dehydrogenase/reductases (SDRs), which aid in managing peroxidase stress, in vitro detoxification as well as in vivo survival of Mtb , drug resistance and overall homeostasis . The SDRs are one of the largest protein families, distributed widely in several organisms and classified into seven families such as classical, atypical, extended, intermediate, divergent, complex and unassigned . SDRs can utilize a range of diverse substrates, such as alcohols, sugars and aromatic compounds . SDRs represent various enzyme classes such as isomerases, lyases and oxidoreductases, although the majority of the SDRs are oxidoreductases . These SDRs share an NAD(P) + Rossmann-fold domain for substrate binding and use NADH or NADPH as its electron donors . Moreover, SDRs are involved in metabolic pathways such as detoxification, drug resistance, steroid metabolism, and biosynthetic pathways of bacteria . In the context of Mtb infection, specific SDRs have been implicated in modulating the activation or inactivation of certain drugs used in TB treatment, highlighting the involvement of SDRs in bacterial physiology and survival . The genome of Mtb possess 23 SDRs, namely Rv0148, Rv0484c, Rv0687, Rv0851c, Rv0927c, Rv0945, Rv1144, Rv1245c, Rv1865c, Rv1882c, Rv1928c, Rv1941, Rv2214c, Rv2509, Rv2766c, Rv2857c, Rv3085, Rv3174, Rv3224, Rv3485c, Rv3502c, Rv3548c and Rv3549c (as listed in Mycobrowser) and only a few of these are characterized .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
PMC11277061_p2
|
PMC11277061
|
sec[0]/p[2]
|
1. Introduction
| 4.316406 |
biomedical
|
Study
|
[
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[
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Strikingly, a transposon mutant of Rv0687, SDR has also shown to be attenuated for growth in both mice and primate models using the DeADMAn approach (designer arrays for defined mutant analysis) . Though, the DeADMAn approach has limitations such as the polar effect due to the inactivation of downstream genes in operon. Therefore, we investigated the role of Rv0687 in Mtb pathogenesis and survival. To avoid any polar effects that leads to inactivation of downstream genes, this study involves generating precise knockout in-frame deletions using the strategy described by Jain et al. 2014 . Rv0687 has homologs in other mycobacteria that are characterized as mycofactocin-associated dehydrogenases with non-exchangeable NAD-cofactors and as a putative carveol dehydrogenase . Notably, Rv0687 is a non-essential in the Mtb genome, with a gene length of 828 base pairs, encoding a 275 amino acid protein with a molecular mass of 28 kDa. In the current study, we generated a knockout mutant of Rv0687 using specialized transduction and characterized the phenotype of the mutant in comparison with the wildtype (WT) and complemented Mtb strains . Our results demonstrate that Mtb requires Rv0687 in vitro to cope up with the oxidative stress, nitrosative stress and for survival as well as growth in the mouse bone marrow-derived macrophage (BMDM) infection assays. Moreover, it is required by Mtb for survival and proliferation in vivo in immunocompetent and immunocompromised mice models of infection.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277061_p3
|
PMC11277061
|
sec[1]/sec[0]/p[0]
|
2.1. Growth Kinetics of RvΔ0687 in Broth Media
| 4.113281 |
biomedical
|
Study
|
[
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[
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RvΔ0687 deletion strain and its complemented strains were generated and confirmed as mentioned in the Section 4 . To assess the role of Rv0687 in Mtb survival WT, RvΔ0687 and C-Rv0687 strains were grown in both 7H9 complete and 7H9 dextrose media lacking OADC, and their survival was monitored via CFU assay for 12 days. In 7H9 complete media, the survival of RvΔ0687 showed no significant differences compared to the WT and C-Rv0687 at any of the tested time points . Whereas in 7H9-dextrose media, all the strains were able to sustain without any change in survival until day 4, followed by a gradual decline in CFUs, which is not statistically significant at day 8. However, no CFUs were obtained for the RvΔ0687 at day 12, whereas WT and C-Rv0687 were able to persist in the 7H9 dextrose media and form CFUs . The 7H9 dextrose media showed the difference in growth response among the Mtb strains.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999999 |
PMC11277061_p4
|
PMC11277061
|
sec[1]/sec[1]/p[0]
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2.2. RvΔ0687 Is Sensitive to ROS and Nitrite Stress Generating Agents
| 4.1875 |
biomedical
|
Study
|
[
0.99951171875,
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[
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As we have seen differences in growth response among the Mtb strains in 7H9 dextrose media, we evaluated the response of WT, RvΔ0687 and C-Rv0687 to in vitro ROS using H 2 O 2 and tBooth. In our pilot experiment , we used 7H9 complete media supplemented with glycerol, tyloxapol and OADC, treated with 5 mM H 2 O 2 to perform the stress response studies, and we did not observe significant growth defect between the WT, RvΔ0687 and C-Rv0687 strains. However, the RvΔ0687 showed sensitivity to exposure of 5 mM and 10 mM H 2 O 2 as determined by the reduction in CFUs, compared to the WT and C-Rv0687 in 7H9 dextrose media lacking catalase. At 5 mM H 2 O 2 exposure, a significant reduction in RvΔ0687 CFUs was observed only at 96 h, whereas at 10 mM H 2 O 2 exposure, at 24 h, a decrease in CFUs was observed, but at 48 h, the CFUs of RvΔ0687 dropped by 2 logs compared to WT and C-Rv0687 strains. Furthermore, RvΔ0687 was so susceptible to H 2 O 2 stress that we recovered no CFUs at 96 h post-treatment compared to the WT and C-Rv0687 . In addition, the RvΔ0687 was sensitive to 2 mM tBOOH at 24 h, with significantly declined CFUs compared to WT and the C-Rv0687 strains. However, none of the strains were able to sustain their growth after 48 h, as observed by the lack of CFUs in the presence of 2 mM tBOOH . The nitrite stress response was assessed using 2 mM SNAP and 2 or 10 mM SN. At 72 and 96 h post-treatment, we observed a log decrease in the RvΔ0687 compared to WT and the C-Rv0687 strains in response to 2 mM SNAP treatment . However, neither of the tested bacterial strains showed any difference in CFUs on treatment with 2 or 10 mM SN or in response to metal ions (copper, zinc, and iron) stress at any of the tested time points after post-treatment .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p5
|
PMC11277061
|
sec[1]/sec[2]/p[0]
|
2.3. Deletion of Rv0687 Gene Does Not Impact the Mtb NADH, and NAD + and NADH/NAD + Ratio In Vitro
| 4.054688 |
biomedical
|
Study
|
[
0.99951171875,
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The NADH and NAD + ratios have a crucial role in the redox homeostasis of a bacterial cell. Consequently, we measured the concentrations of NADH and NAD + as well as the NADH/NAD + ratio for the WT, RvΔ0687 and C-Rv0687 strains. Based on our observations, it is evident that the deletion of Rv0687 has no significant impact on modulating the NADH or NAD + levels and redox homeostasis of Mtb in aerobic conditions.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277061_p6
|
PMC11277061
|
sec[1]/sec[2]/p[1]
|
2.3. Deletion of Rv0687 Gene Does Not Impact the Mtb NADH, and NAD + and NADH/NAD + Ratio In Vitro
| 4.097656 |
biomedical
|
Study
|
[
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The WT, RvΔ0687 and C-Rv0687 were grown to 10 7 bacteria and the cells were pelleted and lysed using NADH extraction buffer. NAD/NADH were extracted using 10 kD spin column and the resulting concentrations (pmol) were estimated at 1, 2, 3 and 4 h by measuring the absorbance at 450 nm after addition of NADH developer. The change in the spectral readings at various timepoints probably represents reactivity of proteins formation of measurable product due to interaction between NADH, NAD + and NADH developer. Based on the readings, the concentration of NADH and NAD + were estimated. Concentration of NAD + was calculated by subtracting NADH from NAD + (represents total conc of NAD + and NADH). In addition, ratio of NADH/NAD + is calculated using the formula NADH/NAD + . The experiment was performed in triplicates with biological samples.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p7
|
PMC11277061
|
sec[1]/sec[3]/p[0]
|
2.4. Deletion of Rv0687 Enhances Susceptibility of Mtb to Antimycobacterial Drugs Delamanid and NMR711
| 4.136719 |
biomedical
|
Study
|
[
0.99951171875,
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[
0.99951171875,
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0.00007206201553344727
] |
To assess the sensitivity of WT, RvΔ0687 mutant and C-Rv0687 strains to Isoniazid (INH), Delamanid (DEL), Rifampicin (RIF), Bedaquiline (BDA) and NMR711, an Alamar Blue assay was performed. The RvΔ0687 displayed increased susceptibility to DEL at concentrations of 0.062–1 mg/L, which is lower than the survival rates observed for the WT and the C-Rv0687 strains . In our previously published studies, we discovered that small compound NMR711 has anti-mycobacterial activity against Mtb at a concentration of 50 mg/L . Interestingly, we observed that RvΔ0687 also displayed reduced survival for NMR711 at tested concentrations of 0.39–50 mg/L compared to WT and the C-Rv0687 strains . Conversely, RvΔ0687 exhibited similar susceptibility to antimycobacterial drugs BDA and RIF as the WT and C-Rv0687 strains .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277061_p8
|
PMC11277061
|
sec[1]/sec[4]/p[0]
|
2.5. RvΔ0687 Is Required for Intracellular Replication and Suppression of Early Inflammatory Immune Response in Macrophages
| 4.144531 |
biomedical
|
Study
|
[
0.99951171875,
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[
0.99951171875,
0.0001653432846069336,
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0.00006151199340820312
] |
The in vitro survival of WT, RvΔ0687 and C-Rv0687 strains were tested using mouse BMDMs. As shown in , no significant difference was observed in the uptake of the WT, RvΔ0687 and C-Rv0687 strains by BMDMs at day 0. Interestingly at day 1, though, we observed small differences in the CFUs between WT and RvΔ0687 strains, but the differences were significant at later stages at day 3, 5 and 7 post infections . We found that Rv0687 is required for the intracellular survival of Mtb , as the deletion mutant of Rv0687 is attenuated for survival in the macrophages. It was noted that there was a slight change in the survival of C-Rv0687 compared to WT at 5 and 7 days post-infection, which is not statistically significant. We further assessed the production of RNS (Griess assay) and ROS (NBT assay) by BMDMs in response to infection with WT, RvΔ0687, and C-Rv0687 strains. Though there was a significant change in RNS and ROS production by WT-infected vs. uninfected BMDMs, we did not observe any significant change in the production of ROS or RNS by BMDMs during infection with either WT, RvΔ0687, and C-Rv0687 strains .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999999 |
PMC11277061_p9
|
PMC11277061
|
sec[1]/sec[4]/p[1]
|
2.5. RvΔ0687 Is Required for Intracellular Replication and Suppression of Early Inflammatory Immune Response in Macrophages
| 4.113281 |
biomedical
|
Study
|
[
0.99951171875,
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[
0.99951171875,
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0.00006014108657836914
] |
Furthermore, we measured the cytokines to determine the activation status of the macrophages. The expression levels of TNF-α, IL-1β, IP-10 and MIP-1α cytokines were analyzed in the WT, RvΔ0687 and C-Rv0687 strains in the cell-free post-infected supernatants of infected BMDMs at 1–5 days post-infection. Within 3 h of infection, compared to uninfected, WT-infected BMDMs showed 5-fold increased secretion of TNF-a, IL-1β and ~10-fold increase in MIP-1a and IP-10. Interestingly, RvΔ0687-infected BMDMs showed a significantly higher expression of TNF-α and MIP-1α at d1 and d5 days compared to WT- and C-Rv0687-infected BMDMs . We have not observed any significant change in the secretion of IL-1β and IP-10 by RvΔ0687-infected BMDMs compared to that of WT- and C-Rv0687-infected BMDMs .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p10
|
PMC11277061
|
sec[1]/sec[5]/p[0]
|
2.6. RvΔ0687 Is Attenuated In Vivo in Immunocompromised and Immunocompetent Mice
| 4.183594 |
biomedical
|
Study
|
[
0.99951171875,
0.0003333091735839844,
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[
0.99951171875,
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] |
The in vivo survival of WT, RvΔ0687 and C-Rv0687 strains were analyzed in immunocompromised (Rag −/− ) and immunocompetent (C57BL/6) mice . Rag −/− mice were infected via aerosol with a dose of 200–250 CFU/mice. Mice were sacrificed at day 0, W4 and W8 to determine the CFU burden in the lungs and spleen. A group of mice infected with each strain were kept for survival studies. At W4 and W8 post-infection, WT, RvΔ0687 and C-Rv0687 showed similar bacterial burden in the lungs of Rag −/− mice , but in spleen at W4 the growth of RvΔ0687 was 8-fold lower than WT and C-Rv0687 strains . Furthermore, we compared the survival of Rag −/− mice after infection with WT, C-Rv0687 or RvΔ0687 strains. Interestingly, the WT or C-Rv0687 infected group started to die between 60 and 70 days post-infection, while the RvΔ0687 infected animals survived until 125 days post-infection . This suggests that RvΔ0687 is impaired in virulence and plays a significant role in the in vivo survival of Mtb in Rag −/− mice.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p11
|
PMC11277061
|
sec[1]/sec[5]/p[1]
|
2.6. RvΔ0687 Is Attenuated In Vivo in Immunocompromised and Immunocompetent Mice
| 4.109375 |
biomedical
|
Study
|
[
0.99951171875,
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[
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To further evaluate the role of Rv0687 in Mtb pathogenesis in vivo, we infected C57BL/6 mice and euthanized them at 4 and 8 weeks post-infection. Lungs and spleen were harvested, plated and CFUs were enumerated. In lungs, though at W4 CFUs were similar for WT, RvΔ0687 and C-Rv0687 strains, differences were observed during late infection at W8 and enumerated CFUs for RvΔ0687 were 3-fold lower than WT or C-Rv0687 . Furthermore, in spleen, compared to WT or C-Rv0687 infected mice at W4 and W8, RvΔ0687-infected mice displayed ~4-fold or 2.5-fold lower CFU’s, respectively .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p12
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PMC11277061
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sec[2]/p[0]
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3. Discussion
| 4.25 |
biomedical
|
Study
|
[
0.99951171875,
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] |
[
0.9990234375,
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In this study, we investigated the functional role of Mtb Rv0687 that codes for a putative short-chain dehydrogenase. In bacteria including Mtb , it is reported that short-chain dehydrogenase/reductases (SDRs) aid in managing peroxidase stress, detoxification, in vitro, in vivo survival, drug resistance and overall homeostasis . Better understanding of role of these SDR’s in Mtb pathogenesis will lead to the development of drugs and therapeutics in future to combat tuberculosis (TB). To understand the functional role of Mtb Rv0687, we created a gene knockout mutant of Rv0687 , its complemented strain and characterized the role of Rv0687 in Mtb survival under various stress conditions including, oxidative, nitrite and drug-mediated stress. We also investigated the function of Rv0687 in Mtb infection in BMDMs and mice models. Overall, our study suggests that Rv0687 has a substantial role in Mtb pathogenesis in combating oxidative and nitrite stress, drug tolerance and host immunity and is indispensable for pathogenicity of Mtb in macrophages and mice models of infection.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p13
|
PMC11277061
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sec[2]/p[1]
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3. Discussion
| 4.28125 |
biomedical
|
Study
|
[
0.99951171875,
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[
0.9990234375,
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Bacterial growth and survival are influenced by the composition of the growth media . The OADC supplement is commonly added to Mtb growth media. Since Mtb growth can produce toxic by-products, such as ROS through metabolic processes, the presence of OADC (catalase) aids in neutralizing these toxic compounds by catalyzing the decomposition of hydrogen peroxide. This enzymatic activity supports better bacterial growth in OADC-containing media . While performing survival kinetics, we observed that in nutrient-rich conditions and in the presence of catalase, the growth of RvΔ0687 was indistinguishable from that of WT and C-Rv0687 under in vitro conditions. We further used 7H9 media supplemented with glycerol, tyloxapol and 0.2% dextrose (7H9-dextrose) and found that WT, RvΔ0687 and C-Rv0687 strains were able to survive normally until day 8 and declined gradually until day 12, suggesting the susceptibility of the strain to media lacking the catalase and BSA . Taken together, our findings highlight the contribution of the Rv0687 gene in the adaptation of Mtb to grow under stress conditions.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277061_p14
|
PMC11277061
|
sec[2]/p[2]
|
3. Discussion
| 4.269531 |
biomedical
|
Study
|
[
0.99951171875,
0.0003287792205810547,
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[
0.99951171875,
0.0002856254577636719,
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0.00009834766387939453
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Since tolerance to ROS and nitrite stress plays an essential role in bacterial pathogenesis, we further investigated the response of Mtb strains against stress-generating agents . In our pilot experiment , we used 7H9 complete media supplemented with glycerol, tyloxapol and OADC, treated with 5 mM H 2 O 2 to perform the stress response studies, and we did not observe significant growth defect between the WT, RvΔ0687 and C-Rv0687 strains. In contrast, when we tested the phenotype of the Mtb strains in 7H9 dextrose media lacking OADC, we found that the RvΔ0687 was more sensitive to ROS-generating agents, as evidenced by a decline in its growth compared to WT and C-Rv0687 . To conclude, the failure of RvΔ0687 growth might be due to enhanced oxidative stress mediated due to the absence of catalase for bacterial survival. Our results showed that RvΔ0687 is more sensitive to ROS (H 2 O 2 and tBOOH). Our results are further supported by previous microarray studies that shows that Rv0687 is upregulated on Mtb treatment with H 2 O 2 . Like Mtb sigJ, mel2 and sod that are known to play a role in providing resistance against ROS and Mtb pathogenesis , Rv0687 might also help Mtb in coping up the ROS and is required for Mtb pathogenesis.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277061_p15
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PMC11277061
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sec[2]/p[3]
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3. Discussion
| 4.550781 |
biomedical
|
Study
|
[
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[
0.99853515625,
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0.00018465518951416016
] |
In addition, we investigated the response of RvΔ0687 to nitrite stress by treating bacterial cultures with SNAP and SN. The increased sensitivity of RvΔ0687 to SNAP treatment suggests that the Rv0687 gene may play a role in Mtb defense against nitrosative stress induced by SNAP, which is a nitric oxide donor, as has been shown for the gene knockout mutant of Rv2617c of Mtb involved in virulence . Our findings also suggest that RvΔ0687 can tolerate exposure to SN up to 10 mM concentrations; this was like the findings of a study by Suwarna et al., which showed that Mtb can resist and survive in the presence of a nitrite concentration up to 10 mM in broth media . Furthermore, other possible explanation is that we have not observed any effect of sodium nitrite on the viability of the bacterial strains as we used sodium nitrite at physiological pH, as a donor of reactive nitrogen species (RNS), and it dissociates into reactive nitrogen species at acidic pH ; therefore, the acidification of growth media prior to NaNO2 addition would have been better for RNS studies. Additionally, we tested the response of RvΔ0687 to exposure to transitional metals such as copper, zinc and iron, which can induce oxidation reactions. However, we could not find a significant difference in the survival between the RvΔ0687 and WT or complemented strains. The Mtb , being an intracellular pathogen, grows inside the phagosomes of macrophages, where oxygen and nitrogen intermediates are produced; having such evasion mechanisms would enable Mtb to survive under stress conditions. SDRs in Mtb are involved in regulating peroxidase stress and detoxification. Interestingly, a homolog of Rv0687 in other mycobacterial species is characterized as a mycofactocin-associated dehydrogenase with non-exchangeable NAD-cofactors . The homolog of Rv0687 in M. paratuberculosis is a putative carveol dehydrogenase, and its molecular structure has been reported. Accordingly, carveol has been shown to have an antioxidant role . This suggests that Rv0687 might also be involved in redox reactions or synthesis of cellular metabolites that can play an important role in the detoxification of ROS or RNS in Mtb and can be further explored in the future. Therefore, understanding the specific mechanisms by which the Rv0687 gene confers protection against ROS and RNS could provide valuable insights into the bacterial stress response relating to the pathogenesis of Mtb .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999999 |
PMC11277061_p16
|
PMC11277061
|
sec[2]/p[4]
|
3. Discussion
| 4.34375 |
biomedical
|
Study
|
[
0.99951171875,
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] |
[
0.9990234375,
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] |
We also investigated the impact of Rv0687 on the redox homeostasis of Mtb . In general, redox homeostasis, involving the balance between reduced NADH and oxidized NAD + , plays a key role in maintaining cellular redox equilibrium, which is crucial for bacterial survival and adaptation to various environmental stresses, including oxidative and nitrosative stresses. . Hence, any disruption in redox homeostasis can have a significant impact on bacterial physiology and survival . We found that the NADH and NAD + concentrations, as well as the NADH/NAD + ratio, were comparable between RvΔ0687 and the WT and C-Rv0687 strains. These results were consistent with the phenotype of the DosR regulon mutant of Mtb known to assist Mtb in metabolic homeostasis and recovery from dormancy, but the DosR mutant of Mtb exhibited similar NADH and NAD + levels as the WT during aerobic growth conditions at early time points. There is a possibility of plasticity in SDRs that can compensate for the function of Rv0687, as it has been shown that deletion of a single succinate dehydrogenase (sdhA) gene, which is an SDR, does not have major redox potential in Mtb . These observations suggest that Rv0687 might not be directly involved in regulating the balance between NADH and NAD + in Mtb under the conditions tested. This could be due to the presence of redundant or alternative SDR enzymes that ensure the stability of the NADH/NAD + ratio in the absence of RvΔ0687 under the tested conditions. Further research is required to elucidate the precise conditions in which Rv0687 might play direct or indirect role in balancing NADH/NAD + ratio by interacting with other redox-related components of Mtb .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p17
|
PMC11277061
|
sec[2]/p[5]
|
3. Discussion
| 4.25 |
biomedical
|
Study
|
[
0.99951171875,
0.0003437995910644531,
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] |
[
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] |
Rv0148 is an SDR of Mtb and the deletion mutant of Rv0148 has been associated with the sensitivity to antibiotics . Therefore, we assessed the sensitivity of WT, RvΔ0687 and C-Rv0687 strains against anti- Mtb antibiotics, using a Alamar Blue assay. The results demonstrated distinct drug susceptibility patterns for the RvΔ0687 mutant. Notably, compared to the WT and C-Rv0687 strains, the RvΔ0687 showed increased susceptibility to two drugs: NMR711 and DEL. Conversely, the RvΔ0687 strain exhibited no significantly different sensitivity than the WT and C-Rv0687 strains against INH probably due to interference of Alamar Blue with INH . We also did not observe significant differences in bacterial survival between WT and C-Rv0687 strains against BDA and RIF. This indicates that the Rv0687 plays a role in bacterial susceptibility towards antibiotics, particularly to DEL and NMR711 . It correlates with our unpublished data that Rv0687 was upregulated in Mtb samples treated with NMR711. There is possibility that it might work synergistically with delamanid, a recently approved TB drug but for which additional studies are required. Further studies are also needed to elucidate the underlying mechanisms by which Rv0687 influences the bacterial susceptibility to these drugs.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277061_p18
|
PMC11277061
|
sec[2]/p[6]
|
3. Discussion
| 4.164063 |
biomedical
|
Study
|
[
0.99951171875,
0.0003070831298828125,
0.00019025802612304688
] |
[
0.99951171875,
0.00016891956329345703,
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] |
During Mtb infection, the recognition of the pathogen by macrophages induces changes in the expression of cytokines that impact the elimination or progression of infection . To assess the infection and in vivo survival of WT, RvΔ0687 and C-Rv0687 strains, we used a mouse BMDMs infection model and monitored the intracellular bacterial survival after infection. We observed that RvΔ0687 was defective for intracellular survival compared to WT and C-Rv0687 strains in BMDMs. The current results were consistent with the findings in another SDR and Rv2159c alkylhydroperoxidase of Mtb . The Rv0148 and Rv2159 mutants were defective for intracellular survival, although it induced proinflammatory cytokines in a macrophage model . Differential survival of C-Rv0687 strain might be attributed to increased hypoxia conditions and the use of mycobacterial vector pMV361 for complementing the Rv0687 gene. Compared to the uninfected, we have observed significant changes in the production of ROS and RNS by infected BMDMs though there were no significant changes among WT vs. RvΔ0687 strains.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277061_p19
|
PMC11277061
|
sec[2]/p[7]
|
3. Discussion
| 4.242188 |
biomedical
|
Study
|
[
0.99951171875,
0.0002694129943847656,
0.00016438961029052734
] |
[
0.99951171875,
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] |
Further, Mtb -infected phagocytes generate ROS and RNS, which is required to control or kill Mtb in infected macrophages. Other than its antimicrobial effect, ROS and RNS generated by macrophages have been shown to influence the activation of T cell responses, production of proinflammatory cytokines, induce autophagy, and apoptosis . Due to the change in the ROS and RNS production during BMDMs infection with Mtb and RvΔ0687, we further hypothesized this could impact the secretion of cytokines or chemokines and analyzed the levels of IL-1β, TNF-α, MIP-1α and IP-10 cytokines in the supernatants of BMDMs infected with Mtb strains. Strikingly, we observed that RvΔ0687-infected BMDMs showed significantly higher TNF-α and MIP-1α levels compared to both WT and C-Rv0687 strains. MIP-1α/CCL3 is a chemotactic chemokine secreted by macrophages to recruit inflammatory cells and maintain effector immune response . This suggests that the Rv0687 gene might influence the host immune response during Mtb infection in macrophages.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p20
|
PMC11277061
|
sec[2]/p[8]
|
3. Discussion
| 4.285156 |
biomedical
|
Study
|
[
0.99951171875,
0.0004036426544189453,
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] |
[
0.9990234375,
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0.00010609626770019531
] |
