Real-time PCR Results Research Articles

An Integrated Profiling of Liver Metabolome and Transcriptome of Pigs Fed Diets with Different Starch Sources

Diets containing higher-amylose-content starches were proved to have some beneficial effects on monogastric animals, such as promoting the proliferation of intestinal probiotics. However, current research on the effects of diets with different starch sources on animals at the extraintestinal level is still very limited. We hypothesized that diets with different starch sources may affect lipid-related gene expression and metabolism in the liver of pigs. This study aimed to use adult pig models to evaluate the effects of diets with different starch sources (tapioca starch, TS; pea starch, PS) on the liver gene expressions and metabolism. In total, 48 growing pigs were randomly assigned to the TS and PS diets with 8 replicate pens/group and 3 pigs per pen. On day 44 of the experiment, liver samples were collected for metabolome and transcriptome analysis. Metabolome data suggested that different starch sources affected (p < 0.05) the metabolic patterns of liver. Compared with the TS diet, the PS diet increased (p < 0.05) some unsaturated fatty acids and several amino acids or peptide levels in the liver of pigs. Moreover, transcriptome data indicated the PS diets elevated (p < 0.05) fatty acid β-oxidation-related gene expression in the liver of pigs, and reduced (p < 0.05) unsaturated fatty acid metabolism-related gene expression. The results of quantitative real-time PCR confirmed that the PS diet upregulated (p < 0.05) the expression of acyl-CoA dehydrogenase very long chain (ACADVL), carnitine palmitoyl transferase (CPT) 1A, and malonyl-CoA decarboxylase (MLYCD), and downregulated (p < 0.05) the expression level of cytochrome P450 2U1 (CYP2U1) and aldehyde dehydrogenase 1B1 (ALDH1B1) in the liver. In addition, the results of a Mantel test indicated the muscle fatty acids were significantly closely correlated (p < 0.05) with liver gene expressions and metabolites. In summary, these findings suggest that diets containing higher amylose starches improved the lipid degradation and the unsaturated fatty acid levels in pig livers, and thus can generate some potential beneficial effects (such as anti-inflammatory and antioxidant) on pig health.

Exploration on the construction of a bladder cancer prognostic model based on disulfidptosis-related lncRNAs and its clinical significance.

Disulfidptosis is a novel programmed cell death mode that has been reported to play a role in oncogenesis. Increasing evidences suggest that the long non-coding RNAs (lncRNAs) play crucial roles in the initiation and progression of bladder cancer (BLCA). However, the role and prognostic value of disulfidptosis-related lncRNAs in BLCA remain unknown.The aim of this study was to construct and validate a disulfidptosis-related lncRNA risk model for predicting the prognosis of BLCA patients. A risk model consisting of 5 disulfidptosis-related lncRNAs was developed to predict the prognosis of BLCA patients. The overall survival (OS) of BLCA patients in the high-risk group was significantly shorter than that in the low-risk group (P < 0.05). The effectiveness of this model was validated using the receiver operating characteristic (ROC) curve analysis, and this model proved superior in prognostic accuracy compared with other clinical features. Furthermore, the tumor immune dysfunction and exclusion (TIDE) score in the high-risk group was significantly higher than that in the low-risk group, suggesting that the high-risk group had a less favorable response to immunotherapy. Simultaneously, patients in the low-risk group exhibited significantly higher sensitivity to CTLA-4 monoclonal antibody therapy compared to those in the high-risk group, suggesting potential benefits of immunotherapy for patients in the low-risk group. The combination of high risk and low tumor mutational burden (TMB) could further shortened the OS of BLCA patients. Lastly, the drug sensitivity analysis revealed that the BLCA cells in the high-risk group showed an increased sensitivity to cisplatin, sunitinib, cetuximab, axitinib, docetaxel, saracatinib, vinblastine and pazopanib compared with those in the low-risk group. According to the Quantitative real time PCR results, we found that five lncRNAs of the risk model were more highly expressed in BCa cell lines than human immortalized uroepithelial cell line. The disulfidptosis-related lncRNA risk model has a valuable effect in assessing the prognosis of BLCA patients.

