Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

<雜録>歐洲ニ於ケル農業的勞働關係

HIV infection provokes a myriad of pathological effects on the immune system where many markers of CD4+ T cell dysfunction have been identified. However, most studies to date have focused on single/double measurements of immune dysfunction, while the identification of pathological CD4+ T cell clusters that is highly associated to a specific biomarker for HIV disease remain less studied. Here, multi-parametric flow cytometry was used to investigate immune activation, exhaustion, and senescence of diverse maturation phenotypes of CD4+ T cells. The traditional method of manual data analysis was compared to a multidimensional clustering tool, FLOw Clustering with K (FLOCK) in two cohorts of 47 untreated HIV-infected individuals and 21 age and sex matched healthy controls. In order to reduce the subjectivity of FLOCK, we developed an "artificial reference", using 2% of all CD4+ gated T cells from each of the HIV-infected individuals. Principle component analyses demonstrated that using an artificial reference lead to a better separation of the HIV-infected individuals from the healthy controls as compared to using a single HIV-infected subject as a reference or analyzing data manually. Multiple correlation analyses between laboratory parameters and pathological CD4+ clusters revealed that the CD4/CD8 ratio was the preeminent surrogate marker of CD4+ T cells dysfunction using all three methods. Increased frequencies of an early-differentiated CD4+ T cell cluster with high CD38, HLA-DR and PD-1 expression were best correlated (Rho = -0.80, P value = 1.96×10-11) with HIV disease progression as measured by the CD4/CD8 ratio. The novel approach described here can be used to identify cell clusters that distinguish healthy from HIV infected subjects and is biologically relevant for HIV disease progression. These results further emphasize that a simple measurement of the CD4/CD8 ratio is a useful biomarker for assessment of combined CD4+ T cell dysfunction in chronic HIV disease

Cerebrovascular Toxicity of PCB153 is Enhanced by Binding to Silica Nanoparticles

Environmental polychlorinated biphenyls (PCBs) are frequently bound onto nanoparticles (NPs). However, the toxicity and health effects of PCBs assembled onto nanoparticles are unknown. The aim of this study was to study the hypothesis that binding PCBs to silica NPs potentiates PCB-induced cerebrovascular toxicity and brain damage in an experimental stroke model. Mice (C57BL/6, males, 12-week-old) were exposed to PCB153 bound to NPs (PCB153-NPs), PCB153, or vehicle. PCB153 was administered in the amount of 5 ng/g body weight. A group of treated animals was subjected to a 40 min ischemia, followed by a 24 h reperfusion. The blood-brain barrier (BBB) permeability, brain infarct volume, expression of tight junction (TJ) proteins, and inflammatory mediators were assessed. As compared to controls, a 24 h exposure to PCB153-NPs injected into cerebral vasculature resulted in significant elevation of the BBB permeability, disruption of TJ protein expression, increased proinflammatory responses, and enhanced monocyte transmigration in mouse brain capillaries. Importantly, exposure to PCB153-NPs increased stroke volume and potentiated brain damage in mice subjected to ischemia/reperfusion. A long-term (30 days) oral exposure to PCB153-NPs resulted in a higher PCB153 content in the abdominal adipose tissue and amplified adhesion of leukocytes to the brain endothelium as compared to treatment with PCB153 alone. This study provides the first evidence that binding to NPs increases cerebrovascular toxicity of environmental toxicants, such as PCB153

Structural evidence for the critical role of the prion protein hydrophobic region in forming an infectious prion

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89-230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion