Evaluation of vaccine candidates against Rhodococcus equi in BALB/c mice infection model: cellular and humoral immune responses.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that mainly causes fatal lung and extrapulmonary abscesses in foals and immunocompromised individuals. To date, no commercial vaccine against R. equi exists. We previously screened all potential vaccine candidates from the complete genome of R. equi using a reverse vaccinology approach. Five of these candidates, namely ABC transporter substrate-binding protein (ABC transporter), penicillin-binding protein 2 (PBD2), NlpC/P60 family protein (NlpC/P60), esterase family protein (Esterase), and M23 family metallopeptidase (M23) were selected for the evaluation of immunogenicity and immunoprotective effects in BALB/c mice model challenged with R. equi. The results showed that all five vaccine candidate-immunized mice experienced a significant increase in spleen antigen-specific IFN-γ- and TNF-α-positive CD4 + and CD8 + T lymphocytes and generated robust Th1- and Th2-type immune responses and antibody responses. Two weeks after the R. equi challenge, immunization with the five vaccine candidates reduced the bacterial load in the lungs and improved the pathological damage to the lungs and livers compared with those in the control group. NlpC/P60, Esterase, and M23 were more effective than the ABC transporter and PBD2 in inducing protective immunity against R. equi challenge in mice. In addition, these vaccine candidates have the potential to induce T lymphocyte memory immune responses in mice. In summary, these antigens are effective candidates for the development of protective vaccines against R. equi. The R. equi antigen library has been expanded and provides new ideas for the development of multivalent vaccines.

Identification of vaccine candidates against rhodococcus equi by combining pangenome analysis with a reverse vaccinology approach.

Rhodococcus equi (R. equi) is a zoonotic opportunistic pathogen that can cause life-threatening infections. The rapid evolution of multidrug-resistant R. equi and the fact that there is no currently licensed effective vaccine against R. equi warrant the need for vaccine development. Reverse vaccinology (RV), which involves screening a pathogen's entire genome and proteome using various web-based prediction tools, is considered one of the most effective approaches for identifying vaccine candidates. Here, we performed a pangenome analysis to determine the core proteins of R. equi. We then used the RV approach to examine the subcellular localization, host and gut flora homology, antigenicity, transmembrane helices, physicochemical properties, and immunogenicity of the core proteins to select potential vaccine candidates. The vaccine candidates were then subjected to epitope mapping to predict the exposed antigenic epitopes that possess the ability to bind with major histocompatibility complex I/II (MHC I/II) molecules. These vaccine candidates and epitopes will form a library of elements for the development of a polyvalent or universal vaccine against R. equi. Sixteen R. equi complete proteomes were found to contain 6,238 protein families, and the core proteins consisted of 3,969 protein families (∼63.63% of the pangenome), reflecting a low degree of intraspecies genomic variability. From the pool of core proteins, 483 nonhost homologous membrane and extracellular proteins were screened, and 12 vaccine candidates were finally identified according to their antigenicity, physicochemical properties and other factors. These included four cell wall/membrane/envelope biogenesis proteins; four amino acid transport and metabolism proteins; one cell cycle control, cell division and chromosome partitioning protein; one carbohydrate transport and metabolism protein; one secondary metabolite biosynthesis, transport and catabolism protein; and one defense mechanism protein. All 12 vaccine candidates have an experimentally validated 3D structure available in the protein data bank (PDB). Epitope mapping of the candidates showed that 16 MHC I epitopes and 13 MHC II epitopes with the strongest immunogenicity were exposed on the protein surface, indicating that they could be used to develop a polypeptide vaccine. Thus, we utilized an analytical strategy that combines pangenome analysis and RV to generate a peptide antigen library that simplifies the development of multivalent or universal vaccines against R. equi and can be applied to the development of other vaccines.

A pairwise and network meta-analysis comparing the efficacy and safety of progestogens in threatened abortion.

BACKGROUND: Progesterone is widely used to prevent threatened miscarriage. OBJECTIVE: To determine the efficacy and safety of progestogens in the treatment of threatened miscarriage. SEARCH STRATEGY: PubMed, Cochrane Library, EMBASE, CNKI, CBM, and WanFang databases were searched for randomized controlled trials (RCTs) published from the date of inception of the database to August 2020. The search terms included "abortion, threatened," "progesterone," and "progestogens." SELECTION CRITERIA: A network meta-analysis was conducted of all the RCTs on threatened abortion so far to compare the efficacy and safety of different progestogens in the treatment of threatened abortion. DATA COLLECTION AND ANALYSIS: Odds ratios for dichotomous data with 95% confidence intervals were calculated and the data were pooled using a random-effects model. The surface under the cumulative ranking area (SUCRA) was calculated for efficacy and safety with different interventions. MAIN RESULTS: A total of 59 RCTs with 10 424 participants were included. Oral dydrogesterone (DYD) had the lowest risk of miscarriage (SUCRA 100.0%), followed by vaginal progesterone (SUCRA 67.9%). Oral micronized progesterone had the highest risk of miscarriage (SUCRA 15.7%). CONCLUSION: Oral DYD is effective in the treatment of threatened miscarriage. The results of the present study can help patients make informed decisions about treatment options for threatened miscarriage.

Chlorogenic acid prevents vancomycin-induced nephrotoxicity without compromising vancomycin antibacterial properties.

Vancomycin (VCM) is an effective chemotherapeutic agent commonly used against gram-positive microorganisms but has serious nephrotoxic side effects that limit its effectiveness. New therapeutics and strategies are urgently needed to combat VCM associated nephrotoxicity. In this study, we determined the protective effect of chlorogenic acid (CA) in a rat model of VCM-induced nephrotoxicity. VCM administration led to markedly elevated blood urea nitrogen and serum creatinine levels that could be prevented with CA co-administration. VCM-mediated oxidative stress was also significantly attenuated by CA as reflected by decreased malondialdehyde and nitric oxide in VCM-treated kidneys. CA administration also prevented the VCM-mediated decrease in the renal antioxidative enzyme activities of glutathione reductase, glutathione peroxidase, and catalase and led to increased levels of reduced glutathione that had been depleted by VCM. Moreover, CA administration clearly inhibited VCM-induced expression of nuclear factor-kappa B, inducible nitric oxide synthase and the downstream pro-inflammatory mediators tumor necrosis factor-α and interleukins 1ß and 6. Apoptotic markers were also markedly down-regulated with CA. Overall, CA treatment mitigated VCM-induced oxidative and nitrosative stresses and countered the apoptotic and inflammatory effects of VCM. Notably, CA did not affect the antibacterial activity of VCM in vitro.

RNA-Seq-based transcriptomic profiling of primary interstitial cells of Cajal in response to bovine viral diarrhea virus infection.

Infections with bovine viral diarrhea virus (BVDV) contribute significantly to health-related economic losses in the beef and dairy industries and are widespread throughout the world. Severe acute BVDV infection is characterized by a gastrointestinal (GI) inflammatory response. The mechanism of inflammatory lesions caused by BVDV remains unknown. The interstitial cells of Cajal (ICC) network plays a pivotal role as a pacemaker in the generation of electrical slow waves for GI motility, and it is crucial for the reception of regulatory inputs from the enteric nervous system. The present study investigated whether ICC were a good model for studying GI inflammatory lesions caused by BVDV infection. Primary ICC were isolated from the duodenum of Merino sheep. The presence of BVDV was detected in ICC grown for five passages after BVDV infection, indicating that BVDV successfully replicated in ICC. After infection with BVDV strain TC, the cell proliferation proceeded slowly or declined. Morphological changes, including swelling, dissolution, and formation of vacuoles in the ICC were observed, indicating quantitative, morphological and functional changes in the cells. RNA sequencing (RNA-Seq) was performed to investigate differentially expressed genes (DEGs) in BVDV-infected ICC and explore the molecular mechanism of underlying quantitative, morphological and functional changes of ICC. Eight hundred six genes were differentially expressed after BVDV infection, of which 538 genes were upregulated and 268 genes were downregulated. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the 806 DEGs were significantly enriched in 27 pathways, including cytokine-cytokine receptor interaction, interleukin (IL)-17 signaling and mitogen-activated protein kinase (MAPK) signaling pathways. The DEGs and raw files of high-throughput sequencing of this study were submitted to the NCBI Gene Expression Omnibus (GEO) database (accession number GSE122344). Finally, 21 DEGs were randomly selected, and the relative repression levels of these genes were tested using the quantitative real-time PCR (qRT-PCR) to validate the RNA-Seq results. The results showed that the related expression levels of 21 DEGs were similar to RNA-Seq. This study is the first to establish a new infection model for investigating GI inflammatory lesions induced by BVDV infection. RNA-Seq-based transcriptomic profiling can provide a basis for study on BVDV-associated inflammatory lesions.

4-Hydroxyphenylacetic Acid Prevents Acute APAP-Induced Liver Injury by Increasing Phase II and Antioxidant Enzymes in Mice.

Acetaminophen (APAP) overdose is the principal cause of drug-induced acute liver failure. 4-hydroxyphenylacetic acid (4-HPA), a major microbiota-derived metabolite of polyphenols, is involved in the antioxidative action. This study seeks to investigate the ability of 4-HPA to protect against APAP-induced hepatotoxicity, as well as the putative mechanisms involved. Mice were treated with 4-HPA (6, 12, or 25 mg/kg) for 3 days, 1 h after the last administration of 4-HPA, a single dose of APAP was intraperitoneally infused for mice. APAP caused a remarkable increase of oxidative stress markers, peroxynitrite formation, and fewer activated phase II enzymes. 4-HPA increased Nrf2 translocation to the nucleus and enhanced the activity of phase II and antioxidant enzymes, and could thereby ameliorate APAP-induced liver injury. Studies reveal that 4-HPA, as an active area of bioactive dietary constituents, could protect the liver against APAP-induced injury, implying that 4-HPA could be a new promising strategy and natural hepatoprotective drug.

Bta-miR-2411 attenuates bovine viral diarrhea virus replication via directly suppressing Pelota protein in Madin-Darby bovine kidney cells.

MicroRNAs (miRNAs) are endogenous ∼22 nt noncoding RNAs that control the translation initiation and stability of target genes in a sequence-specific manner and, thus, play important regulatory roles in animals and plants. Homologs of Dom34, called Pelota or PELO, are broadly conserved in eukaryotes and archaea. Biochemical and genetic studies indicate that eukaryotic Dom34/Pelota plays an important role in cell division, differentiation of germline stem cells, and stem cell self-renewal by controlling the expression of specific genes at the translational level. Additionally, it is reported that Pelota is specifically required for high efficiency synthesis of proteins in numerous viruses. In earlier studies, we found the Bos taurus bta-miR-2411 (shortly miR-2411 herein) was significantly upregulated by more than 2.1 times in bovine viral diarrhea virus (BVDV) strain NADL-infected Madin-Darby bovine kidney (MDBK) cells after 8 h post-infection (pi) compared to normal MDBK cells without BVDV infection. Moreover, miR-2411 overexpression significantly reduced the BVDV E1 mRNA level and viral titer. Nevertheless, the mechanisms of miR-2411 attenuating on viral replication remain unclear. Here, we report that miR-2411 as a novel microRNA regulates BVDV NADL replication via directly targeting the Pelota gene in MDBK cells. We investigated whether the potential target sequences of miR-2411, located in the Pelota 3'UTR, and miR-2411 agomir transfection attenuated Pelota mRNA and protein levels. Indeed, upon miR-2411 overexpression, BVDV NADL replication was prevented. Importantly, BVDV NADL replication levels were reversed to normal levels as a result of the Pelota rescuing experiment even though miR-2411 was existent. Overall, we profiled the unique role of miR-2411 in regulating BVDV NADL replication and provided a novel strategy for generalized inhibition of viral infection.

3,4-Dihydroxyphenylacetic acid, a microbiota-derived metabolite of quercetin, attenuates acetaminophen (APAP)-induced liver injury through activation of Nrf-2.

1. Acetaminophen (APAP) overdose leads to severe hepatotoxicity. 3,4-dihydroxyphenylacetic acid (DOPAC) is a scarcely studied microbiota-derived metabolite of quercetin. The aim of this study was to determine the protective effect of DOPAC against APAP-induced liver injury. 2. Mice were treated intragastrically with DOPAC (10, 20 or 50 mg/kg) for 3 days before APAP (300 mg/kg) injection. APAP alone caused increase in serum aminotransferase levels and changes in hepatic histopathology. APAP also promoted oxidative stress by increasing lipid peroxidation and decreasing anti-oxidant enzyme activities. These events led to hepatocellular necrosis and reduced liver function. DOPAC increased nuclear factor erythroid 2-related factor 2 (Nrf-2) translocation to the nucleus and enhanced the expression of phase II enzymes and anti-oxidant enzymes, and thereby reduced APAP hepatotoxicity and enhanced anti-oxidant ability. 3. Our data provide evidence that DOPAC protected the liver against APAP-induced injury, which is involved in Nrf-2 activation, implying that DOPAC can be considered as a potential natural hepatoprotective agent.

Sulfated cholecystokinin-8 increases ghrelin secretion but does not affect oxyntomodulin in Holstein steers.

The effect of appetite regulatory hormone cholecystokinin (CCK) on the secretions of oxyntomodulin (OXM) and ghrelin, and the effect of ghrelin on the secretions of CCK and OXM were studied in ruminants. Eight Holstein steers, 7 months old, 243 ± 7 kg body weight (BW), were arranged in an incomplete Latin square design (8 animals × 4 treatments × 4 days of sampling). Steers were intravenously injected with 10 µg of sulfated CCK-8/kg BW, 20 µg of acyl ghrelin/kg BW, 100 µg of des-acyl ghrelin/kg BW or vehicle. Blood samples were collected from -60 min to 120 min relative to time of injection. Plasma concentrations of ghrelin, sulfated CCK and OXM were measured by double-antibody radioimmunoassay. Plasma acyl ghrelin was increased to peak level (428.3 ± 6 pg/mL) at 60 min after injection of CCK compared with pre-injected levels (203.3 ± 1 pg/mL). These results showed for the first time, that intravenous bolus injection of CCK increased ghrelin secretion in ruminants. In contrast, injection of ghrelin did not change CCK secretion. Administration of ghrelin or CCK has no effect on plasma OXM concentrations. In conclusion, our results show that administration of CCK increased ghrelin secretion but did not affect OXM release in ruminants. Ghrelin did not affect the secretions of CCK and OXM.

Bombesin-like peptides stimulate growth hormone secretion mediated by the gastrin-releasing peptide receptor in cattle.

This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0 µg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0 µg/kg NMC; (3) six steers were IV injected with 2.5µg/kg bovine gastrin-releasing peptide (GRP), 1.0 µg/kg NMC combined with 20.0 µg/kg [d-Lys(3)]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0 µg/kg NMC combined with 20.0 µg/kg N-acetyl-GRP(20-26)-OCH(2)CH(3) (N-GRP-EE, an antagonist for the GRP receptor), 20.0 µg/kg N-GRP-EE alone, 1.0 µg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0 µg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5 min after NMC injection and the minimal effective dose was 1.0 µg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys(3)]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle.

Sulfated gastrin stimulates ghrelin and growth hormone release but inhibits insulin secretion in cattle.

