Oral Administration of the Antimicrobial Peptide Mastoparan X Alleviates Enterohemorrhagic Escherichia coli-Induced Intestinal Inflammation and Regulates the Gut Microbiota.

The gut microbiota plays an important role in intestinal immune system development and in driving inflammation. Antibiotic administration for therapeutic purposes causes an imbalance in the gut microbiota. Antimicrobial peptides can regulate the gut microbiota and maintain intestinal homeostasis. The aim of this study was to investigate the anti-inflammatory effects and regulation of the gut microbiota by the orally administered antimicrobial peptide mastoparan X (MPX). In this study, Escherichia coli was used to induce intestinal inflammation, and the results showed that MPX+ E. coli alleviated weight loss and intestinal pathological changes in necropsy specimens of E. coli-infected mice. MPX+ E. coli reduced the serum levels of the inflammation-related proteins interleukin-2, interleukin-6, tumour necrosis factor-α, myeloperoxidase, and lactate dehydrogenase on days 7 and 28. Furthermore, MPX+ E. coli increased the length of villi and reduced the infiltration of inflammatory cells into the jejunum and colon post infection. Scanning electron microscopy and transmission electron microscopy results showed that MPX could improve the morphology of jejunum villi and microvilli and increase tight junction protein levels. 16S rRNA sequencing analysis of caecal content samples showed that the species diversity and richness were lower in the E. coli-infected group. At the genus level, MPX+ E. coli significantly reduced the abundance of Bacteroidales and Alistipes and enhanced the relative abundance of Muribaculaceae. Alpha-diversity analyses (Shannon index) showed that MPX significantly increased the microbial diversity of mice. Overall, this study is the first to investigate the effects of oral administration of MPX on intestinal inflammation and the gut microbiota, providing a new perspective regarding the prevention of enteritis and maintenance of intestinal homeostasis.

Antimicrobial peptide MPX attenuates LPS-induced inflammatory response and blood-testis barrier dysfunction in Sertoli cells.

Orchitis accounts for a high proportion of male animal reproductive disorders. Hence, it is urgent to identify drugs for the prevention and treatment of orchitis. Antimicrobial peptides (AMPs) are currently recognized as one of the most promising alternatives to antibiotics. However, the protective effects of AMPs on lipopolysaccharide (LPS)-induced orchitis have not been reported. In this study, we developed an LPS-induced orchitis model in which primary bovine Sertoli cells were used as model cells. MPX was indicated to effectively reduce the inflammatory response of Sertoli cells. MPX attenuated the gene expression of the proinflammatory cytokines TNF-α, IL-6 and IL-1ß by suppressing the MAPK pathway, especially the phosphorylation of p38 and ERK. MPX also decreased the oxidative stress response caused by LPS and upregulated Occludin and Claudin-1 expression, thereby maintaining the integrity of the blood-testis barrier. Moreover, we found that MPX inhibited apoptosis in Sertoli cells. In a mouse model, we found that MPX significantly inhibited the disruptive effects of LPS, reducing seminiferous epithelium damage, vacuolations, hyperplasia, and apoptosis in spermatogenic cells and rescuing spermatogenesis. In addition, the expression of inflammatory factors such as IL-1ß, IL-18, IL-6 and TNF-α was decreased after MPX treatment in the mouse testes. MPX had no effect on other organs in mice, indicating its safety. This study was undertaken to investigate how MPX regulates the inflammatory response in Sertoli cells and provide a reference for the clinical prevention and treatment of male animal orchitis.

Tandem repeat sequence of duck circovirus serves as downstream sequence element to regulate viral gene expression.

Duck circovirus (DuCV) has a small, single-stranded circular DNA genome of approximately 1.99 kb. Through a genome sequence analysis using the dottup program, we found that a quadruple tandem repeat sequence (QTR) in the intergenic region between the rep and cap genes of the DuCV genome, but not in other circoviruses. The QTR was also substantially different and evolutionarily conserved in the genotype 1 and 2 DuCV strains. Furthermore, a luciferase reporter assay demonstrated that QTR functioned as a downstream sequence element (DSE) of polyadenylation signals to enhance mRNA stability, which was dependent on four copies but not the QTR direction. Cap and Rep expression derived by subgenomic constructs also revealed a critical role of QTR in regulating viral gene expression. Finally, a reverse genetic study of a DuCV-based minicircle DNA technique found that a deletion of QTR induced a significant deficiency in viral genes transcription and replication. Our findings were the first to report that QTR only exists in the DuCV genome and serves as a novel molecular marker of DuCV genotyping, and has revealed its crucial biological function in regulating viral gene expression.

Interferon-γ regulates cell malignant growth via the c-Abl/HDAC2 signaling pathway in mammary epithelial cells.

Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.

A corn straw-based diet increases release of inflammatory cytokines in peripheral blood mononuclear cells of dairy cows.

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-ß1 (TGF-ß1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.

Characterization of the complete mitochondrial genome of cape elephant shrew, Elephantulus edwardii.

In this study, the complete mitochondrial genome of cape elephant shrew, Elephantulus edwardii, was determined through sequencing of PCR fragments. The complete mitochondrial genome of E. edwardii was 16,552 bp in length and and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The overall nucleotide composition is: 32.7% A, 29.5% T, 25.0% C, and 12.9% G, with a total G+C content of 37.9%. By phylogenetic analysis using Bayesian method, E. edwardii showed the closest relationship with another Elephantulus speceis (Genbank accession: AB096867.1).

Development of an Indirect Dot-PPA-ELISA using glutamate dehydrogenase as a diagnostic antigen for the rapid and specific detection of Streptococcus suis and its application to clinical specimens.

Streptococcus suis is an important zoonotic pathogen causing infections in pigs and humans. Bacterial surface-related proteins are often explored as potential vaccine candidates and diagnostic antigens. In the present study, glutamate dehydrogenase, a highly conserved immunogenic extracellular protein, was used to establish a dot horseradish peroxidase enzyme-linked staphylococcal protein A immunosorbent assay (Dot-PPA-ELISA) for diagnosis of S. suis infection. The antigen-antibody reaction was optimised through checkerboard titration involving serial dilutions, followed by selective blocking tests and evaluations of cross-reaction, repeatability, and stability. Comparative analysis by using a conventional plate ELISA kit showed that the specificity and sensitivity of the Dot-PPA-ELISA were 97.5 and 96.6%, respectively. Furthermore, dynamic changes in the levels of antibody in rabbits immunised with a propolis inactivated vaccine were monitored by Dot-PPA-ELISA. A total seroprevalence of 73.1% in 305 pig serum samples indicated the method's applicability to detect S. suis infection. Cumulatively, the results suggested that Dot-PAA-ELISA is a convenient, rapid, sensitive, and specific diagnostic method suitable for studying large numbers of samples obtained from clinical and epidemiological studies, thereby helping reduce important economic losses.

Development of drug-in-adhesive transdermal patch for α-asarone and in vivo pharmacokinetics and efficacy evaluation.

A transdermal drug delivery system has been reported that can increase the bioavailability, reduce the administration duration, and maintain the concentration of drug in blood. In the present study, drug-in-adhesive transdermal patches of α-asarone using Eudragit E100 as pressure-sensitive adhesives and oleic acid plus isopropyl myristate as penetration co-enhancers were developed. In vitro permeation, in vivo pharmacokinetics in rabbits, and efficacy in asthmatic rats were evaluated. The results showed that co-enhancers could induce a synergistic effect on α-asarone permeability. In vivo study suggested that the patch can keep a relatively certain blood level of drug within 10-30 h in rabbits. Furthermore, the patch with the size of 4 cm² containing drug 3 mg/cm² showed a noticeable treating effect on asthmatic rats which is equivalent to the effect of dexamethasone, while avoiding the side-effect induced by the corticorsteroid. This suggests that the drug-in-adhesive transdermal patch is a promising delivery system containing α-asarone to be used for asthma treatment.