To investigate the in vivo survival and virulence of RvΔ0687, in comparison to the WT and C-Rv0687 strains, we used two mice models of pulmonary infection. Rag −/− mice, which lack functional T and B cells, is considered to be an ideal model to screen the mutant survival as they will be more vulnerable to infection compared to C57BL/6 mice. In Rag −/− mice, all the tested Mtb strains exhibited similar bacterial burden in the lungs at 4 weeks. However, in the spleen, RvΔ0687 displayed a significantly lower bacterial growth compared to WT and C-Rv0687 at this time. This suggests that Rv0687 may play a role in the dissemination of Mtb primary infection from lung to spleen and early establishment of Mtb infection in the spleen of immunocompromised mice. But at 8 weeks post-infection, all the strains showed a comparable bacterial burden in both the lungs and spleen, indicating that the RvΔ0687 mutant was able to overcome the initial difficulty in spleen colonization. In addition, RvΔ0687 mutant-infected Rag −/− mice survived for a longer duration than the WT-infected mice. There is a possibility that RvΔ0687 might induce lesser lung inflammation and pathology compared to WT, leading to prolonged survival. Furthermore, there is a possibility that there is difference in the natural killer (NK)-mediated response generated by WT vs. RvΔ0687. Interestingly, a study by Feng et al. showed the contribution of interferon-gamma secreting natural killer (NK) in the survival of Rag −/− mice after Mtb infection in the absence of T and B cells compared to p40 −/− Rag −/− mice . However, additional detailed studies are needed to test this hypothesis.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999999 |
PMC11277061_p21
|
PMC11277061
|
sec[2]/p[9]
|
3. Discussion
| 4.3125 |
biomedical
|
Study
|
[
0.99951171875,
0.00033211708068847656,
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] |
[
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] |
Interestingly, in C57BL/6 mice, a similar bacterial load was observed in the lungs of WT, RvΔ0687 and C-Rv0687 strains after 4 weeks of infection. However, at 8 weeks post-infection, RvΔ0687 displayed a 3-fold lower bacterial burden in the lungs and ~3–4 fold reduced bacterial load in the spleen at both 4 and 8 weeks post-infection. As the CFU of WT and RvΔ0687 were similar in the lungs at 4 weeks (an initial time point) we do not expect tremendous differences in pathology of lungs by H&E staining; however, these can be analyzed in our future studies. The observed phenotype for RvΔ0687 at 8 W post infection but not earlier, suggests that possibly Rv0687 gene is required by Mtb to counteract the host adaptive immunity during the chronic stages of infection (i.e., 8 W post infection). Another possibility is the differential adaptive response generated by host in response to infection with RvΔ0687 KO mutant compared to WT Mtb to counteract the host response. Together, these findings shed light on the complex interplay between Mtb and the host immune system, emphasizing the significance of the Rv0687 in modulating Mtb survival in mouse lungs and spleen. Further studies involving biochemical characterization of the SDRs reported in this study and its interaction with other regulatory networks will shed light on its role in bacterial adaptation to the host environment, tolerance to antibiotics and in Mtb pathogenesis.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277061_p22
|
PMC11277061
|
sec[3]/sec[0]/p[0]
|
4.1. Bacterial Strains
| 4.089844 |
biomedical
|
Study
|
[
0.99951171875,
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[
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] |
Wildtype Mtb bacterial strains and vector plasmids (p004s, phAE159, pMV361) used in this study were gifted by Dr. William R Jacob Jr (Albert Einstein College of Medicine) and are listed in Supplementary Materials . All the Mtb strains were grown at 37 °C in Middlebrook 7H9 (Difco, Burlington, MA, USA) media, supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase), 0.5% glycerol and 0.05% tyloxapol designated in the text as 7H9 complete media. Wherever required, Mtb strains were grown in 7H9 media supplemented with dextrose (0.2%), 0.5% glycerol and 0.05% tyloxapol (referred as 7H9 dextrose media) for in vitro ROS and RNS sensitivity studies. To grow Mtb strains carrying antibiotic markers, hygromycin (75 mg/mL) or kanamycin (20 mg/mL) were supplemented to the media wherever needed.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p23
|
PMC11277061
|
sec[3]/sec[1]/p[0]
|
4.2. Gene Knockout Construction and Confirmation
| 4.140625 |
biomedical
|
Study
|
[
0.99951171875,
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[
0.99951171875,
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] |
The Mtb genomic DNA was extracted using CTAB-NaCl method . Gene knockout of Mtb was performed by specialized transduction as described previously . Briefly, the flanking regions of the Rv0687 gene sequence were obtained from Tuberculist and cloned into the p0004-SacB vector, carrying the Hyg R resistance gene for homologous recombination. The recombinant construct was further cloned into phAE159 temperature-sensitive phasmid and a knockout mutant of Rv0687 was generated using specialized transduction. Deletion of Rv0687 was confirmed using 3-primer strategy , and whole genome sequencing was performed on a MiSeq instrument (Illumina, San Diego, CA, USA) following the protocol provided by Illumina . Further, the mutant strain was complemented by integrating the wildtype copy of the gene into the genome of the RvΔ0687 mutant using a pMV361 mycobacterial integrative vector and complementation was confirmed using gene-specific primers and kanamycin primers . The details of the strains, plasmids, phasmids and primers used in the study are provided in Supplementary Tables (Tables S1–S3) .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p24
|
PMC11277061
|
sec[3]/sec[2]/p[0]
|
4.3. Assessing Mtb Response to ROS and Nitrite Stress Generating Agents
| 4.117188 |
biomedical
|
Study
|
[
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The wildtype, RvΔ0687 and C-Rv0687 Mtb strains were grown in 7H9 complete media to OD 600 nm ≈ 0.8–1. Bacterial strains were centrifuged and pellet was washed with PBST to remove the catalase and diluted to 0.05 × 10 7 cells/mL with 7H9 dextrose media to a final volume of 5 mL. Diluted bacterial cultures were treated with ROS; 5 or 10 mM hydrogen peroxide (H 2 O 2 ) and 2 mM tert-butyl hydroperoxide (tBOOH) or nitrite stress; 2 mM S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and 2 and 10 mM sodium nitrite (SN) generating agents; and the cultures were incubated for 24, 48, 72 and 96 h after post-treatment and the colony forming units (CFUs) were determined by plating the serially diluted bacterial culture onto 7H10 plates and incubating the plates at 37 °C for 4–6 weeks.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p25
|
PMC11277061
|
sec[3]/sec[3]/p[0]
|
4.4. Quantification of Mtb NADH and NAD + Levels
| 4.21875 |
biomedical
|
Study
|
[
0.99951171875,
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[
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] |
To quantify the NADH and NAD + levels, we used the calorimetric kit from abcam and protocol was followed as recommended by manufacturer. The wildtype, RvΔ0687 and C-Rv0687 bacterial strains were grown in 7H9 complete media to OD 600 ≈ 0.5–0.8 and the cultures were centrifuged at 4000 rpm for 5 min. Pellets were washed with PBST. Pellet was suspended in NADH/NAD extraction buffer (Abcam, Cambridge, UK) with addition of protein inhibitor cocktail (Sigma, St. Louis, MO, USA) and lysed using silica beads in bead-beater for 10 cycles (<60 s) with 1 min intervals. Crude lysates were centrifuged at 10,000 rpm for 10 min at 4 °C and supernatants were collected and filtered using 10 kD spin column to eliminate proteins that can temper with NADH. Filtrates containing NADH as well as NAD + were divided into two reaction sets (1) to measure NAD + (contains both NAD + and NADH) (2) to measure NADH from which NAD + is removed by heating at 60 °C for 30 min to decompose NAD + while NADH remained intact. Afterwards, sample (1) containing NAD + + NADH, sample (2) containing NADH and the standards were transferred to a 96-well plate, this was followed by addition of reaction mix (NAD cycling buffer and NAD cycling enzyme mix, proprietary but just enhances the sensitivity of assay and converts free NAD to NADH). In the end, NADH developer was added and the colorimetric signals was measured at 450 nm using spectrophotometer (Spectramax, Waltham, MA, USA) at 1 h, 2 h, 3 h and 4 h. Individual concentrations of NADH/NAD + were estimated by considering the standard values as reference. Concentation of NAD + was calculated by subtracting concentrations of NAD + -NADH that allowed the calculation of the NADH/NAD + ratio .
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277061_p26
|
PMC11277061
|
sec[3]/sec[4]/p[0]
|
4.5. Response of Mtb to Mycobacterial Drugs
| 4.105469 |
biomedical
|
Study
|
[
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[
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The wildtype, RvΔ0687 and C-Rv0687 bacterial strains were grown in 7H9 complete media to an OD 600 ≈ 0.5–0.8. Final OD 600 was adjusted to 0.1 (~1 × 10 7 CFU/mL). To determine the survival of Mtb, the bacterial strains were exposed to Isoniazid (4–0.007 mg/L), Delamanid (4–0.007 mg/L), NMR711 (50–0.390 mg/L), Rifampicin (8–0.062 mg/L) and Bedaquiline (4–0.015 mg/L). The drugs were serially diluted 2-fold in 7H9 media to reach the least concentration gradient. A total of 100 mL of bacteria (~1 × 10 6 CFU) was added into the wells containing 80 mL of the drug followed by the addition of 20 mL of Alamar Blue to bring the final volume to 200 uL. The plates were incubated at 37 °C for 1–7 days and at the indicated time points, the plates were assessed for bacterial growth and read in a plate reader at OD 590 nm. The survival of bacteria is assessed by plotting the OD 590 nm values on day 7 against drug concentration using GraphPad prism 10 software.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p27
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PMC11277061
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sec[3]/sec[5]/p[0]
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4.6. Infection of Bone Marrow Derived Macrophages (BMDMs)
| 4.152344 |
biomedical
|
Study
|
[
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[
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] |
The C57BL6 mice were euthanized, and the femurs and tibias were separated and flushed thoroughly with Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen) to obtain cells. After lysing of red blood cells, the cells were maintained in DMEM, containing 10% FBS and 30% L929 supernatant (complete media) . Cells were cultured in tissue culture flasks for 7 days and seeded at 0.25 × 10 6 cells per well into 24-well plates. The bacterial strains were grown at 37 °C to OD 600 nm ≈ 0.8–1. After washing with phosphate-buffered saline with tyloxapol (PBST), the bacterial suspensions were diluted in DMEM and used to infect BMDMs cells for 3 h at 37 °C in 5% CO 2 at a multiplicity of infection of 5 (MOI 1:5). Following infection at 3 h, the media were removed, and the wells were washed with PBST before treating with gentamycin for 1 h. Subsequently, the antibiotic media was removed, and the wells were washed with PBST and replenished with fresh DMEM media (complete media). At every time point tested, the infected BMDMs were lysed with 0.01% Triton X-100, and the lysates were serially diluted in PBST and plated on 7H10 agar media to determine the number of colony-forming units (CFUs). The post-infected cell free supernatants were collected before lysis of the cells and analyzed for TNF-α, MIP-1α, IL-1β and IP-10 cytokines.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277061_p28
|
PMC11277061
|
sec[3]/sec[6]/p[0]
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4.7. Measurement of ROS and RNS in BMDMs during Infection
| 4.109375 |
biomedical
|
Study
|
[
0.99951171875,
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[
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To assess the levels of ROS generated during BMDM infection, 0.1% Nitroblue Tetrazolium Chloride (NBT; Sigma) is added to wildtype, RvΔ0687 and C-Rv0687 infected BMDMs in the 96-well plate and incubated at 37 °C for 30 min. After incubation, the NBT solution was removed, and the cells were suspended in 2 M potassium hydroxide to stabilize the color. Dimethyl Sulfoxide (DMSO) was then added to solubilize the formazan. The amount of formazan, which represents the ROS levels, was quantified by measuring the optical density at 570 nm against DMSO as the reference.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
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https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p29
|
PMC11277061
|
sec[3]/sec[6]/p[1]
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4.7. Measurement of ROS and RNS in BMDMs during Infection
| 4.09375 |
biomedical
|
Study
|
[
0.99951171875,
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[
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0.000056684017181396484
] |
To assess the levels of RNS, post-infected supernatants from wildtype-, RvΔ0687- and C-Rv0687-infected BMDMs were collected, and the assay was performed using the Griess Reagent Kit (Biotium, Fremont, CA, USA) following the guidelines as provided in the kit. Various concentrations of sodium nitrite references were prepared to create a standard curve, which was used to measure the absorbance at 570 nm. Post-infected supernatants were mixed with deionized water and Griess reagent, and the reaction mixture was incubated for ≥30 min at room temperature. The absorbance was then measured at 570 nm, and the optical density reading represented the nitrite concentration in the samples. These values were normalized to the reference values.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277061_p30
|
PMC11277061
|
sec[3]/sec[7]/p[0]
|
4.8. Mouse Infection Experiments
| 4.113281 |
biomedical
|
Study
|
[
0.99951171875,
0.0002677440643310547,
0.00015783309936523438
] |
[
0.9990234375,
0.0005402565002441406,
0.0003044605255126953,
0.00007092952728271484
] |
The wildtype, RvΔ0687 and C-Rv0687 strains were cultured to an OD 600nm ≈ 0.8–1. The bacteria were washed twice with PBST. Subsequently, the samples were sonicated in a Branson cup-horn sonicator, twice for 10 s each time, and then diluted to achieve the desired cell densities of ~1 × 10 6 CFUs/mL. Female C57BL/6 mice (6–8 weeks old) were obtained from the Jackson Laboratory, and female Rag −/− mice (6–8 weeks old) were obtained from the in-house breeding facility. The mice were infected with a dose of Mtb strains (200–250 CFUs per lung) using a Glass-Col chamber as described previously and were euthanized at indicated time points. Lung and spleen were harvested and homogenized with PBST. The CFUs were performed by serially diluting the lysate and plated onto 7H10 plates and colonies were enumerated after 4–6 weeks of incubation at 37 °C and CFUs were estimated per lung and spleen. The animal protocol was approved by the University of Texas at El Paso Institutional Animal Care and Use Committee (IACUC) protocol, A-202004-3 as per NIH principles for animal usage. All the animals were maintained according to the guidelines of IACUC.
