Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.     

Impacts of glutathione peroxidase-1 knockout on the protection by injected selenium against the pro-oxidant-induced liver aponecrosis and signaling in selenium-deficient mice

Previous research has suggested that repletion of cellular glutathione peroxidase (GPX1) activity by a single injection of Se was dissociated from the Se protection against the pro-oxidant-induced liver necrosis in Se-deficient rodents. Using the GPX1 knockout (GPX1-/-) mice, TUNEL assay, and apoptosis gene expression microarray, we have demonstrated strikingly different impacts of GPX1 knockout on hepatotoxicity and the related signaling induced by an intraperitoneal injection of 12.5 mg paraquat/kg body weight (b.wt.). In both Se-deficient GPX1-/- and wild-type (WT) mice, the paraquat did not induce typical liver necrosis, rather aponecrosis or necrapoptosis, a syncretic process of cell death sharing characteristics of both apoptosis and necrosis. The severity of liver aponecrosis and the associated mortality were reduced to a much greater extent by an injection of Se (ip, 50 microg/kg b.wt. as Na2SeO3) prior to paraquat stress in the WT mice, compared with the GPX1-/- mice. The induced liver aponecrosis seemed to be more apoptotic in the GPX1-/- mice but more necrotic in the WT mice. The paraquat-mediated gene or protein expression of proapoptotic Bax, Bcl-w, and Bcl-X(S), cell survival/death factors GADD45, MDM2, c-Myc, and caspase-3 was upregulated, but that of antiapoptotic Bcl-2 was downregulated in the GPX1-/- mice vs. the WT mice. Overall, these differences between the two groups of mice were related to a low level of liver GPX1 activity in the WT mice that represented < 4% of the normal physiological level. Therefore, the low level of GPX1 activity in the Se-deficient mice can exert a potent role in defending against liver aponecrosis induced by moderate oxidative stress.     

Aspirin Use and Lung Cancer Risk: A Possible Relationship? Evidence from an Updated Meta-Analysis

Background and Purpose

Growing evidence has emerged and controversial results reported on possible relationship between aspirin use and lung cancer risk. We, therefore, conducted this updated and comprehensive meta-analysis to evaluate this issue, with focus on dose-risk and duration-risk relationships.

Methods

We searched electronic databases including PUBMED, EMBASE and Cochrane library to identify eligible studies. Relative risk (RR) and its 95% confidence interval (CI) were used for cohort studies, while odds ratio (OR) were employed for case-control studies. The random effects and fixed effects models were used for analyses.

Results

18 studies were identified including 19835 lung cancer cases, which were eligible for inclusion in the present meta-analysis. Pooled data from case-control studies showed a significant inverse association between regular aspirin use and lung cancer risk. But for cohort studies, insignificant association was detected with little evidence of heterogeneity (RR: 1.05, 95%CI: 0.95 – 1.16; I²: 10.3%, p value: 0.351). In case-control studies, standard aspirin use (>325mg) was related to lower lung cancer incidence, compared with low-dose aspirin use (75–100mg). A similar trend was observed in cohort studies. Besides, when analysis was restricted to long time regular aspirin use (>5 years), insignificant results were reported in both cohort and case-control studies. Finally, regular aspirin use might result in higher reduction of non-small cell lung cancer incidence among men.

Conclusions

Our findings do not support the protective effect of regular aspirin use on lung cancer risk. Long time aspirin use, sex, dose and type of lung cancer might alter the effect of aspirin use on lung cancer risk. More well-designed studies are needed to further clarify these associations.     

Differential Phosphorylation of Smad1 Integrates BMP and Neurotrophin Pathways through Erk/Dusp in Axon Development

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Stable Li Metal Anode Enabled by Space Confinement and Uniform Curvature through Lithiophilic Nanotube Arrays

The application of lithium (Li) metal anodes in rechargeable batteries is primarily restricted by Li dendrite growth on the metal's surface, which leads to shortened cycle life and safety concerns. Herein, well‐spaced nanotubes with ultrauniform surface curvature are introduced as a Li metal anode structure. The ultrauniform nanotubular surface generates uniform local electric fields that evenly attract Li‐ions to the surface, thereby inducing even current density distribution. Moreover, the well‐defined nanotube spacing offers Li diffusion pathways to the electroactive areas as well as the confined spaces to host deposited Li. These structural attributes create a unique electrodeposition manner; i.e., Li metal homogenously deposits on the nanotubular wall, causing each Li nanotube to grow in circumference without obvious sign of dendritic formation. Thus, the full‐cell battery with the spaced Li nanotubes exhibits a high specific capacity of 132 mA h g^?1 at 1 C and an excellent coulombic efficiency of ≈99.85% over 400 cycles.     

Discovery and SAR analysis of phenylbenzo[d][1,3]dioxole-based proprotein convertase subtilisin/kexin type 9 inhibitors

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapeutic target for the development of cholesterol-lowering drugs. In the discovery of PCSK9/LDLR (low-density lipoprotein receptor) protein-protein interaction (PPI) impairing small molecules, a total of 47 phenylbenzo[d][1,3] dioxole-based compounds were designed and synthesised. The result revealed that the 4-chlorobenzyl substitution in the amino group is important for the PPI disrupting activity. In the hepatocyte-based functional tests, active compounds such as A12, B1, B3, B4 and B14, restored the LDLR levels on the surface of hepatic HepG2 cells and increased extracellular LDL uptake in the presence of PCSK9. It is notable that molecule B14 exhibited good performance in all the evaluations. Collectively, novel structures targeting PCSK9/LDLR PPI have been developed with hypolipidemic potential. Further structural modification of derived active compounds is promising in the discovery of lead compounds with improved activity for the treatment of hyperlipidaemia-related disorders.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Three lysine residues in the common β chain of the interleukin-5 receptor are required for Janus kinase (JAK)-dependent receptor ubiquitination, endocytosis, and signaling

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory diseases including allergic asthma and hypereosinophilic syndrome. Eosinophil physiology is critically dependent on IL-5 and the IL-5 receptor (IL-5R), composed of a ligand binding α chain (IL-5Rα), and a common β chain, βc. Previously, we demonstrated that the βc cytoplasmic tail is ubiquitinated and degraded by proteasomes following IL-5 stimulation. However, a complete understanding of the role of βc ubiquitination in IL-5R biology is currently lacking. By using a well established, stably transduced HEK293 cell model system, we show here that in the absence of ubiquitination, βc subcellular localization, IL-5-induced endocytosis, turnover, and IL-5R signaling were significantly impaired. Whereas ubiquitinated IL-5Rs internalized into trafficking endosomes for their degradation, ubiquitination-deficient IL-5Rs accumulated on the cell surface and displayed blunted signaling even after IL-5 stimulation. Importantly, we identified a cluster of three membrane-proximal βc lysine residues (Lys(457), Lys(461), and Lys(467)) whose presence was required for both JAK1/2 binding to βc and receptor ubiquitination. These findings establish that JAK kinase binding to βc requires the presence of three critical βc lysine residues, and this binding event is essential for receptor ubiquitination, endocytosis, and signaling.     

Endoplasmic reticulum stress induces inverse regulations of major functions in portal myofibroblasts during liver fibrosis progression

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis.