Physiological Effects on the Expression of Aquaporin 1-Like HC-1 in Cope\u27s Gray Tree Frog, \u3cem\u3eHyla Chrysoscelis\u3c/em\u3e

Aquaporin 1 is a cell membrane integral protein, which functions for water transport through the hydrophobic cell membrane to the cytoplasm. HC-1 is an aquaporin 1-like protein expressed in Hyla chrysoscelis and amphibian that survives at sub-freezing temperatures in its natural habitat. The goal of the current study was to investigate the expression of HC-1 during changing physiological conditions. The expression level of HC-1 was determined in skin, muscle, liver, kidney, and intestines of warm, cold, frozen, and dehydrated frogs using Western blot and immunohistofluorescence. Although HC-1 was widely distributed, the expression level varied among tissues and physiological conditions. In addition, HC-1 was present in both glycosylated and deglycosylated forms, the pattern of glycosylation also varied with tissues and physiological conditions. Thus, I conclude that HC-1 is widely expressed in tissues of Hyla chrysoscelis, and that it is regulated in these tissues according to physiological demands

Modeling and Analysis of the Spread of Japanese Encephalitis with Environmental Effects

A nonlinear mathematical model for the spread of Japanese Encephalitis, caused by infected mosquito feeding on susceptible human population incorporating demographic and environmental factors is proposed and analyzed. In the modeling process, it is assumed that the growth rates of reservoir animal population and vector mosquito population are enhanced due to environmental discharges caused by human population related factors. The model is analyzed by stability theory of differential equations and computer simulation. Both the disease-free and the endemic equilibria are found and their stability is investigated. It is found that whenever the disease-free equilibrium is locally asymptotically stable, the endemic equilibrium does not exist. The analysis of the model shows that if the growth rates of reservoir animal population and vector mosquito population caused by environmental factors increase, the spread of Japanese Encephalitis increases and the disease becomes more endemic due to human immigration. Numerical simulations are also carried out to investigate the influence of certain parameters on the spread of disease, to support the analytical results and illustrate possible behavioral scenario of the model

Modeling the Effect of Environmental Factors on the Spread of Bacterial Disease in an Economically Structured Population

We have proposed and analyzed a nonlinear mathematical model for the spread of bacterial disease in an economically structured population (rich and poor) including the role of vaccination. It is assumed that rich susceptible get infected through direct contact with infectives in the same class and with infectives from the poor class who work as service providers in the houses of rich people, living in much cleaner environment. The susceptible in the poor class are assumed to become infected through direct contact with infectives in the same class as well as by bacteria present in their own environment, degraded due to unhygienic environmental conditions. It is further assumed that the bacteria population affects only the population in the degraded environment of the poor class but does not survive in the clean environment of rich people. The density of bacteria population is assumed to be governed by a logistic model and is dependent on environmental discharges conducive to the growth of bacteria population. The cumulative density of environmental discharges depends upon the human population related factors of the poor class. The model analysis shows that the increased growth rate of environmental discharges increases the bacteria population density in the poor class due to unhygienic environmental conditions leading to increase the infectives in the poor class i.e., service providers. As a consequence, due to interaction with these service providers the spread of disease increases in the rich class. The improved environmental conditions of the region inhabited by service providers along with suitable vaccination strategy can be helpful in reducing the spread of the disease

Analysis of a Vaccination Model for Carrier Dependent Infectious Diseases with Environmental Effects

We have proposed and analyzed a nonlinear mathematical model for the spread of carrier dependent infectious diseases in a population with variable size structure including the role of vaccination. It is assumed that the susceptibles become infected by direct contact with infectives and/or by the carrier population present in the environment. The density of carrier population is assumed to be governed by a generalized logistic model and is dependent on environmental and human factors which are conducive to the growth of carrier population. The model is analyzed using stability theory of differential equations and numerical simulation. We have found a threshold condition, in terms of vaccine induced reproduction number R(φ) which is, if less than one, the disease dies out in the absence of carriers provided the vaccine efficacy is high enough, and otherwise the infection is maintained in the population. The model also exhibits backward bifurcation at R(φ) = 1. It is also shown that the spread of an infectious disease increases as the carrier population density increases. In addition, the constant immigration of susceptibles makes the disease more endemic