|
[
"Gunapati Bhargavi",
"Mohan Krishna Mallakuntla",
"Deepa Kale",
"Sangeeta Tiwari"
] |
https://doi.org/10.3390/ijms25147862
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277073_p0
|
PMC11277073
|
sec[0]/p[0]
|
1. Introduction
| 3.601563 |
biomedical
|
Other
|
[
0.9921875,
0.005279541015625,
0.0023517608642578125
] |
[
0.0074005126953125,
0.96484375,
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0.0011930465698242188
] |
Physical performance tests (PPTs) are commonly used by physiotherapists to assess lower limb muscle function after injury, to monitor the progress of treatment and rehabilitation, for screening, or to assess the fitness of athletes of various skill levels throughout pre-season preparation . The advantages of PPTs include easy protocols requiring no specialized knowledge, quick implementation, and being able to perform the test without expensive measurement equipment .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277073_p1
|
PMC11277073
|
sec[0]/p[1]
|
1. Introduction
| 3.898438 |
biomedical
|
Study
|
[
0.99853515625,
0.0006661415100097656,
0.0009245872497558594
] |
[
0.338623046875,
0.325927734375,
0.334228515625,
0.0012226104736328125
] |
Isokinetic dynamometry is considered the gold standard for assessing the value of extensor force moments developed at the knee joint . Similar sensitivity in detecting asymmetry of knee joint extensor force moments using isometric dynamometry at 90 degrees, and at 30 degrees of lower leg flexion at the knee joint, has been shown compared to isokinetic testing . The vast majority of clinicians and trainers do not have access to this equipment, so PTTs are an alternative for assessing and monitoring athletes’ performance, as well as their recovery from injury .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277073_p2
|
PMC11277073
|
sec[0]/p[2]
|
1. Introduction
| 4.058594 |
biomedical
|
Review
|
[
0.9970703125,
0.0009717941284179688,
0.0019245147705078125
] |
[
0.3779296875,
0.12890625,
0.491943359375,
0.001064300537109375
] |
An asymmetry index (ASI) based on PPT allows for the assessment of the progress of treatment, readiness to return to activity and sports participation, and athletic performance in people following surgical procedures or injuries to the lower limbs. LSI and ASI are most commonly used to evaluate the knee joint . The literature identifies 12 different formulas for calculating the indices of symmetry and/or asymmetry, most of which require indicating the stronger limb. The four formulas allow for the determination of the stronger or weaker limb, but they yield different values, making it impossible to compare the results. When choosing a formula for calculating the LSI or ASI, it is important to consider the nature of the task, i.e., if it is unilateral or bilateral . The ( A − B ) M a x ( A , B ) × 100 (with A meaning left limb value and B meaning right limb value) formula is recommended for the evaluation of asymmetry based on unilateral tests because it involves normalization of the absolute difference to the value of the stronger limb .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277073_p3
|
PMC11277073
|
sec[0]/p[3]
|
1. Introduction
| 2.669922 |
biomedical
|
Other
|
[
0.99365234375,
0.001659393310546875,
0.004924774169921875
] |
[
0.02899169921875,
0.96875,
0.0019254684448242188,
0.0005602836608886719
] |
It is important to distinguish between intra-limb asymmetry, i.e., different results for the same limb in the same test, and inter-limb asymmetry, and, therefore, different results for the right and left limbs in the same test .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277073_p4
|
PMC11277073
|
sec[0]/p[4]
|
1. Introduction
| 4.132813 |
biomedical
|
Study
|
[
0.9990234375,
0.0006337165832519531,
0.00030159950256347656
] |
[
0.9990234375,
0.0002734661102294922,
0.0005288124084472656,
0.00008052587509155273
] |
Many studies that analyzed indices of symmetry and asymmetry have used the single-leg long jump test (SLLJT) . Isokinetic dynamometers and isometric dynamometers that take into account the joint flexion angle are used to study the symmetry and asymmetry of the values of the peak moment of force of the knee joint extensors . Objective testing of knee extensor strength without specialized equipment available only in research laboratories is a major challenge for clinicians and coaches. Clinical evaluation is increasingly using the 30 s skater squat functional test (30 s SSFT) to calculate LSI or ASI. One article presented the technique of performing the skater squat functional test and its progressions and regressions . There are studies available that examine the correlation of the asymmetry, dynamic strength, and isometric force of lower limbs with different functional tests . No studies comparing LSI/ASI based on 30 s SSFT or another timed squat test with LSI/ASI values based on muscle strength measured using isometric dynamometry were found in the available literature. Our hypothesis of the study was that evaluating limb asymmetry between right and left knee extensors according to the 30 s SSFT can successfully prove lower limb muscle weakness. The purpose of this study was to calculate and compare the asymmetry index using the 30 s skater squat functional test, the single-leg long jump test, and with isometric measurement of knee extensor strength. With this study, the utility of another functional test to monitor progress in the process of training as well as rehabilitation was evaluated.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277073_p5
|
PMC11277073
|
sec[1]/p[0]
|
2. Material and Methods
| 1.210938 |
biomedical
|
Other
|
[
0.83203125,
0.002536773681640625,
0.1656494140625
] |
[
0.0362548828125,
0.96240234375,
0.0007390975952148438,
0.0007119178771972656
] |
The research received approval from the Senate’s Research Bioethics Committee on 10 February 2023. All participants gave informed written consent to participate in the study after they were informed about the purpose and procedure of the experiment and the possibility of withdrawal from the study.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277073_p6
|
PMC11277073
|
sec[1]/sec[0]/p[0]
|
2.1. Participants
| 4.101563 |
biomedical
|
Study
|
[
0.99755859375,
0.0023517608642578125,
0.0003058910369873047
] |
[
0.99853515625,
0.0008225440979003906,
0.00026345252990722656,
0.00017583370208740234
] |
The target population of the study is young, physically active adults, and twenty-seven people were enrolled in the study, of whom two were excluded (one due to limited range of motion in the ankle joint, and the other due to an ankle injury 3 weeks before the date of the study). Twenty-five men aged 23 ± 3.17 years old, with a body height 182 ± 7.49 cm, body weight 79 ± 9.43 kg and a BMI 24 ± 2.14, were studied. The study group was recruited on a convenience sampling basis. Among the participants, all participated in regular physical activity (weight training and/or other sports for more than 6 months, a minimum of 2 times a week). Inclusion criteria for the study were age between 18 and 35 years, consent to participate in the study, no weight training for lower limbs 72 h before the study, no medical contraindications to exercise, no musculoskeletal injuries in the lower limbs and trunk (within the last 3 months: fractures, sprains, dislocations, or mechanical instability of the ankle, knee, hip, or trunk). Exclusion criteria were female gender, current musculoskeletal injuries to the lower limb or trunk (within the past 3 months: fractures, sprains, dislocations, or mechanical instability of the ankle, knee, hip, or trunk), reported femoroacetabular impingement (FAI), and heel detachment during the skater squat test.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
PMC11277073_p7
|
PMC11277073
|
sec[1]/sec[1]/p[0]
|
2.2. Procedures
| 4.042969 |
biomedical
|
Study
|
[
0.99658203125,
0.0030612945556640625,
0.0005059242248535156
] |
[
0.99755859375,
0.00189971923828125,
0.00046372413635253906,
0.00020813941955566406
] |
Before lower limb asymmetry measurements, anthropometric measurements and a 5 min warm-up (conducted under the supervision of a personal trainer with 2 years of professional experience) were performed. The warm-up was developed based on the literature and the clinical experience of the authors of this study and physiotherapist and consisted of A-skips over 20 m, C-skips over 20 m, 20 bodyweight squats, and 10 hops . As a form of familiarization with the test (familiarization session) , two sets of skater squats and single-leg long jump test were performed, with five repetitions of each exercise (with a one-minute break between sets). The familiarization session was preceded by playing the author’s video of the correct execution of the tests. Additional verbal instructions were given if the participant significantly modified the performance of the test. Then, the measurements proper were carried out.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999994 |
PMC11277073_p8
|
PMC11277073
|
sec[1]/sec[2]/p[0]
|
2.3. Measurements
| 4.117188 |
biomedical
|
Study
|
[
0.9970703125,
0.00205230712890625,
0.0007233619689941406
] |
[
0.98876953125,
0.01042938232421875,
0.00044727325439453125,
0.00038623809814453125
] |
The measurement using the 30 s skater squat functional test (30 s SSFT) was as follows: withe foot of the supporting limb was parallel to the sagittal plane, the non-tested limb should be flexed at the knee joint and during the squat, and the tibial tuberosity touched the step (with adjustable height), so that the angle of flexion at the knee joint tested was 90 degrees (measurement was performed using a Saehan 15 cm goniometer (SAEHAN, GripTM, Rulong, Belgium). Furthermore, one should pay attention to the adherence of the heel of the supporting limb to the ground and the lack of contact between the foot of the non-supporting limb and the ground . The correct positioning of the limbs and step during the 30 s SSFT is illustrated in Figure 1 . The verbal instruction before the test was as follows: “Do as many repetitions as possible in 30 s. Do not touch the ground with the foot of the non-tested limb. Do not detach the heel of the tested limb.” With the “Stop” command, time measurement was stopped, followed by a 3 min break before the next test. The procedure was recorded for later verification of the number of repetitions.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277073_p9
|
PMC11277073
|
sec[1]/sec[2]/p[1]
|
2.3. Measurements
| 4.046875 |
biomedical
|
Study
|
[
0.994140625,
0.002742767333984375,
0.003200531005859375
] |
[
0.97705078125,
0.0220489501953125,
0.0004968643188476562,
0.00043511390686035156
] |
The single-leg long jump test (SLLJT) was carried out as follows: the front of the shoe was placed on the starting line and additional verbal instruction was given: “Perform the longest possible one-legged jump, land on the take-off leg, and maintain balance for 3 s. Don’t prop yourself up after you land.” The distance achieved in each test was marked with a marker, measured from the starting line to the back of the heel. The test included two correctly performed tests, of which the result with the higher value was used for later analysis. In the case of three incorrectly performed jumps in a row, the test was not scored, the measurement was not taken into account, and the test set had to be repeated after a break (not on the same day). There was no such case in this experiment. Failure to pass the test occurred in the event of loss of balance within the first 3 s after landing and propping up within the first 3 s with the non-tested lower limb or the upper limb . The correct technique for performing the single-leg long jump test is shown in Figure 2 .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
PMC11277073_p10
|
PMC11277073
|
sec[1]/sec[2]/p[2]
|
2.3. Measurements
| 4.140625 |
biomedical
|
Study
|
[
0.9990234375,
0.0005688667297363281,
0.0002777576446533203
] |
[
0.99853515625,
0.000896453857421875,
0.00030994415283203125,
0.00011307001113891602
] |
The peak moment of force of the knee joint extensors was applied according to the maximal voluntary contraction method on a JBA Zbigniew Staniak ® measuring station (“JBA” Zb. Staniak, Warsaw, Poland) . The peak moment of force of the knee joint extensors was measured in static measurements on a TBK3 stand designed by JBA Zb. Staniak. The parameter has a measurement range of 1200 Nm with a maximum measurement error of 1%. The vertical design ensures the optimal alignment of the trunk, knee, and hip joints in the seated position (the standard used in biomechanics laboratories). The axis of torque measurement is parallel to the axis of rotation of the joint. In the measurements, the maximum energy is delivered in 1.5–3.0 s. The maximum of the three highest torque values is recorded as the final measurement result . Measurements were taken in a sitting position on a TBK3 JB Staniak stand, with stabilization on the lumbar spine and thighs. The upper limbs were crossed on the chest. The axis of the torque head was aligned with the axis of rotation of the knee joint. The resistance roller (gauge) was placed on the anterior surface of the lower leg above the talocrural joint. The position for measuring the peak moment of force of the knee joint extensors in isometric condition is shown in Figure 3 .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277073_p11
|
PMC11277073
|
sec[1]/sec[3]/p[0]
|
2.4. Statistics
| 4.089844 |
biomedical
|
Study
|
[
0.9990234375,
0.00045013427734375,
0.0002982616424560547
] |
[
0.99951171875,
0.0003058910369873047,
0.0003211498260498047,
0.00006973743438720703
] |
Statistical analysis was performed using Statistica 13.3 (TIBCO Software, Palo Alto, CA, USA). Descriptive statistics were used to characterize the study participants by calculating mean values and their standard deviations. The normality of the distributions was verified using the Shapiro–Wilk test, and all group data had normal distributions. Using a simple linear correlation (Person’s r), the relationship between 30 s SSFT, single-leg long jump test, and knee extensor strength values was calculated using all the variables together for the right and left lower limbs. A simple linear correlation (Person’s r) was used to test whether there was a reciprocal relationship between the ASI calculated from functional tests and the values of knee extensor strength. The statistical significance of differences was set at p < 0.05. The correlation coefficient (r) was interpreted as follows, based on the classification described by J. Cohen: 0–0.4 (weak), 0.4–0.7 (moderate), and 0.7–1.0 (strong) . The ASI index was calculated from the formula proposed by Parkinson et al. ASI = ( A − B ) M a x ( A , B ) × 100 , based on the number of repetitions in the 30 s SSFT, with A meaning the number of repetitions for the left lower limb and B meaning the number of repetitions for the right lower limb; based on the best distance obtained in the SLLJT, with A meaning the jump distance on the left lower limb and B on the right; and based on the highest value from three measurements of the peak moment of force of the knee joint extensors, with A denoting the measurement value for the left lower limb and B for the right.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277073_p12
|
PMC11277073
|
sec[2]/p[0]
|
3. Results
| 4.148438 |
biomedical
|
Study
|
[
0.99951171875,
0.0003142356872558594,
0.0003724098205566406
] |
[
0.99951171875,
0.00017774105072021484,
0.0002830028533935547,
0.00005131959915161133
] |
Analysis of the relationships between the results of 30 s skater squat functional test (30 s SSFT) (n = 50; 26.34 ± 6.11 reps; for right and left lower limb, respectively: n = 25; 26.56 ± 5.81 reps and n = 25; 26.12 ± 6.50 reps), single-leg long jump test (SLLJT) (n = 50; 182.17 ± 21.41 cm; for right and left lower limb, respectively: n = 25, 183.54 ± 20.56 cm and n = 25, 180.80 ± 22.56 cm) and values of the strength of knee extensors (KES) (n = 50; 231.56 ± 40.98 Nm; for right and left lower limb, respectively: n = 25 237.44 ± 42.63 Nm and n = 25, 225.68 ± 39.23 Nm) showed a moderate correlation (r = 0.540) between 30 s TFSS and SLLJT, and a moderate correlation (r = 0.533) between SLLJT and KES. Detailed results are shown Table 1 and Figure 4 .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
PMC11277073_p13
|
PMC11277073
|
sec[2]/p[1]
|
3. Results
| 4.113281 |
biomedical
|
Study
|
[
0.9990234375,
0.00037384033203125,
0.00047278404235839844
] |
[
0.99951171875,
0.00018513202667236328,
0.00024437904357910156,
0.00004476308822631836
] |
Statistical analysis of the relationship between the asymmetry index (ASI) values calculated from 30 s SSFT (2.38 ± 10.98), SLLJT (1.59 ± 4.70), and KES (4.56 ± 8.84) showed a moderate correlation (r = 0.501) between the ASI calculated from the 30 s SSFT test and SLLJT. Detailed results are shown in Table 2 and Figure 5 .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277073_p14
|
PMC11277073
|
sec[2]/p[2]
|
3. Results
| 3.427734 |
biomedical
|
Study
|
[
0.998046875,
0.0006155967712402344,
0.0015001296997070312
] |
[
0.9990234375,
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0.0001583099365234375,
0.00007218122482299805
] |
The value of the ASI calculated from the functional tests was lower compared to the value of the ASI calculated using the values in the strength of knee extensors. The ASI value calculated based on SLLJT was lower compared to the ASI calculated using 30 s SSFT. Table 3 shows the mean values and standard deviations of the ASI calculated from the functional tests and KES.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277073_p15
|
PMC11277073
|
sec[3]/p[0]
|
4. Discussion
| 3.445313 |
biomedical
|
Study
|
[
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] |
[
0.9990234375,
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0.000054717063903808594
] |
The present study is one the first to address skater squat problems and the first in the world to calculate the ASI index from a timed single-leg squat test. To date, there have been no studies comparing the ASI 30 s SSFT index with other functional tests or the peak moment of force of the knee joint extensors. The ASI index for the 30 s SSFT test was shown to detect larger average asymmetries than SLLJT.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277073_p16
|
PMC11277073
|
sec[3]/p[1]
|
4. Discussion
| 4.214844 |
biomedical
|
Study
|
[
0.9990234375,
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[
0.99560546875,
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0.00009310245513916016
] |
There was a moderate correlation between 30 s SSFT and SLLJT and between SLLJT and strength of knee extensors. Furthermore, there was no correlation between 30 s SSFT and strength of knee extensors (shown in the results section, Table 1 ). No articles were found in the literature to examine the relationships between the timed single-leg squat test and the values of peak moment of force of the knee joint extensors. Only two reports were available to examine the relationships between SLS depth and the strength of knee extensors measured with an isokinetic dynamometer. Batty et al. found a weak linear correlation between squat depth and the strength of knee extensors, while Östenberg et al. found no relationship between the strength of knee extensors and squat depth. Reports on the relationships of SLLJT with objective measurements of knee extensor strength in healthy populations are inconsistent. Östenberg et al. and Vassis et al. showed no relationship between the distance in the SLLJT test and the strength of knee extensors. Other researchers have shown moderate to strong correlations between SLLJT and isokinetic measurements of the strength of knee extensors . In another study, Vassis et al. found that the strength of knee extensors measured under isokinetic conditions was a significant predictor of TSJNO, explaining 40.4% of the variance for 180°/s and 45.3% of the variance for 60°/s angular velocities. The results of the study should be interpreted with caution, as results of about 40% do not explain 100% of the variation and do not explain more than half of the variance. This means that the strength of knee extensors measured with an isokinetic dynamometer was not the only variable affecting SLLJT test results. Other causes that may affect test results should be considered, e.g., neuromuscular control. It is claimed that no other simple measurement is as strongly related to strength of knee extensors as the SLLJT. One study was found describing squat depth as a predictor of knee strength in patients 6 and 12 months after ACL reconstruction. Batty et al. demonstrated that an LSI of <90% (ASI > 10%) between the operated and non-operated limb is a predictor of an LSI < 90% (ASI > 10%) of the strength of knee extensors measured using an isokinetic dynamometer. The sensitivity of this test was determined to be 34.7 at 6 months and 17.5 at 12 months following the ACL reconstruction. Specificity was defined as 80 at 6 months and 79.1 at 12 months following the ACL reconstruction. There are many indications that maximum arbitrary flexion angle returns to symmetry >90% faster than the strength of knee extensors. Due to the high specificity of the maximum flexion test during the single-leg squat, it has clinical value and can complement the functional evaluation of patients after ACL reconstruction.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277073_p17
|
PMC11277073
|
sec[3]/p[2]
|
4. Discussion
| 4.195313 |
biomedical
|
Study
|
[
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[
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There was a statistically significant moderate correlation between the ASI for lower limbs calculated from the 30 s SSFT and the SLLJT. There was no statistically significant correlation between the ASI calculated from the functional tests results and the value of peak moment of force of the knee joint extensors. There are discrepancies in the literature regarding the methods of calculating LSI and ASI indices, with the antinomic (apparently correct, but leading to contradictions) terms of symmetry and asymmetry being used interchangeably, which obliges the reader to be careful and vigilant in interpreting the results. This is because 0% asymmetry is equivalent to 100% symmetry, which means no asymmetry and, therefore, full symmetry . Another problem that arises when interpreting the results of studies on the symmetry and asymmetry of lower limbs is the different methods of calculating their indices. Twelve different formulas for calculating symmetry and asymmetry indices have been found in the literature, some of which require choosing the stronger lower limb in advance, which does not work when testing a healthy population . The asymmetry index ASI = ( A − B ) M a x ( A , B ) × 100 , which was used in the present study and does not require a prior reference to a stronger limb, has also been used in previous studies, . In this study, ASI was calculated for the first time using a 30 s single-leg squat test, but there are no studies with could be used for comparison. Many authors suggest that functional tests underestimate the level of asymmetry (overestimate the level of symmetry) compared to those using dynamometers . In the present study, it was observed that the mean asymmetry results obtained from the functional tests were lower compared to the results of the peak moment of force of the knee joint extensors (ASI—SLLJT 1.6 ± 4.7, ASI—30 s SSFT 2.4 ± 11.0 and ASI SLLJT 4.6 ± 8.8), while the mean asymmetry scores obtained from the 30 s SSFT were higher than those obtained from the SLLJT test (ASI—SLLJT 1.6 ± 4.7 and ASI—30 s SSFT 2.4 ± 11.0). Available scientific evidence shows that an LSI > 90% calculated from three jump tests (including SLLJT) may be an insufficient criterion for safe participation in physical activity and competitive sports . Based on the available scientific evidence and the clinical experience of physiotherapy specialists, physicians, and coaches, the current criteria for returning to participation in sports after injury include such aspects as no swelling, a full range of motion of the knee joint, no joint instability, and thigh circumference measured 15 cm above the patella not less than 1.5 cm compared to the non-operated lower limb. The symmetry index for the strength of knee extensors measured using an isokinetic dynamometer was above 90% (ASI < 10%), the scoring on the FMS test was 14 or higher, and a symmetry index of more than 90% was obtained in four jump tests and landing after a jump using the landing error scoring system .