This study was designed to determine the effects of gastrin on the circulating levels of ghrelin, growth hormone (GH), insulin, glucagon and glucose in ruminants. Two experiments were done in eight Holstein steers. Animals were randomly assigned to receive intravenous bolus injections: (1) 0.1% bovine serum albumin in saline as vehicle, 0.8, 4.0 and 20.0 µg/kg body weight (BW) of bovine sulfated gastrin-34; (2) vehicle, 0.53 µg/kg BW of bovine sulfated gastrin-17 alone or combined with 20.0 µg/kg BW of [D-Lys(3)]-GHRP-6, the selective antagonist of GHS-R1a. Blood samples were collected from -10 to 150 min relative to injection time. Concentrations of acyl and total ghrelin in response to gastrin-34 injection were significantly increased in a dose-dependent manner. Concentrations of GH were also markedly elevated by gastrin-34 injection; however, the effect of 20.0 µg/kg was weaker than that of 4.0 µg/kg. The three doses of gastrin-34 equally decreased insulin levels within 15 min and maintained the level until the time of last sampling. Gastrin-34 had no effect (P > 0.05) on the levels of glucagon and glucose. Levels of acyl ghrelin increased after administration of gastrin-17 alone or combined with [D-Lys(3)]-GHRP-6; however, [D-Lys(3)]-GHRP-6 did not block the elevation of GH by gastrin-17. The present results indicate that sulfated gastrin stimulates both ghrelin and GH release, but the GHS-R1a may not contribute to the release of GH by gastrin. Moreover, sulfated gastrin seems to indirectly maintain the homeostasis of blood glucose through the down-regulation of insulin in ruminants.

In vivo wound healing and antibacterial performances of electrospun nanofibre membranes.

In this work, nanofibre membranes have been produced from polyvinyl alcohol (PVA), polycaprolactone (PCL), polyacrylonitrile (PAN), poly (vinylidene fluoride-co-hexafluoropropene) (PVdF-HFP), and polymer blend of PAN and polyurethane (PEU) using an electrospinning technique, and wound healing performance of the as-spun nanofibre membranes was examined in vivo using female Sprague-Dawley rats. To understand the nutrition effect, a wool protein was coated on PVA and PCL nanofibres and incorporated into PVA nanofibres via coelectrospinning of a PVA solution containing the wool protein. Silver nanoparticles were also applied to PVA nanofibres to improve antibacterial activity. It was found that the wound healing performance is mainly influenced by the porosity, air permeability, and surface wettability of the nanofibre membranes. A nanofibre membrane with good hydrophilicity and high porosity considerably facilitates the healing of wound especially at the early healing stage. However, the fiber diameter and antibacterial activity have little effect on the wound healing efficiency. As pores in nanofibre membranes are typically smaller than that of conventional cotton gauze, the nanofibre membrane should be able to decontaminate and prevent exogenous infections via sieve effect. This work provides basic understanding of material structure-property relationship for further design of efficient nanofibre-based wound dressing materials.

Involvement of endothelin B receptors in the endothelin-3-induced increase of ghrelin and growth hormone in Holstein steers.

The present study was designed to determine the dose-dependent effects of endothelin-3 (ET-3) on the secretion of ghrelin and growth hormone (GH) and characterize the receptors involved in these effects. Eight Holstein steers were randomly assigned to receive intravenous bolus injections of vehicle (0.1% bovine serum albumin in saline), bovine ET-3 (0.1, 0.4, 0.7 and 1.0microg/kg), IRL1620 (selective ET(B) receptor agonist, 2.0microg/kg), [d-Lys(3)]-GHRP-6 (GH secretagogue receptor type 1a [GHS-R1a] antagonist, 20.0microg/kg) and bovine ET-3 (1.0microg/kg) combined with [d-Lys(3)]-GHRP-6 (20.0microg/kg), respectively. Blood samples were collected at -30, -15, 0, 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60min relative to injection time. Concentrations of acyl ghrelin, total ghrelin (acyl and des-acyl ghrelin) and GH in plasma were analyzed by a double antibody radioimmunoassay system. Concentrations of acyl and total ghrelin were significantly increased by ET-3 in a dose-dependent manner. Concentrations of GH were markedly elevated by administration of 0.4, 0.7 and 1.0microg/kg of ET-3, and the effect of 0.7microg/kg was greater than that of 1.0microg/kg. The minimum effective dose of ET-3 in the secretion of ghrelin and GH was 0.4microg/kg. IRL 1620 mimicked the effects of ET-3 on the secretion of ghrelin and GH in plasma. ET-3-induced elevation of plasma GH was blocked by [d-Lys(3)]-GHRP-6. These results indicate that ET-3 dose-dependently stimulates ghrelin release, and ET(B) receptors involve in these processes. Moreover, this study shows that endogenous ghrelin response to ET-3 increases GH secretion through GHS-R1a.