Canine Distemper Virus in Tigers (Panthera tigris) and Leopards (P. pardus) in Nepal

From wild dogs (Lycaon pictus) in the Serengeti to tigers (Panthera tigris altaica) in the Russian Far East, canine distemper virus (CDV) has been repeatedly identified as a threat to wild carnivores. Between 2020 and 2022, six Indian leopards (P. pardus fusca) presented to Nepali authorities with fatal neurological disease, consistent with CDV. Here, we report the findings of a serosurvey of wild felids from Nepal. A total of 48 serum samples were tested, comprising 28 Bengal tigers (P. t. tigris) and 20 Indian leopards. Neutralizing antibodies were identified in three tigers and six leopards, equating to seroprevalences of 11% (CI: 2.8–29.3%, n = 28) and 30% (CI: 12.8–54.3%, n = 20), respectively. More than one-third of seropositive animals were symptomatic, and three died within a week of being sampled. The predation of domestic dogs (Canis lupus familiaris) has been posited as a potential route of infection. A comparison of existing diet studies revealed that while leopards in Nepal frequently predate on dogs, tigers do not, potentially supporting this hypothesis. However, further work, including molecular analyses, would be needed to confirm this

The EYA Tyrosine Phosphatase Activity Is Pro-Angiogenic and Is Inhibited by Benzbromarone

Eyes Absents (EYA) are multifunctional proteins best known for their role in organogenesis. There is accumulating evidence that overexpression of EYAs in breast and ovarian cancers, and in malignant peripheral nerve sheath tumors, correlates with tumor growth and increased metastasis. The EYA protein is both a transcriptional activator and a tyrosine phosphatase, and the tyrosine phosphatase activity promotes single cell motility of mammary epithelial cells. Since EYAs are expressed in vascular endothelial cells and cell motility is a critical feature of angiogenesis we investigated the role of EYAs in this process. Using RNA interference techniques we show that EYA3 depletion in human umbilical vein endothelial cells inhibits transwell migration as well as Matrigel-induced tube formation. To specifically query the role of the EYA tyrosine phosphatase activity we employed a chemical biology approach. Through an experimental screen the uricosuric agents Benzbromarone and Benzarone were found to be potent EYA inhibitors, and Benzarone in particular exhibited selectivity towards EYA versus a representative classical protein tyrosine phosphatase, PTP1B. These compounds inhibit the motility of mammary epithelial cells over-expressing EYA2 as well as the motility of endothelial cells. Furthermore, they attenuate tubulogenesis in matrigel and sprouting angiogenesis in the ex vivo aortic ring assay in a dose-dependent fashion. The anti-angiogenic effect of the inhibitors was also demonstrated in vivo, as treatment of zebrafish embryos led to significant and dose-dependent defects in the developing vasculature. Taken together our results demonstrate that the EYA tyrosine phosphatase activity is pro-angiogenic and that Benzbromarone and Benzarone are attractive candidates for repurposing as drugs for the treatment of cancer metastasis, tumor angiogenesis, and vasculopathies

Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

Abstract Background Multicellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. Here we exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia. Methods Activation of DNA damage repair markers, proliferation, cell death, glycogen accumulation and developing hypoxia were investigated using immunofluorescence, immuno-histochemistry, EdU incorporation, Western blots, COMET assays, and pharmacological agents in A673 Ewing sarcoma spheroids and monolayer cultures. Results DNA damage marker γ-H2AX is observed in the hypoxic, peri-necrotic region of growing spheroids. While most proliferating cells are seen on the spheroid surface, there are also a few Ki-67 positive cells in the hypoxic zone. The hypoxia-induced phosphorylation of H2AX to form γ-H2AX in spheroids is attenuated by the ATM inhibitor KU55933, but not the ATR inhibitor VE-821. Conclusion Tumor spheroids mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable, proliferating, and undergoing DNA damage repair processes under these different micro-environmental conditions. ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 spheroids. Spheroids could offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment

Glycerol Uptake by Erythrocytes from Warm- and Cold-Acclimated Cope’s Gray Treefrogs

