Chen Lv

Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter

The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.

Novel short antimicrobial peptide isolated from Xenopus laevis skin: Novel Short Antimicrobial Peptide

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright

The effect of heat stress on bull sperm quality and related HSPs expression

Heat stress dramatically decreases bull sperm quality and has recently received more attention due to the warmer global climate and more intensive production. However, no data exist regarding sperm quality or the related molecular mechanisms under heat stress. Recent studies showed that inducible heat shock proteins (HSPs) play an important role in the dairy heat stress regulation. In this article, to investigate the impacts of heat stress on sperm quality and the associated molecular mechanisms, sperm quality and enzyme activities concerning acrosome reaction were assessed in Simmental, Limousin and Yanbian bulls under heat stress. Subsequently, changes in heat shock protein expression profiles of Simmental bulls were observed, because we observed that sperm quality of these bulls was most sensitive to heat stress. Finally, the relationship between sperm quality and heat shock protein expression under heat stress was analyzed. The results show that summer heat stress decreased the sperm quality of the three bull breeds significantly. Moreover, different levels of heat stimulation induced various enzyme activity changes, among which the activity change in acrosomal enzyme was the most remarkable. Furthermore, the expression of heat shock proteins in the sperm was influenced by the imposed heat stress, among which the expression levels of HSP60 and HSP70 were increased while HSP90 decreased. In summary, our data show that heat stress seriously affects sperm quality and that HSP90 was most sensitive, although it should be noted that seasonal effects may confound these results. This change in heat shock protein expression may be the major factor that affected the sperm quality of the bulls. The findings may provide a new hypothesis for how heat stress impacts reproduction mechanistically.

Influence Factors of Remediation of Benzene in Groundwater by Air Sparging

Medium sand was selected as simulation medium. Benzene was chosen as the target pollutant. Soil column simulation experiments were carried out to investigate the effects of influencing factors such as aeration rate, medium permeability and air injection mode on benzene removal efficiency in groundwater. The results showed that the aeration rate and medium permeability affected AS remediation efficiency significantly. The benzene removal efficiency increased with the aeration rate. When the aeration rate exceeded 300 mL•min-1, the removal efficiency did not increase any more. The bigger the hydraulic conductivity was, the higher removal efficiency of benzene was. In the same operation condition, pulsed intermittent air injection had advantages over continuous air injection for benzene •remediation in the medium sand with low hydraulic conductivity.

Equivalent Circuit Model of Top-Emitting OLED for the Designing of OLED-on-Silicon Microdisplay

In the commonly-used EDA software, there is no equivalent circuit model of top-emitting OLED for SPICE, so it is difficult while the OLED pixel driving circuits are designed such as in microdisplay applications. To solve the problem, its SPICE model of top-emitting OLED was studied in this paper. Three models including the diode-connected NMOS model, the single-diode model, and the double-diode paralleled model are discussed and their simulation results from EDA software HSPICE are compared. Results show that the fitted errors obtained from the diode-connected NMOS model is the smallest, but its simulated errors are the largest. The fitted errors and the simulated errors obtained from the single-diode model are also large. However, small fitted errors and simulated errors got from double-diode paralleled model show that this model satisfies the application of the designing for OLED pixel driving circuits.

Pseudo‑Ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway

Ginsenoside Rh2, a major effective constituent of ginseng, has been suggested to have a pro-apoptotic effect in a variety of cancer cells. Pseudo-Ginsenside-Rh2 (pseudo-G-Rh2) is a novel derivative of ginsenoside Rh2. The aim of the present study was to evaluate the effect of pseudo-G-Rh2 on the apoptosis of lung adenocarcinoma A549 cells. The cytotoxicity of pseudo-G-Rh2 on A549 cells was evaluated using an MTT assay. Apoptosis was detected using DAPI staining and flow cytometry. The expression of apoptosis associated proteins was identified by western blot analysis. The results demonstrated that pseudo-G-Rh2 inhibits the proliferation of A549 cells in a dose-dependent manner. DAPI staining revealed topical morphological changes in apoptotic bodies following pseudo-G-Rh2 treatment. Flow cytometric analysis revealed that the percentage of Annexin V-fluorescein isothiocyanate-positive cells, which are apoptotic, increased with pseudo-G-Rh2 treatment in a dose-dependent manner. Furthermore, treatment with pseudo-G-Rh2 increased the level of reactive oxygen species in A549 cells as well as the activation of caspase-9, caspase-3 and poly ADP-ribose polymerase. Pseudo-G-Rh2 treatment was observed to induce mitochondrial membrane potential loss. Furthermore, the results of western blotting revealed that B-cell lymphoma 2 (Bcl-2) expression was significantly decreased while Bcl-2-associated X protein expression was significantly upregulated in A549 cells with pseudo-G-Rh2 treatment. Pseudo-G-Rh2-induced apoptosis was accompanied by sustained phosphorylation of Ras, Raf, extracellular signal-regulated kinase (ERK) and p53. In conclusion, the results of the present study suggest that pseudo-G-Rh2 induces mitochondrial apoptosis in A549 cells and is responsible for excessive activation of the Ras/Raf/ERK/p53 pathway.

The Mechanism of Dietary Protein Modulation of Bone Metabolism via Alterations in Members of the GH/IGF Axis

Dietary protein intake as a critical regulatory factor of bone metabolism is a vital element to regulate nutritional status of mammals. Under the action of protease, dietary protein is digested into peptides and free amino acids (FAAs). Then, the metabolites are absorbed by enterocytes and metabolized in various organs of mammals. The dietary protein intake regulates bone metabolism generally via two aspects, dietary itself and signaling transduction. At the dietary level, different kinds of amino acids (AAs) of dietary protein may affect various protein metabolism of bone by regulating proteasome depending on proteolysis and protein synthesis. In addition, dietary protein from multiple sources such as animal, vegetal and healthcare products, presents distinct influences on bone metabolism via regulating calcium balance; At the cellular level, these products can regulate several biological functions via regulating signaling transduction. For example, the significant member of growth hormone/insulin-like growth factor (GH/IGF) axis can be regulated by dietary protein, which has an influence on bone metabolism through different approaches. This review mainly discusses the relationship between dietary protein and GH/IGF axis and illustrates the regulation of bone metabolism in mammals by dietary protein and its signaling transduction.

Effects of GnRHR polymorphisms on sperm quality in Chinese water buffalo

Gonadotropin-releasing hormone receptor (GnRHR) plays a critical physiological role in animal reproduction and is a potential marker for improving sperm quality. In the present study, eight SNPs (g.539T>C, g.640A>G, g.655T>C, g.707T>C, g.812A>G, g.18951A>T g.16867T>C and g.18953Indel GGCAAAGTAA) were detected in the GnRHR gene from one-hundred-sixty-five water buffalo by direct sequencing and identification of overlapping peaks. All SNPs were associated significantly with the ejaculate volume and two genes (g.655T>C and g.707T>C) were correlated with sperm abnormalities. Furthermore, three haplotypes (H1:TAI, H2:CT-, and H3:TT-) were identified by linkage disequilibrium analysis and were composed of four combined genotypes. Notably, buffalo with the combined genotypes H1H2 and H1H3 had the higher ejaculate volume compared to the other combined genotypes. Among the eight SNPs and four combined genotypes, the deletion of GGCAAAGTAA at position 18953bp in GnRHR was associated significantly with a higher ejaculate volume. Moreover, the GGCAAAGTAA deletion may lead to the miR8661 binding failure and subsequent changes in GnRHR gene expression. In the present study, we demonstrate that there is a significant association between SNPs in the GnRHR gene and the sperm ejaculate volume of Chinese water buffalo. To the best of our knowledge, this study is the first to address the association between the SNPs in the GnRHR gene and the sperm quality of Chinese buffalo.

Amplification Experiment on Quick Start-up of Filter for Removing Manganese by Contact Oxidation Method

Based on the pilot experiment on quick start-up of filter for removing manganese, amplification experiment was carried out. High concentration aeration and low concentration running were used in this experiment as starting mode. The maturity period of the filter for manganese removal is 9 days. Through the research on the distribution of manganese ions in filter bed by different filtration rate, the result shows a bigger depth of the filter bed is needed with the increase of filtration rate. For raw water with manganese ions concentration of 1.0-1.5 mg•L-1, the highest flow rate that can be handled by this equipment is 0.7 m•h-1. The blockages in filter bed can be washed out under the condition that the back washing intensity is 7.81 L•s-1•m-2 and the back washing time is 5 min. The back washing period of the filter is 13 days.