The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target

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Molecular epidemiology of plasmid mediated resistance to fosfomycin among bacteria isolated from different environments

In this report we present data on the dispersion of a gene responsible for the plasmid-mediated resistance to fosfomycin among bacteria isolated from four hospitals and seven geographically separated domestic sewage treatment plants. Colony hybridization experiments, using a 0.85 kbp DNA fragment which carried the fosfomycin resistance determinant, have shown that the gene was present in isolates from three hospitals and six sewage plants although with different incidence and that it is carried by species of Enterobacteriaceae, Pseudomonas, Acinetobacter, Staphylococcus and Bacillus .     

Intracellular calcium release mediated by two muscarinic receptor subtypes

Four subtypes of muscarinic acetylcholine receptor (mAChR) were stably expressed in neuroblastoma-glioma hybrid cells (NG108-15). By combining fluorescent indicator dye (fura-2) studies with electrophysiological measurements it is shown that stimulation of mAChR I and mAChR III readily leads to release of calcium from intracellular stores and to associated conductance changes, whereas stimulation of mAChR II and mAChR IV exerts no such effect. Dose-response curves describing the amplitude or the delay of the calcium rise induced by acetylcholine suggest that the apparent affinity of mAChR III for its agonist is higher by about one order of magnitude than that of mAChR I. Ionic substitution experiments and current fluctuation analysis indicate that calcium activates a K⁺-specific conductance of ‘small’ single-channel amplitude similar to the SK type [1]. Furthermore, an outward current (M current) suppressed by activation of mAChR I and mAChR III has a single-channel amplitude corresponding to a conductance of approximately 3 pS.     

Comparing Stochastically Restricted Linear Estimators in a Regression Model

Conditions for superiority of the minimum dispersion estimator over another with respect to the covariance matrix are derived when the vector parameter of a regression model is subject to competing stochastic restrictions. The restrictions may also consist both of a deterministic part and a stochastic part.     

Discovery of dual ZAP70 and Syk kinases inhibitors by docking into a rare C-helix-out conformation of Syk

The non-receptor tyrosine kinase Syk (spleen tyrosine kinase) is a pharmaceutical relevant target because its over-activation is observed in several autoimmune diseases, allergy, and asthma. Here we report the identification of two novel inhibitors of Syk by high-throughput docking into a rare C-helix-out conformation published recently. Interestingly, both compounds are slightly more active on ZAP70 (Zeta-chain-associated protein kinase 70), which is the kinase closest to Syk in the phylogenetic tree of human kinases. Taken together, the docking pose and experimental results suggest that the higher affinity of the inhibitors for ZAP70 than Syk originates from a more populated C-helix-out conformation in ZAP70. The latter observation is congruent with the 100-fold lower intrinsic activity of ZAP70 than Syk, as the C-helix-out conformation is inactive. The pharmacophore features of DFG-in, C-helix-out compounds are analyzed in relation to DFG-out inhibitors.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

Water and ion permeability of a native collagen membrane

With the help of a ribonucleoprotein it is possible to precipitate collagen in a layer of fibers with a 700 Å period. As collagen is a constituent of many membrane systems in the body, it seemed interesting to investigate the permeability of ions and water through a native collagen membrane.The experiments were carried out with the help of an acryl glass apparatus, where an osmotic pressure, a hydrostatic pressure difference or both can be maintained between the two bulk phases separated by the membrane. The diffusion coefficients for NaCl and KCl were found to be comparable with those in other biological membranes (D_s = 9 · 10⁻⁷cm² · s⁻¹) whereas there is difference of more than three orders of magnitude in the hydraulic permeability (L_p = 6 cm⁴ · J⁻¹ · s⁻¹).Volume flow measurements caused by an osmotic gradient indicated that the reflection coefficient for NaCl and KCl is very small. In hydrostatic pressure experiments, the membrane shows a preferred direction for volume flows which seems to have something to do with the mode of preparation of the membrane.