Cope’s gray treefrogs, Hyla chrysoscelis, accumulate glycerol during the period of cold acclimation that leads to the development of freeze tolerance. Glycerol must cross cell membranes in numerous processes during this time, including exit from hepatocytes where glycerol is synthesized and entry into other tissues, where glycerol is cryoprotective. Thus, we hypothesized that erythrocytes fromH. chrysoscelis would be permeable to glycerol and that that permeability would be up-regulated during cold acclimation. Further, we hypothesized that glycerol permeability would be associated with the expression of aquaporins, particularly those from the glyceroporin sub-family. Erythrocytes from warm-acclimated treefrogs had high glycerol permeability at 20°C, as assessed by the time required for osmotic lysis following suspension in 0.2 M glycerol. That osmotic lysis, as well as uptake of radio-labeled glycerol, was inhibited by 0.3 mM HgCl3. Permeability assessed via osmotic lysis was markedly reduced at 5°C. These properties were similar in animals deriving from northern (Ohio) and southern (Alabama) populations, although suggestive (through statistical interactions) of greater glycerol permeability in northern animals. Erythrocytes expressed mRNA and protein for a previously described glyceroporin, HC-3. In cold-acclimated animals, HC-3 protein expression was up-regulated, but we could not detect a concomitant enhancement of glycerol permeability

Structure-activity relationships of benzbromarone metabolites and derivatives as EYA inhibitory anti-angiogenic agents.

The tyrosine phosphatase activity of the phosphatase-transactivator protein Eyes Absent (EYA) is angiogenic through its roles in endothelial cell migration and tube formation. Benzbromarone, a known anti-gout agent, was previously identified as an inhibitor of EYA with anti-angiogenic properties. Here we show that the major metabolite of BBR, 6-hydroxy benzbromarone, is a significantly more potent inhibitor of cell migration, tubulogenesis and angiogenic sprouting. In contrast, other postulated metabolites of BBR such as 5-hydroxy benzbromaorne and 1'-hydroxy benzbromarone are less potent inhibitors of EYA tyrosine phosphatase activity as well as being less effective in cellular assays for endothelial cell migration and angiogenesis. Longer substituents at the 2 position of the benzofuran ring promoted EYA3 binding and inhibition, but were less effective in cellular assays, likely reflecting non-specific protein binding and a resulting reduction in free, bio-available inhibitor. The observed potency of 6-hydroxy benzbromarone is relevant in the context of the potential re-purposing of benzbromarone and its derivatives as anti-angiogenic agents. 6-hydroxy benzbromarone represents a metabolite with a longer half-life and greater pharmacological potency than the parent compound, suggesting that biotransformation of benzbromarone could contribute to its therapeutic activity

BAZ1B is dispensable for H2AX phosphorylation on Tyrosine 142 during spermatogenesis

Meiosis is precisely regulated by the factors involved in DNA damage response in somatic cells. Among them, phosphorylation of H2AX on Serine 139 (γH2AX) is an essential signal for the silencing of unsynapsed sex chromosomes during male meiosis. However, it remains unknown how adjacent H2AX phosphorylation on Tyrosine 142 (pTyr142) is regulated in meiosis. Here we investigate the meiotic functions of BAZ1B (WSTF), the only known Tyr142 kinase in somatic cells, using mice possessing a conditional deletion of BAZ1B. Although BAZ1B deletion causes ectopic γH2AX signals on synapsed autosomes during the early pachytene stage, BAZ1B is dispensable for fertility and critical events during spermatogenesis. BAZ1B deletion does not alter events on unsynapsed axes and pericentric heterochromatin formation. Furthermore, BAZ1B is dispensable for localization of the ATP-dependent chromatin remodeling protein SMARCA5 (SNF2h) during spermatogenesis despite the complex formation between BAZ1B and SMARCA5, known as the WICH complex, in somatic cells. Notably, pTyr142 is regulated independently of BAZ1B and is dephosphorylated on the sex chromosomes during meiosis in contrast with the presence of adjacent γH2AX. Dephosphorylation of pTyr142 is regulated by MDC1, a binding partner of γH2AX. These results reveal the distinct regulation of two adjacent phosphorylation sites of H2AX during meiosis, and suggest that another kinase mediates Tyr142 phosphorylation