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277073_p18
|
PMC11277073
|
sec[3]/p[3]
|
4. Discussion
| 4.066406 |
biomedical
|
Review
|
[
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[
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It is important to emphasize the value of functional tests which aim to evaluate factors, such as muscle strength, joint stability, neuromuscular control, and overall health . Given the need to develop the most effective methods of verification and precise criteria for return to sport after injury (e.g., ACL), it is reasonable to attempt to include the 30 s SSFT test as another criterion for return to sport. This test is quick to perform and provides additional information on the functional performance of the limb after reconstruction surgery. The list of requirements before returning to sport after an ACL injury is long and requires access to specialized and expensive equipment. In addition to the criteria of muscle strength and fitness for a safe return to full participation, the athlete must meet appropriate psychological criteria, and appropriate athlete management should be implemented to reduce risk factors for re-injury . Meeting all the conditions on the list still does not eliminate the risk of re-injury and does not accurately identify patients at increased risk of ACL re-injury . This shows how complex the topic of ACL reconstruction and subsequent rehabilitation is. New surgical techniques and rehabilitation protocols are being developed all the time for a safe return to activity and participation, but confirmation of their effectiveness requires randomized controlled clinical trials with a long follow-up period.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999994 |
PMC11277073_p19
|
PMC11277073
|
sec[3]/p[4]
|
4. Discussion
| 4.074219 |
biomedical
|
Study
|
[
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[
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The 30 s SSFT results obtained in the paper do not correlate with those of objective measurements, i.e., peak muscle force measured with isometric dynamometers. This does not mean that this test cannot be applied in practice. In order for the 30 s SSFT to find use among clinicians, using standardized procedures and adherence to them during implementation are required. It is also not suitable for all patients, as the movement task of the single-leg squat requires adequate motor coordination, muscle strength, and mobility in the peripheral joints of the ACC. The 30 s TFSS can find application in athletes and physically active people as one of the components of assessing overall fitness. Furthermore, the introduction of regular standardized testing in sports teams could help clinicians more accurately determine the function of a limb before injury. A major challenge for physiotherapists and coaches is to estimate the function of the lower limb before the injury. Sports differ in the specifics of their movement tasks, which can translate into greater or lesser levels of asymmetry. In sports with unilateral asymmetry, e.g.,: long jump, with athletes preferring one of the limbs during the run-up, asymmetry in knee extensor strength may be a natural adaptation to the sport . Comparison of limb strength and functional performance after injury is fraught with errors. First, it is unclear which lower limb was stronger before the injury. Secondly, as a result of the break required for recovery, there is a high probability that the strength and function of the unused limb have deteriorated, which is the determinant when calculating the symmetry or asymmetry indices. Regular functional fitness tests, such as the 30 s SSFT and the cluster of jump tests, can serve as a baseline for the assessment of lower limb injuries. If possible, it is worth extending the testing of athletes with regular measurements of the peak muscle force in the lower limb.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277073_p20
|
PMC11277073
|
sec[3]/p[5]
|
4. Discussion
| 4.097656 |
biomedical
|
Study
|
[
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This study assessed the utility of the 30 s SSFT as a functional test to monitor exercise and rehabilitation progress. Despite its novel approach to the research problem, the study has minor limitations that are worth pointing out and addressing in future research. First, measurements of the strength of knee extensors were assessed using an isometric dynamometer. In the literature, testing the strength of knee extensors with an isokinetic dynamometer is considered the gold standard. Second, a sample size analysis was not performed, as there have been no results of studies on 30 s SSFT to date. Third, it is worth including female participants in future studies, and assessing the impact of confounding variables within this group of participants. Another aspect worth including in future studies would be to perform an internal and external concordance analysis for the 30 s SSFT visual assessment performed by different researchers. The practical significance of this study was that its results could play an important role in the training process and monitoring the return to sports after a possible injury.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
PMC11277073_p21
|
PMC11277073
|
sec[4]/p[0]
|
5. Conclusions
| 3.447266 |
biomedical
|
Study
|
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The asymmetry index calculated based on functional tests and values of knee extensor strength in a group of young men should not be used interchangeably. The asymmetry index calculated from the 30 s skater squat functional test detects greater differences in knee extensor strength than the ASI index calculated from the single-leg long jump test in a group of young male athletes.
|
[
"Mateusz Kamiński",
"Anna Katarzyna Cygańska"
] |
https://doi.org/10.3390/jcm13144017
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
39057274_p0
|
39057274
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|
1. Introduction
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biomedical
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Study
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The acoustic system (AS) (signals, auditory organs, and stridulatory devices) is a key innovation that evolved independently in many insect groups . Around 92% of over 195,000 described insect species produce mainly vibrational signals and other forms of mechanical signaling involved in different intra– and interspecific functions , such as disturbance and alarm, aggregation, aggression, courtship, copulatory, postcopulatory, and aggressive mimicry . Yet, the AS does not consist of a set of evolutionarily independent components, as they are likely to be functionally related to components of other communication systems, such as chemical and visual . The importance of each system depends on the insect group and on the environment in which they live .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057274_p1
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39057274
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1. Introduction
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biomedical
|
Other
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[
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The pattern of sound signals in insects is varied, complex, and generally species–specific . Sound signals are continuous or discontinuous vibrations with physical properties dependent on its dispersion across a medium and its reception by auditory organs . The most common signaling device in insects is an intersegmental stridulatory apparatus, followed by mesonotal–pronotal and other less common apparatuses, such as elytro–tergal, vertex–pronotal, and gula–prosternal .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
39057274_p2
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39057274
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1. Introduction
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|
Study
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[
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Bark beetles (Curculionidae: Scolytinae) are a diverse group of herbivorous insects that play an important role in the ecosystem because they contribute to the structure, composition, dynamics, and vitality of the forests by colonizing and killing old, damaged, or physiologically weakened trees . Moreover, bark beetles create food webs by generating habitats by killing trees, which promotes biodiversity (fungi, mites, bacteria, and many other invertebrates and vertebrates use this new resource) . Furthermore, these beetles have established complex ecological interactions with their symbionts, thereby constituting a true holobiont . Yet, they are also considered important disturbance agents, because the outbreaks of some bark beetle species can kill thousands of healthy trees and cause a negative impact on ecosystem services for humans and significant economic losses to timber producers .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p3
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39057274
|
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|
1. Introduction
| 2.441406 |
biomedical
|
Other
|
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[
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Bark beetles are essentially olfactory insects, since they use specific mixtures of semiochemicals to differentiate, select, and colonize host trees and attract mates and conspecifics . Inside plants, bark beetles live in a subcortical environment where they build their galleries, mate, and grow their offspring . This is a dark and chemically saturated environment where a chemical communication system could not be reliable . In the subcortical environment, the AS may be of paramount importance, since wood is a good mediator of sonic signal transmission for communication .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057274_p4
|
39057274
|
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|
1. Introduction
| 4.007813 |
biomedical
|
Study
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[
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[
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Studies of the AS in bark beetles have been carried out mainly in species of the genera Dendroctonus , Ips , Hylurgus and Polygraphus . From these studies, the manner in which sound is produced in these beetles has been delineated (e.g., elytro–tergal, vertex–pronotal, and gula–prosternal); specifically in the Dendroctonus –bark beetles, the mechanism found is the elytro–tergal, which is composed of two structures: the pars stridens located on the inner face of the declive elytral and the plectrum, situated at the seventh abdominal tergite, which generates sound sequences or stridulations . Moreover, a general catalogue of sounds under stress conditions has been outlined , as well as the spectral and temporal characteristics of sound in different biological contexts (e.g., stress, courtship, and female–male interactions) and the effect that some morphological features (e.g., beetle size) have on the sound type produced during courtship .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057274_p5
|
39057274
|
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|
1. Introduction
| 3.515625 |
biomedical
|
Study
|
[
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[
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The roundheaded pine beetle, D . adjunctus Blandfort, is an aggressive species that colonize around 18 pine species, preferentially Pinus hartwegii , across its distribution range from the United States to Central America . Studies of acoustic communication in this bark beetle are scarce and were conducted under a stress condition; in addition, dimorphism was observed in the sound production because only males produced it . Based on this information, we investigated the variability and specific degree of the spectral and temporal features of male calls. We first described the morphology of the stridulatory apparatus using optical and scanning electron microscopy and, later, we recorded the call types produced by males, as well as temporal and spectral features of sounds under different behavioral conditions, such as stress, female–male, and male–male interactions.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999994 |
39057274_p6
|
39057274
|
sec[1]/p[0]
|
2. Materials and Methods
| 3.546875 |
biomedical
|
Study
|
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Pinus hartwegii trees infested and non–infested by the roundheaded pine beetle were felled in May 2023 at the Parque Nacional Volcán Nevado de Colima, Jalisco State, Mexico . Trees of each condition were sectioned into logs (20 cm length × 30 cm diameter) and sealed at the ends with paraffin to avoid dehydration. Once in the laboratory, uninfected logs were stored in the freezer at 4 °C, while infested logs were enclosed in cloth bags and stored at room temperature. Emerged adults were collected daily, placed in Magenta TM vessels GA 7 (Magenta Corp, Sigma–Aldrich–Merck, Darmstadt, Germany) containing wet filter paper, and stored at 4 °C to keep them alive until needed. The sex of the organisms was ascertained by the shape of the seventh abdominal tergite .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999999 |
39057274_p7
|
39057274
|
sec[1]/sec[0]/p[0]
|
2.1. Optical and Scanning Electron Microscopy of Stridulatory Structures
| 4.242188 |
biomedical
|
Study
|
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The seventh abdominal tergite and the left elytra from 30 males were removed using a pair of fine forceps and fine needles. Both structures were cleared by incubating them for 3 h at 70 °C in a 10% KOH solution. Thereafter, structures were immersed in a 10% acetic acid solution to neutralize the KOH solution, rinsed with 96% alcohol, and later mounted on slides in Hoyer’s medium . The pars stridens on the left elytra and the plectrum in the seventh tergite were observed by optical microscopy (Prime Star 1, Carl Zeiss, Jena, Germany), and the length and width of both structures were measured with a graduated eyepiece and a 0.01 mm calibration slide (Walfront, Micrometer Calibration Slide). The variables measured of these structures were as follows: The length of the pars stridens was the distance between the ridge closest to the anterior edge of the elytra to the ridge farthest from the posterior edge ; the width was the distance from the midpoint of the pars stridens closest to the sutural margin to the farthest midpoint of this structure ; and the ridge width was the distance between grooves, calculated from the beginning of one groove to the beginning of the next . The external width of the plectrum was the distance between the outer edge of the right lobe to the outer edge of the left lobe in the anteroposterior position of the insect body . The internal width was the distance between the inner edge of the right lobe to the inner edge of the left lobe , and the lobe width was the distance between the edges of the right lobe . In addition, the stridulatory apparatus of males was observed and photographed in a variable pressure scanning electron microscope under low vacuum (0.6 mbar) and acceleration voltage of 5 kV (FEI Quanta 250 ESEM, FEI Company, Hillsboro, OR, USA) at the Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
39057274_p8
|
39057274
|
sec[1]/sec[1]/p[0]
|
2.2. Sound Recording
| 3.818359 |
biomedical
|
Study
|
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In this study, we referred to the sounds produced by insects as calls , which present different temporal and spectral features depending on the type of interaction. To avoid noise disturbance, all calls produced by the roundheaded pine beetle under stress, female–male, and male–male interactions were recorded at night, inside a purpose–built soundproofed wooden box (width × length × depth, 20 × 40 × 30 cm) to minimize stray noise. Calls were recorded with an omnidirectional condenser microphone model ECM8000 (Behringer, Willich, German) and an audio interface model U–PHORIA UM2B (Behringer, Willich, German) in an ASUS laptop, using the virtual audio editor Audacity at 96 kHz, 48 dB gain, and 24 bit sampling rate.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p9
|
39057274
|
sec[1]/sec[1]/p[1]
|
2.2. Sound Recording
| 4.121094 |
biomedical
|
Study
|
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Stress calls of thirty males were recorded while holding them with the thumb and forefinger for 2 min and placing the microphone at a 5 mm distance, leaving the elytra free, and lightly pressing their head and pronotum. Female–male interaction calls were repeated 24 times. For this assay, a 0.9 mm diameter hole was drilled in the non–infested P . hartwegii logs bark with a drill and a 5/16*4′′ drill bit to go through the bark and avoid reaching the phloem. Subsequently, each female was confined in logs for 1 h by placing an empty gel capsule over the hole. Once the female began to build a gallery, which was confirmed by the accumulation of frass in the entrance hole, the gel capsule was removed and a randomly selected unmated male was placed at the entrance of the gallery . Male–male interaction was repeated 15 times. For this assay, a circular arena of 2 cm diameter was made in the bark of non–infested logs with the help of a knife and a drill with a 5/16 drill bit. Subsequently, the first male was placed in the arena with the help of fine–tipped tweezers. After 1 min, the second male was quickly placed in the arena, covering it with a 0.2 mm mesh to avoid escape. Calls from female–male and male–male interactions were recorded for two minutes or until no signals were detected; the microphone was placed at 2 cm from the hole and arena, respectively .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p10
|
39057274
|
sec[1]/sec[2]/sec[0]/p[0]
|
Spectro–Temporal Features
| 2.232422 |
biomedical
|
Study
|
[
0.92333984375,
0.0013065338134765625,
0.0755615234375
] |
[
0.7177734375,
0.279296875,
0.0017175674438476562,
0.0012111663818359375
] |
From the sounds file, call types (single or multiple notes), ratio, and spectro–temporal features were measured using spectrograms and waveforms in Raven Pro™ 1.4 following the nomenclature from .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p11
|
39057274
|
sec[1]/sec[2]/sec[0]/p[1]
|
Spectro–Temporal Features
| 4.164063 |
biomedical
|
Study
|
[
0.9931640625,
0.0003745555877685547,
0.006317138671875
] |
[
0.99951171875,
0.00036215782165527344,
0.00019860267639160156,
0.00004035234451293945
] |
A sound call is a train or set of stridulatory impulses provoked by the tooth strike, that is, the rubbing of the plectrum against a tooth of the pars stridens. In bark beetles, single–note calls are characterized by a series of regularly spaced strikes and multiple–noted calls by two or more pulses spaced by brief periods of silence . In this study, multiple–noted calls were considered as sound spacings with a duration longer than two and a half milliseconds, and at least three strikes in each part of the train. The call ratio was calculated as the relationship between single–noted calls or multiple–noted calls with respect to the total number of calls observed; this feature was expressed as a percentage. The temporal features recorded included the tooth strike rate (estimated as the strike number delivered per second), the intertooth strike interval (calculated as the silence time at milliseconds between each tooth strike), and the call duration (estimated as the time that elapsed between the first and last tooth strike from a call). The spectral features evaluated include the maximum frequency (measured as the highest number of repeated waves with respect to time), the minimum frequency (measured as the lowest number of repeated waves), and the dominant frequency (calculated as the overlapping of the frequency with the highest amplitude) .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p12
|
39057274
|
sec[1]/sec[2]/sec[0]/p[2]
|
Spectro–Temporal Features
| 3.888672 |
biomedical
|
Study
|
[
0.98876953125,
0.0003571510314941406,
0.010833740234375
] |
[
0.9990234375,
0.0006546974182128906,
0.0002486705780029297,
0.00004696846008300781
] |
The spectro–temporal features of the single–noted and multiple–noted calls of the three conditions assayed were compared using a paired t–test for independent data and different sample sizes. To estimate significant differences of the spectro–temporal features of single–noted and multiple–noted calls among the three conditions assayed (stress, male–male interaction, and female–male), a one–way ANOVA and post hoc test of Tukey–Kramer were conducted. All analyses were performed with Past 4.03.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999999 |
39057274_p13
|
39057274
|
sec[2]/sec[0]/p[0]
|
3.1. Stridulatory Apparatus of D. adjunctus
| 3.994141 |
biomedical
|
Study
|
[
0.9501953125,
0.0004916191101074219,
0.0494384765625
] |
[
0.998046875,
0.0017986297607421875,
0.00016772747039794922,
0.00006389617919921875
] |
The shape of the pars stridens is a file of ridges arranged longitudinally with respect to the inner margin of the elytra. The file has ridges aligned perpendicular to the margin of the elytra, which are highly developed on the left elytra and marginally on the right; in fact, when the right elytra is closed, it overlaps the left . The pars stridens is characterized by most of its ridges being continuous, with only a minority of them observed as fused at some points . The size of this structure was variable among individuals, with a length of 550–690 ± 7.1 µm, width of 170–260 ± 4.2 µm, and a crest width of 6.25–8.3 ± 0.1 µm ( Table 1 ).
