Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

Effects of Methane-Rich Saline on the Capability of One-Time Exhaustive Exercise in Male SD Rats

Purpose

To explore the effects of methane-rich saline (CH₄ saline) on the capability of one-time exhaustive exercise in male SD rats.

Methods

Thirty rats were equally divided into to three groups at random: control group (C), placebo group (P) and methane saline group (M). Rats in M group underwent intraperitoneal injection of CH₄ saline, and the other two groups simultaneously underwent intraperitoneal injection of normal saline. Then, the exercise capability of rats was tested through one-time exhaustive treadmill exercise except C group. Exercise time and body weight were recorded before and after one-time exhaustive exercise. After exhaustive exercise, the blood and gastrocnemius samples were collected from all rats to detect biochemical parameters in different methods.

Results

It was found that the treadmill running time was significantly longer in rats treated with CH₄ saline. At the same time, CH₄ saline reduced the elevation of LD and UN in blood caused by one-time exhaustive exercise. The low level of blood glucose induced by exhaustive exercise was also normalized by CH₄ saline. Also CH₄ saline lowered the level of CK in plasma. Furthermore, this research indicated that CH₄ saline markedly increased the volume of T-AOC in plasma and alleviated the peak of TNF-α in both plasma and gastrocnemius. From H&E staining, CH₄ saline effectively improved exercise-induced structural damage in gastrocnemius.

Conclusions

CH₄ saline could enhance exercise capacity in male SD rats through increase of glucose aerobic oxidation, improvement of metabolic clearance and decrease of exhaustive exercise-induced gastrocnemius injury.     

Prevalence and phylogenetic characterization of canine coronavirus from diseased pet dogs in Beijing,China

正Dear Editor,Canine coronavirus including canine coronavirus(CCoV)and canine respiratory coronavirus(CRCoV)have been recognized as pathogenic viruses to dogs worldwide,causing enteric and respiratory issues(Buonavoglia et al.,2006).Zoonotic transfer of the viruses from the animal kingdom to humans has been repeatedly observed over the past decade,     

A leaf shape mutant provides insight into PINOID Serine/Threonine Kinase function in cucumber(Cucumis sativus L.)

Optimizing leaf shape is a major challenge in efforts to develop an ideal plant type. Cucumber leaf shapes are diverse; however, the molecular regulatory mechanisms underlying leaf shape formation are unknown. In this study, we obtained a round leaf mutant(rl) from an ethyl methanesulfonate-induced mutagenesis population. Genetic analysis revealed that a single recessive gene, rl, is responsible for this mutation. A modified Mut Map analysis combined linkage mapping identified a single nucleotide polymorphism within a candidate gene,Csa1 M537400, as the mutation underlying the trait.Csa1 M537400 encodes a PINOID kinase protein involved in auxin transport. Expression of Csa1 M537400 was significantly lower in the rl mutant than in wild type, and it displayed higher levels of IAA(indole-3-acetic acid) in several tissues. Treatment of wild-type plants with an auxin transport inhibitor induced the formation of round leaves,similar to those in the rl mutant. Altered expression patterns of several auxin-related genes in the rl mutant suggest that rl plays a key role in auxin biosynthesis,transport, and response in cucumber. These findings provide insight into the molecular mechanism underlying the regulation of auxin signaling pathways in cucumber,and will be valuable in the development of an ideal plant type.     

Benchmarking the Activity,Stability, and Inherent Electrochemistry of Amorphous Molybdenum Sulfide for Hydrogen Production

Anodically electrodeposited amorphous molybdenum sulfide (AE‐MoS_x) has attracted significant attention as a non‐noble metal electrocatalyst for its high activity toward the hydrogen evolution reaction (HER). The [Mo₃S₁₃]^2? polymer‐based structure confers a high density of exposed sulfur moieties, widely regarded as the HER active sites. However, their intrinsic complexity conceals full understanding of their exact role in HER catalysis, hampering their full potential for water splitting applications. In this report, a unifying approach is adopted accounting for modifications in the inherent electrochemistry (EC), HER mechanism, and surface species to maximize the AE‐MoS_x electroactivity over a broad pH region (0–10). Dramatic enhancements in HER performance by selective electrochemical cycling within reductive (overpotential shift, η_HER ≈ ?350 mV) and electro‐oxidative windows (η_HER ≈ ?290 mV) are accompanied by highly stable performance in mildly acidic electrolytes. Joint analysis of X‐ray photoelectron spectroscopy, Raman, and EC experiments corroborate the key role of bridging and terminal S ligands as active site generators at low pH, and reveal molybdenum oxysulfides (Mo⁵⁺O_xS_y) to be the most active HER moiety in AE‐MoS_x in mildly acidic‐to‐neutral environments. These findings will be extremely beneficial for future tailoring of MoS_x materials and their implementation in commercial electrolyzer technologies.     

Structural and functional characterization of a novel acidophilic 7α‐hydroxysteroid dehydrogenase

7α‐Hydroxysteroid dehydrogenase (7α‐HSDH) is an NAD(P)H‐dependent oxidoreductase belonging to the short‐chain dehydrogenases/reductases. In vitro, 7α‐HSDH is involved in the efficient biotransformation of taurochenodeoxycholic acid (TCDCA) to tauroursodeoxycholic acid (TUDCA). In this study, a gene encoding novel 7α‐HSDH (named as St‐2‐1) from fecal samples of black bear was cloned and heterologously expressed in Escherichia coli. The protein has subunits of 28.3 kDa and a native size of 56.6 kDa, which suggested a homodimer. We studied the relevant properties of the enzyme, including the optimum pH, optimum temperature, thermal stability, activators, and inhibitors. Interestingly, the data showed that St‐2‐1 differs from the 7α‐HSDHs reported in the literature, as it functions under acidic conditions. The enzyme displayed its optimal activity at pH 5.5 (TCDCA). The acidophilic nature of 7α‐HSDH expands its application environment and the natural enzyme bank of HSDHs, providing a promising candidate enzyme for the biosynthesis of TUDCA or other related chemical entities.     

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment

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