Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

Introduction The aim of the present study was to describe autonomic urethral sphincter (US) innervation using specific muscular and neuronal antibody markers and 3D reconstruction. Material and methods We performed en‐bloc removal of the entire pelvis of three male human fetuses between 18 and 40 weeks. Serial whole mount sections (5 μm intervals) were stained and investigated. The sections were stained with Masson's trichrome and Eosin Hematoxylin, and immunostained with: anti‐SMA antibody for smooth muscle; anti‐S100 antibody for all nerves; and anti‐PMP22 antibody, anti‐TH antibody, anti‐CGRP antibody, anti‐NOS antibody for somatic, adrenergic, sensory and nitrergic nerve fibers, respectively. The slides were digitized for 3D reconstruction to improve topographical understanding. An animated reconstruction of the autonomic innervation of the US was generated. Results The external and internal US are innervated by autonomic nerves of the inferior hypogastric plexus (IHP). These nerves are sympathetic (positive anti‐TH antibody), sensory (positive anti‐CGRP antibody), and nitrergic (positive anti‐NOS antibody). Some autonomic fibers run within the neurovascular bundles, posterolaterally. Others run from the IHP to the posteromedial aspect of the prostate apex, above an through the rectourethral muscle. The external US is also innervated by somatic nerves (positive anti‐PMP22 antibody) arising from the pudendal nerve, joining the midline but remaining below the rectourethral. Conclusions This study provides anatomical evidence of an autonomic component in the innervation of the external US that travels in the neurovascular bundle. During radical prostatectomy, the rectourethral muscle and the neurovascular bundles are to be preserved, particularly during apical dissection.

No abstract

The sustained immunosuppression associated with severe sepsis favors an increased susceptibility to secondary infections and remains incompletely understood. Plasmablast and plasma cell subsets, whose primary function is to secrete antibodies, have emerged as important suppressive populations that expand during sepsis. In particular, sepsis supports CD39hi plasmablast metabolic reprogramming associated with adenosine‐mediated suppressive activity. Arginine deficiency has been linked to an increased risk of secondary infections in sepsis. Overcoming arginine shortage by citrulline administration efficiently improves sepsis‐induced immunosuppression and secondary infections in the cecal ligation and puncture murine model. Here, we aimed to determine the impact of citrulline administration on B cell suppressive responses in sepsis. We demonstrate that restoring arginine bioavailability through citrulline administration markedly reduces the dominant extrafollicular B cell response, decreasing the immunosuppressive LAG3+ and CD39+ plasma cell populations, and restoring splenic follicles. At the molecular level, the IRF4/MYC‐mediated B cell reprogramming required for extrafollicular plasma cell differentiation is shunted in the splenic B cells of mice fed with citrulline. Our study reveals a prominent impact of nutrition on B cell responses and plasma cell differentiation and further supports the development of citrulline‐based clinical studies to prevent sepsis‐associated immune dysfunction.

Viper venom as a hemolytic biochemical “cocktail” of toxins, primarily cause to the systemic alteration of blood cells. In the sixties and seventies, human erythrocytes were extensively studied, but the mechanical and chemical stresses commonly exerted on red blood cells continue to attract interest of scientists for the study of membrane structure and function. Here we monitor the effect of Vipera latifi venom on human erythrocytes ghost membranes using phase contrast microscopy and changes in ATPase activity under snake venom influence in vitro. The ion pumps (Na+, K+)-ATPase and (Ca2+, Mg2+)-ATPase plays a pivotal role in the active transport of certain cations and maintenance of intracellular electrolyte homeostasis. We also describe the action of Vipera latifi venom on the freeradical processes in the membrane of erythrocyte ghosts and changes of activity of superoxide dismutase in course of envenomation.

Increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of snake venoms. However, the complexity of animal venoms and the extreme synergy of the venom components during envenomation calls for critical review and analysis. The epithelium is a primary target for injected viper venom’s toxic substances, and therefore, is a focus in modern toxinology. We used the Vero epithelial cell line as a model to compare the actions of a crude Macrovipera lebetina obtusa (Levantine viper) venom with the actions of the same venom with two key enzymatic components inhibited (specifically, phospholipase A2 (PLA2) and metalloproteinases) in the bioenergetic cellular response, i.e., oxygen uptake and reactive oxygen species generation. In addition to the rate of free-radical oxidation and lipid peroxidation, we measured real-time mitochondrial respiration (based on the oxygen consumption rate) and glycolysis (based on the extracellular acidification rate) using a Seahorse analyzer. Our data show that viper venom drives an increase in both glycolysis and respiration in Vero cells, while the blockage of PLA2 or/and metalloproteinases affects only the rates of the oxidative phosphorylation. PLA2-blocking in venom also increases cytotoxic activity and the overproduction of reactive oxygen species. These data show that certain components of the venom may have a different effect within the venom cocktail other than the purified enzymes due to the synergy of the venom components.

The purpose of this work is to reveal the transnational multi-layered political role of the diaspora, particularly the main actor of its political activity - the institution of ethnic lobbying, which implies dual relations between the homeland and the host state. The article aims to understand the place and role of ethnic lobbying as a transnational political factor in modern international relations. Historical-comparative, structural-operational and behavioral methods of scientific analysis were used to achieve the goal of the research and the solution of the set problems. It was concluded that, the activity of ethnic lobby groups is aimed not only at supporting the homeland, but also at influencing. In addition, ethnic lobby groups can also be adversely affected by both the home state and the host state. In that context, however, lobby groups try to maintain their relatively independent role.

No abstract