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p14
|
39057274
|
sec[2]/sec[0]/p[1]
|
3.1. Stridulatory Apparatus of D. adjunctus
| 4.050781 |
biomedical
|
Study
|
[
0.994140625,
0.00030493736267089844,
0.005435943603515625
] |
[
0.9990234375,
0.0009188652038574219,
0.0001131296157836914,
0.0000540614128112793
] |
The plectrum is in the middle part of the posterior margin of the seventh tergite; it consists of two conspicuous lobes projecting towards the eight tergite that, when rubbed voluntarily by friction against the pars stridens, produces the sound . The plectrum was also variable, with an external width of 57–71 ± 0.7 μm, an internal width of 26–31 ± 0.6 µm, and a lobular width of 15–21 ± 0.4 μm ( Table 1 ).
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p15
|
39057274
|
sec[2]/sec[1]/sec[0]/p[0]
|
3.2.1. Stress Calls
| 4.152344 |
biomedical
|
Study
|
[
0.99462890625,
0.0005383491516113281,
0.0045928955078125
] |
[
0.99951171875,
0.00020694732666015625,
0.0002448558807373047,
0.000047266483306884766
] |
Stressed males emitted a call train at an average rate of 2.25 ± 0.07 calls/s and a broadband output of 10–60 dB until the insect was released. The call type was predominantly single–noted in most of the insects (30 specimens) analyzed; however, some of them (13 specimens) also emitted multiple–noted calls . The total call ratio was 90% single–noted and 10% multiple–noted ( Table 2 ). The tooth strike number and tooth strike rate per second were higher in single–noted calls than multiple–noted calls; the intertooth strike interval was greater for multiple–noted calls than for single–noted calls. The duration of single–noted calls was slightly shorter than multiple–noted calls ( Table 2 ). Meanwhile, the minimum frequency of single–noted calls was approximately half that of multiple–noted calls, and the maximum and dominant frequencies were similar between both noted calls ( Table 2 ). The plot of the power spectrum showed a single–noted call with a maximum amplitude (dB) between 1 and 9 kHz .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
39057274_p16
|
39057274
|
sec[2]/sec[1]/sec[1]/p[0]
|
3.2.2. Female–Male Interaction
| 4.128906 |
biomedical
|
Study
|
[
0.9892578125,
0.0007462501525878906,
0.00994873046875
] |
[
0.99951171875,
0.00027680397033691406,
0.0002053976058959961,
0.00005453824996948242
] |
In the female–male interaction, twenty four beetles produced single–noted calls, but twelve of them also produced multiple–noted calls in this context ( Table 2 ) . The call rate was 74% single–noted and 26% multiple–noted ( Table 2 ). During courtship, the male initially emitted single–noted calls, but as it approached the female, it produced multi–noted calls. The call train emitted by males was at an average rate of 7.8 ± 0.27 calls/s and an output of 10–65 dB. The number of tooth strikes, the tooth strike per second, and the intertooth strike interval were higher in multiple–noted calls than single–noted calls. The duration of both types of calls was double in multiple–noted calls compared to single–noted calls ( Table 2 ); the minimum, the maximum, and dominant frequencies of spectral parameters were similar between single–noted and multiple–noted calls ( Table 2 ). The plot of the power spectrum showed a single–noted call with an amplitude (dB) between 2 and 10 kHz .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p17
|
39057274
|
sec[2]/sec[1]/sec[2]/p[0]
|
3.2.3. Male–Male Interaction
| 4.101563 |
biomedical
|
Study
|
[
0.99609375,
0.0005235671997070312,
0.003574371337890625
] |
[
0.99951171875,
0.00019407272338867188,
0.00020623207092285156,
0.000044226646423339844
] |
Males displayed two behaviors during the interaction: Both males stayed in the arena and later one of them left; the latter behavior was observed in most insects (ten out of fifteen), and the time in which the males retreated from the arena was between 30 to 60 s. The males that remained in the arena emitted a call train at an average rate of 3.7 ± 0.27 calls/s, while males withdrawing emitted a call train at an average rate of 2.56 ± 0.22 calls/s. Calls in both behaviors showed a range of amplitude of 10–60 dB. Single–noted calls were the primary call type in male–male interactions ( Table 2 ) . Males that remained in the arena also produced multiple–noted calls . For males that remained in the arena, their temporal and spectral characteristics were maintained during the interaction ( Table 2 ). These temporal and spectral characteristics were like those of the males that remained in the arena until one withdrew from the arena. However, during the insect retraction, the amplitude of the single–noted call decreased significantly.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p18
|
39057274
|
sec[2]/sec[1]/sec[2]/p[1]
|
3.2.3. Male–Male Interaction
| 4.152344 |
biomedical
|
Study
|
[
0.99365234375,
0.00045299530029296875,
0.00604248046875
] |
[
0.99951171875,
0.0001932382583618164,
0.0002789497375488281,
0.00003910064697265625
] |
Significant differences were found between several spectral and temporal features of single–noted and multiple–noted calls generated in each assayed condition. The features tooth strike ( t = 3.32, p = 0.001), tooth strike rate ( t = 4.68, p < 0.05), inter–tooth strike interval ( t = 5.42, p < 0.05), and minimum frequency ( t = 3.75, p < 0.05) were different in the stress condition; tooth strikes ( t = 3.68, p = 0.001), tooth hit rate ( t = 11.44, p < 0.05), intertooth hit interval ( t = 17. 63, p < 0.05), call duration ( t = 15.54, p = 0.04), minimum frequency ( t = 8.41, p < 0.05), and maximum frequency ( t = 4.83, p < 0.05) in the female–male interaction; and only call duration ( t = 2.11, p = 0.04) and dominant frequency ( t = 2.65, p < 0.05) in the male–male interaction ( Table 2 ). ANOVA and the Tukey–Kramer test showed significant differences in the temporal features of the tooth strikes ( F = 153.0, p = 0.001), the frequency of the tooth strikes ( F = 76.4, p = 0.001), the interval between tooth strikes ( F = 78.58, p = 0.001), and the call duration ( F = 12.29, p = 0.001), as well as in the spectral features of minimum ( F = 68.26, p = 0.001) and maximum ( F = 37.59, p = 0.001) frequency both of single and multiple notes among stress, female–male, and male–male conditions ( Table 2 ).
