Trypan Blue Dye Enters Viable Cells Incubated with the Pore-Forming Toxin HlyII of Bacillus cereus

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability

Thermalized Displaced and Squeezed Number States in Coordinate Representation

Within the framework of thermofield dynamics, the wavefunctions of the thermalized displaced number and squeezed number states are given in the coordinate representation. Furthermore, the time evolution of these wavefunctions is considered by introducing a thermal coordinate representation, and we also calculate the corresponding probability densities, average values and variances of position coordinate, which are consistent with results in the literature.Comment: 12 pages, no figures, Revtex. v3: substantially revise

Bacillus anthracis Protease InhA Increases Blood-Brain Barrier Permeability and Contributes to Cerebral Hemorrhages

Hemorrhagic meningitis is a fatal complication of anthrax, but its pathogenesis remains poorly understood. The present study examined the role of B. anthracis-secreted metalloprotease InhA on monolayer integrity and permeability of human brain microvasculature endothelial cells (HBMECs) which constitute the blood-brain barrier (BBB). Treatment of HBMECs with purified InhA resulted in a time-dependent decrease in trans-endothelial electrical resistance (TEER) accompanied by zonula occluden-1 (ZO-1) degradation. An InhA-expressing B. subtilis exhibited increased permeability of HBMECs, which did not occur with the isogenic inhA deletion mutant (ΔinhA) of B. anthracis, compared with the corresponding wild-type strain. Mice intravenously administered with purified InhA or nanoparticles-conjugated to InhA demonstrated a time-dependent Evans Blue dye extravasation, leptomeningeal thickening, leukocyte infiltration, and brain parenchymal distribution of InhA indicating BBB leakage and cerebral hemorrhage. Mice challenged with vegetative bacteria of the ΔinhA strain of B. anthracis exhibited a significant decrease in leptomeningeal thickening compared to the wildtype strain. Cumulatively, these findings indicate that InhA contributes to BBB disruption associated with anthrax meningitis through proteolytic attack on the endothelial tight junctional protein zonula occluden (ZO)-1

Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes.In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas

Role of Myosin Va in the Plasticity of the Vertebrate Neuromuscular Junction In Vivo

Background: Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ). Methodology/Principal Findings: Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins. Conclusions/Significance: In summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to th

Expression and function of G-protein-coupled receptorsin the male reproductive tract

This review focuses on the expression and function of muscarinic acetylcholine receptors (mAChRs), α1-adrenoceptors and relaxin receptors in the male reproductive tract. The localization and differential expression of mAChR and α1-adrenoceptor subtypes in specific compartments of the efferent ductules, epididymis, vas deferens, seminal vesicle and prostate of various species indicate a role for these receptors in the modulation of luminal fluid composition and smooth muscle contraction, including effects on male fertility. Furthermore, the activation of mAChRs induces transactivation of the epidermal growth factor receptor (EGFR) and the Sertoli cell proliferation. The relaxin receptors are present in the testis, RXFP1 in elongated spermatids and Sertoli cells from rat, and RXFP2 in Leydig and germ cells from rat and human, suggesting a role for these receptors in the spermatogenic process. The localization of both receptors in the apical portion of epithelial cells and smooth muscle layers of the vas deferens suggests an involvement of these receptors in the contraction and regulation of secretion.Esta revisão enfatiza a expressão e a função dos receptores muscarínicos, adrenoceptores α1 e receptores para relaxina no sistema reprodutor masculino. A expressão dos receptores muscarínicos e adrenoceptores α1 em compartimentos específicos de dúctulos eferentes, epidídimo, ductos deferentes, vesícula seminal e próstata de várias espécies indica o envolvimento destes receptores na modulação da composição do fluido luminal e na contração do músculo liso, incluindo efeitos na fertilidade masculina. Além disso, a ativação dos receptores muscarínicos leva à transativação do receptor para o fator crescimento epidermal e proliferação das células de Sertoli. Os receptores para relaxina estão presentes no testículo, RXFP1 nas espermátides alongadas e células de Sertoli de rato e RXFP2 nas células de Leydig e germinativas de ratos e humano, sugerindo o envolvimento destes receptores no processo espermatogênico. A localização de ambos os receptores na porção apical das células epiteliais e no músculo liso dos ductos deferentes de rato sugere um papel na contração e na regulação da secreção.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de FarmacologiaUNIFESP, EPM, Depto. de FarmacologiaSciEL

Maternal and neonatal health expenditure in Mumbai slums (India): A cross sectional study

Background: The cost of maternity care can be a barrier to access that may increase maternal and neonatal mortality risk. We analyzed spending on maternity care in urban slum communities in Mumbai to better understand the equity of spending and the impact of spending on household poverty.Methods: We used expenditure data for maternal and neonatal care, collected during post-partum interviews. Interviews were conducted in 2005-2006, with a sample of 1200 slum residents in Mumbai (India). We analysed expenditure by socio-economic status (SES), calculating a Kakwani Index for a range of spending categories. We also calculated catastrophic health spending both with and without adjustment for coping strategies. This identified the level of catastrophic payments incurred by a household and the prevalence of catastrophic payments in this population. The analysis also gave an understanding of the protection from medical poverty afforded by coping strategies (for example saving and borrowing).Results: A high proportion of respondents spent catastrophically on care. Lower SES was associated with a higher proportion of informal payments. Indirect health expenditure was found to be (weakly) regressive as the poorest were more likely to use wage income to meet health expenses, while the less poor were more likely to use savings. Overall, the incidence of catastrophic maternity expenditure was 41%, or 15% when controlling for coping strategies. We found no significant difference in the incidence of catastrophic spending across wealth quintiles, nor could we conclude that total expenditure is regressive.Conclusions: High expenditure as a proportion of household resources should alert policymakers to the burden of maternal spending in this context. Differences in informal payments, significantly regressive indirect spending and the use of savings versus wages to finance spending, all highlight the heavier burden borne by the most poor. If a policy objective is to increase institutional deliveries without forcing households deeper into poverty, these inequities will need to be addressed. Reducing out-of-pocket payments and better regulating informal payments should have direct benefits for the most poor. Alternatively, targeted schemes aimed at assisting the most poor in coping with maternal spending (including indirect spending) could reduce the household impact of high costs

Modulation of summer monsoon sub-seasonal surface air temperature over India by soil moisture-temperature coupling

The influence of soil moisture on the sub-seasonal warmer surface air temperature anomalies during drier soil conditions associated with break spells in the Indian summer monsoon precipitation is explored using observations. The multi-model analysis of land surface states and fluxes available from the Second Global Soil Wetness Project (GSWP-2) are found useful in understanding the mechanism for this soil moisture-temperature coupling on subseasonal timescales. The analysis uses a soil moisture-temperature coupling diagnostic computed based on linear correlations of daily fields. It is shown that the summer surface air temperature variations are linked to intraseasonal variations of the Indian monsoon precipitation, which control the land-climate coupling by modulating the soil moisture variations. Strong coupling mainly occurs during dry soil states within the summer monsoon season over the transition zones between wet and dry climates of central to north-west India. In contrast, the coupling is weak for constantly wet and energy-limited evaporative regimes over eastern India during the entire summer monsoon season. This observational based analysis provided a better understanding of the linkages between the sub-seasonal dry soil states and warm conditions during the Indian summer monsoon season. A proper representation of these aspects of land-atmosphere interactions in weather and climate models used for sub-seasonal and seasonal monsoon forecasting could be critical for several applications, in particular agriculture. The soil moisture-temperature coupling diagnostic used in this study will be a useful metric for evaluating the performance of weather and climate models