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
39057274_p19
|
39057274
|
sec[3]/p[0]
|
4. Discussion
| 4.121094 |
biomedical
|
Study
|
[
0.986328125,
0.0003936290740966797,
0.01329803466796875
] |
[
0.99951171875,
0.00038814544677734375,
0.000164031982421875,
0.00004607439041137695
] |
This is the first report of the acoustic diversity of male D . adjunctus under stress conditions, and female–male and male–male interactions. The roundheaded pine beetle has a stridulatory elytro–tergal apparatus whose general morphological organization aligns with the description of males from D . frontalis , D . pseudotsugae , D . brevicomis , D . ponderosae , D . rufipennis , D . valens , D . terebrans , D . approximatus , as well as D . rhizophagus , D . mexicanus , D . mesoamericanus , and D . vitei . No sound was recorded in females of the roundheaded pine beetle under stress conditions, which agrees with the report by for this same species, but not in the female–male interaction. Previous studies have demonstrated that other female Dendroctonus have a second stridulatory apparatus called the “terminal abdominal sternite” located in the wall of posterior margin of the last sternite , from which, it was assumed, they can generate sounds.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
39057274_p20
|
39057274
|
sec[3]/p[1]
|
4. Discussion
| 4.050781 |
biomedical
|
Study
|
[
0.96923828125,
0.0006098747253417969,
0.029937744140625
] |
[
0.9990234375,
0.0005817413330078125,
0.00021517276763916016,
0.000057578086853027344
] |
To confirm the presence or not of the terminal abdominal sternite, we analyzed 30 females of the roundheaded pine beetle. Interestingly, females have this structure, which is in agreement with that reported for other species of Dendroctonus , but they also have a structure analogous to the male stridulatory apparatus on the left elytra consisting of a file whose ridges are apparently not well developed and arranged as in males . This structure is also present in females of other Dendroctonus species, but it is not known whether they produce sounds with this structure as a true stridulation mechanism as has been reported for D. terebrans . It remains to be resolved whether the sounds produced by females are true calls or are acoustic reminiscences of an atrophied morphological structure in the course of evolution, which could be associated with the ecological role that females play as the sex that initiates host tree selection and colonization, as previously suggested .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057274_p21
|
39057274
|
sec[3]/p[2]
|
4. Discussion
| 2.705078 |
biomedical
|
Study
|
[
0.67431640625,
0.0009813308715820312,
0.324462890625
] |
[
0.9736328125,
0.02532958984375,
0.0006270408630371094,
0.0002677440643310547
] |
Acoustic sound generation by male D. adjunctus includes single– and multi–noted calls in stress tests and female–male and male–male interactions, which are consistent with studies of other Dendroctonus bark beetles under the same conditions . In both call types of this bark beetle, the significant differences observed in spectral and temporal features indicate that these sounds are linked to specific behaviors, as suggested in other Dendroctonus species .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p22
|
39057274
|
sec[3]/p[3]
|
4. Discussion
| 4.109375 |
biomedical
|
Study
|
[
0.95166015625,
0.0008106231689453125,
0.047393798828125
] |
[
0.99951171875,
0.0002942085266113281,
0.0002237558364868164,
0.000049054622650146484
] |
Calls produced in stress conditions by the roundheaded pine beetle were mainly 90% single–noted and 10% multiple–noted; these serve as evidence of two different morphotypes: two–noted and three–noted calls, which is consistent with that reported for other Dendroctonus bark beetles , including D. approximatus, which apparently only generated single–noted calls in stress , but our spectrograms indicate that they can also produce multiple–noted distress calls . In addition, our findings also showed that the intraspecific variation of spectro–temporal features in the roundheaded pine beetle was low and independent of note type and multi–note morphotypes. However, significant differences were found between spectro–temporal features in both call types ( Table 2 ), except maximum and dominant frequencies, and call duration. Furthermore, the interspecific comparison of some spectral–temporal features (e.g., maximum, minimum, and dominant frequencies) of different species generated in stress ( Table 3 ) suggest that the patterns of single–noted distress calls are similar among them, except for the call duration records reported for D . approximatus and D . terebrans . Unfortunately, the absence of multi–noted call data does not allow us to evaluate whether the spectro–temporal features of these call types are similar or different between species.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
39057274_p23
|
39057274
|
sec[3]/p[4]
|
4. Discussion
| 3.248047 |
biomedical
|
Study
|
[
0.93115234375,
0.0006866455078125,
0.0679931640625
] |
[
0.998046875,
0.00164794921875,
0.00022041797637939453,
0.00007981061935424805
] |
Significant differences between the spectro–temporal features of single–noted and multiple–noted calls produced by D . adjunctus males in the female–male interaction and in the stress condition indicate that the general pattern of these calls corresponds with different biological behaviors. Our results show a higher prevalence of single notes than multiple notes in this female–male interaction, which does not coincide with that observed in D . ponderosae and D . approximatus where multiple–note calls are predominant . Unfortunately, it is also not possible to compare spectral and temporal features between species, as these data have not always been reported.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057274_p24
|
39057274
|
sec[3]/p[5]
|
4. Discussion
| 3.224609 |
biomedical
|
Study
|
[
0.8212890625,
0.0006818771362304688,
0.17822265625
] |
[
0.927734375,
0.06500244140625,
0.00696563720703125,
0.0003724098205566406
] |
It has been suggested that the production of interrupted calls is indicative of the vigor and fitness of males, which determines their preference for females . Nevertheless, the ratio of both types of calls, the emission pattern, and their temporal combination or alternation may also be a mechanism of intraspecific recognition and of interspecific reproductive isolation, especially when several species coexist in sympatry or syntropy. This is because it is widely recognized that the Dendroctonus species can coexist in space and time in the same locality and tree. Some studies have shown that males approaching the female’s gallery produce calls that cause the female to stop producing aggregation pheromones . This may be associated with the colonization and mass attack strategy of these species.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
39057274_p25
|
39057274
|
sec[3]/p[6]
|
4. Discussion
| 4.09375 |
biomedical
|
Study
|
[
0.97900390625,
0.0005774497985839844,
0.0204010009765625
] |
[
0.99951171875,
0.00029349327087402344,
0.00028228759765625,
0.00004398822784423828
] |
Male–male interaction is a special case of stress to avoid physical aggression, which can impede the access of another male to the female or to block his entry into the gallery (territoriality). Although the proportion of single and multiple calls between the stress condition (90–10%) and the male–male interaction (85–15%) was similar in this study, significant differences between both conditions were mainly concentrated in temporal characteristics (e.g., rate of tooth strikes, interval between tooth strikes, and call duration), suggesting the configuration of different calling patterns in these two stress conditions. The pattern of calls in the male–male interaction could be due to combinations of intimidation or deterrence to avoid physical contact. In fact, the proportion of single– and multi–note calls was similar in males that remained in the arena but not in males that withdrew, which were only single–note. Similar results have been obtained in D. ponderosae and were interpreted as a characteristic of rivalry between individuals of the same sex, regardless of the presence of a female or of maintaining or blocking the entrance to the gallery when occupied by a pair . In contrast, in D . valens , the sound pattern produced by males was related to their body size, as males competing for females displayed two types of calls, the first was to prevent direct fighting with potential competitors of equivalent size, while the second was to scare away and deter small–sized competitors .
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999998 |
39057274_p26
|
39057274
|
sec[3]/p[7]
|
4. Discussion
| 3.957031 |
biomedical
|
Study
|
[
0.962890625,
0.0007338523864746094,
0.0362548828125
] |
[
0.9990234375,
0.0005221366882324219,
0.0002605915069580078,
0.000057578086853027344
] |
In the male–male interaction, both males of D . adjunctus produced both types of calls while staying together, but when one of the males withdrew, not only did its aggressive behavior change, but the call sound also changed to being 100% one–noted. The minimum frequency of retreat calls was twice that of calls made by males while interacting in territoriality, the maximum frequency was lower in retreat calls compared to the calls of males that stayed together, and the dominant frequency was very similar across all interactions ( Table 2 ). Differences between the calls of males that remained and those that withdrew showed a wider repertoire of signals compared to those known so far, introducing a new type of sound, the “withdrawal calls”. These calls, when exhibited in conjunction with flight behavior, may be an indicator of surrender by the male. Future studies may provide clarity on the behavioral implications of these types of calls.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
39057274_p27
|
39057274
|
sec[3]/p[8]
|
4. Discussion
| 3.970703 |
biomedical
|
Study
|
[
0.99609375,
0.00015294551849365234,
0.003978729248046875
] |
[
0.986328125,
0.005268096923828125,
0.0084686279296875,
0.00010287761688232422
] |
Lastly, while the AS (signals, auditory organs, and stridulatory devices) in other insect orders (e.g., Orthoptera, Hymenoptera, and Hemiptera) have been widely studied and associated with a wide range of biological behaviors and environmental factors, in bark beetles (Coleoptera: Curculionidae: Scolytinae), its integration with the chemical communication system, behavior, and reproductive ecology has received little attention . In particular, it would be desirable for future studies focus on aspects related to acoustic signals, which, being apparently species–specific, could be involved in isolation and reproductive behavior, as well as in pheromone synthesis, especially in species that produce these compounds de novo.
|
[
"León L. Cerrillo-Mancilla",
"Claudia Cano-Ramírez",
"Gerardo Zúñiga"
] |
https://doi.org/10.3390/insects15070542
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999995 |
PMC11277097_p0
|
PMC11277097
|
sec[0]/p[0]
|
1. Introduction
| 4.851563 |
biomedical
|
Study
|
[
0.998046875,
0.0008978843688964844,
0.00095367431640625
] |
[
0.736328125,
0.0024261474609375,
0.260498046875,
0.00106048583984375
] |
Prokineticin receptors (PKRs, namely PKR1 and PKR2) are G protein-coupled receptors (GPCRs) that specifically bind prokineticin 1 (PK1) and prokineticin 2 (PK2) ligands. PKRs are able, by coupling all subtypes of G proteins such as Gαq/11, Gαs and Gαi, to mediate various signalling pathways, including protein kinase C, mitogen-activated protein kinase (ERK), phosphoinositide 3-kinase, STAT3 and protein kinase B . In addition, PKRs interact constitutively with β-arrestin-2, although this interaction does not trigger internalisation of the receptor . The complexity resulting from the presence of different isoforms of ligands and receptors and from the ability of the receptors to couple different G proteins , dimerise and interact with accessory proteins allows the prokineticin system to perform specific functions in different tissues. The physiological functions of prokineticins include neurogenesis, angiogenesis, pain perception, and circadian rhythm regulation . PK2 has also been shown to act as an adipokine. Initially, it was demonstrated that PK2 binding to PKR1 reduces food intake, stimulating the release of α-MSH and adipose tissue proliferation . Recently, evidence has shown that inhibition of food intake by PK2 occurs through activation of amygdala PKR2 neurons .
|
[
"Maria Rosaria Fullone",
"Daniela Maftei",
"Martina Vincenzi",
"Roberta Lattanzi",
"Rossella Miele"
] |
https://doi.org/10.3390/ijms25147816
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277097_p1
|
PMC11277097
|
sec[0]/p[1]
|
1. Introduction
| 4.324219 |
biomedical
|
Study
|
[
0.99951171875,
0.00030303001403808594,
0.0001804828643798828
] |
[
0.9990234375,
0.000274658203125,
0.00048351287841796875,
0.00009328126907348633
] |
The activation and localisation of PKRs is modulated by the accessory melanocortin receptor protein 2 (MRAP2), a single transmembrane protein that regulates several GPCRs involved in energy homeostasis . The inhibitory effect of MRAP2 on the PKRs has been demonstrated both in vitro and in vivo, highlighting that the anorexigenic effect of PK2 is enhanced in mrap2 -deficient mice characterised by severe obesity . There are three different MRAPs in zebrafish: zMRAP, which is related to the tetrapod MRAP1, and zMRAP2a and zMRAP2b, which are classified as paralogues of mammalian MRAP2 . Similarly, the prokineticin system has been identified in the zebrafish genome: two receptors corresponding to mammalian PKR1 and PKR2, named zPKR1 and zPKR2, respectively, and two ligands corresponding to mammalian PK1 and PK2, named zPK1 and zPK2 . The aim of this study is to investigate the ability of zMRAP2a to interact with zPKR1 and to modulate its activity in order to use zebrafish as a model system for the study of diseases caused by dysregulation of the prokineticin system. Due to their rapid development and reproductive rate, zebrafish offer a unique opportunity for high-throughput drug screening to discover new compounds for the treatment of human diseases.
|
[
"Maria Rosaria Fullone",
"Daniela Maftei",
"Martina Vincenzi",
"Roberta Lattanzi",
"Rossella Miele"
] |
https://doi.org/10.3390/ijms25147816
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999996 |
PMC11277097_p2
|
PMC11277097
|
sec[1]/sec[0]/p[0]
|
2.1. Analysis of the mrap2a and mrap2b Genes
| 4.261719 |
biomedical
|
Study
|
[
0.99951171875,
0.00019359588623046875,
0.00046443939208984375
] |
[
0.98779296875,
0.011260986328125,
0.0007538795471191406,
0.00021564960479736328
] |
Zebrafish MRAP2a and MRAP2b, similarly to mammalian MRAP2s, have three distinct domains, each encoded by one exon. The three domains are a tyrosine-rich N-terminal region containing the conserved YEYY motif, a putative hydrophobic transmembrane domain of 23 amino acids (residues 34–56), and a C-terminal domain .
|
[
"Maria Rosaria Fullone",
"Daniela Maftei",
"Martina Vincenzi",
"Roberta Lattanzi",
"Rossella Miele"
] |
https://doi.org/10.3390/ijms25147816
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
PMC11277097_p3
|
PMC11277097
|
sec[1]/sec[0]/p[1]
|
2.1. Analysis of the mrap2a and mrap2b Genes
| 4.460938 |
biomedical
|
Study
|
[
0.99951171875,
0.00031375885009765625,
0.00034928321838378906
] |
[
0.998046875,
0.0014314651489257812,
0.0006060600280761719,
0.00015532970428466797
] |
The zebrafish mrap2a gene consists of three exons and two introns. The first exon contains a 5′ UTR (untranslated region) sequence and a sequence encoding the N-terminal region, which spans the first 41 amino acids; the second exon encodes the transmembrane region, which extends from the amino acid at position 42 to the amino acid at position 73; and the third exon contains a region encoding the C-terminal domain, which consists of 144 amino acids and the 3′ UTR sequence . The zebrafish mrap2b gene also consists of three exons and two introns. As in mrap2a , the three exons encode the three N-terminal, the transmembrane and the C-terminal domains, but unlike the mrap2a exons, they do not contain the 5′ regions and the 3′ UTR. A synteny analysis was performed between the zebrafish mrap2a and mrap2b genes and the human mrap2 and mouse mrap2 genes. In humans and mice, the genes close to the mrap2 gene are RIPPLY2 and cep162 . Interestingly, the situation in zebrafish is in between: mrap2a is located next to cep162 on chromosome 16 and mrap2b is next to RIPPLY2 on chromosome 4 .
|
[
"Maria Rosaria Fullone",
"Daniela Maftei",
"Martina Vincenzi",
"Roberta Lattanzi",
"Rossella Miele"
] |
https://doi.org/10.3390/ijms25147816
|
N/A
|
https://creativecommons.org/licenses/by/4.0/
|
en
| 0.999997 |
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