Replacing Hydrolyzed Soybean Meal with Recombinant β-Glucosidase Enhances Resistance to Clostridium perfringens in Broilers Through Immune Modulation

Aglycone soy isoflavones have notable immune-regulatory bioactivity, while glycosidic forms in soybean meal pose challenges for absorption. β-Glucosidase (EC 3.2.1.21) catalyzes the non-reducing terminal β-d-glucosidic bonds, releasing β-d-glucan and aglycones. This study evaluated the impact of enzymatically hydrolyzed soybean meal (ESM) using recombinant β-glucosidase from Aspergillus niger on the growth performance and intestinal immune function of broilers under Clostridium perfringens infection. Prior to the feeding trial, soybean meal was enzymatically digested with recombinant β-glucosidase, ensuring almost complete conversion of glycosides to aglycones. After a week of pre-feeding, a total 180 healthy AA broilers were randomly assigned to three groups—control, semi-replacement of ESM (50% ESM), and full-replacement of ESM (100% ESM)—with 6 replicates of 10 chickens, and the trial lasted 28 days. On the 36th day, broilers were challenged with 1 mL of 1 × 1010 CFU/mL Clostridium perfringens (Cp) via gavage for 3 days. The results showed that the substitution of ESM had no effect on the body weight gain of broilers but significantly reduced the feed consumption and feed-to-gain ratio (p &lt; 0.01). The study revealed that Cp significantly disrupted jejunal morphology, while ESM significantly mitigated this damage (p &lt; 0.05). Real-time PCR results demonstrated that compared to the Cp group, ESM restored Cp-induced intestinal barrier impairments (e.g., Occludin, Claudin-1, Muc2), normalized aberrant cellular proliferation (PCNA) and apoptosis (Caspase-1 and Caspase-3), and upregulated the expression of anti-inflammatory factor Il-10 while suppressing pro-inflammatory cytokines (Il-1β, Il-6, and Il-8) (p &lt; 0.05). Moreover, flow cytometry analyses demonstrated that ESM promoted Treg cell-derived Il-10, which alleviated macrophage-derived inflammation. Substituting conventional soybean meal with β-glucosidase, enzymatically treated, significantly reduced feed consumption and alleviated the intestinal damage and immune dysfunctions induced by Clostridium perfringens infection in broilers.

Detection by Real-Time PCR of Helicobacter pylori and Clarithromycin Resistance Compared to Histology on Gastric Biopsies

The global rise in Helicobacter pylori (H. pylori)-related gastric complications is largely driven by increasing antimicrobial resistance and treatment failures. As a result, accurate diagnosis followed by effective treatment is crucial. We analyzed 232 gastric biopsy samples from patients undergoing endoscopy during the method validation phase, followed by 502 samples in the routine evaluation phase. Each sample was tested using the Allplex™ H. pylori and ClariR Assay on a CFX96™ real-time PCR (RT-PCR) system, with results processed through Seegene Viewer software. In the validation phase, RT-PCR results were compared to bacterial culture, while in the routine phase, they were compared to histology. The sensitivity and specificity for H. pylori detection were 100% and 96.05% (95% Confidence Interval [CI]: 93.38–98.73), respectively. For clarithromycin resistance detection, the sensitivity and specificity were 100% and 93.33% (95% CI: 84.4–100). Additionally, RT-PCR identified 11 positive samples (10.89%) that histology failed to detect. Incorporating the Allplex™ H. pylori and ClariR Assay into our laboratory workflow improved efficiency, reduced turnaround time (TaT), and proved to be more sensitive than both culture and histology combined.

Experimental and in silico analysis of LINC01279 expression in tumor of patients with breast cancer.

Breast cancer (BC) is characterized by the increase of malignant cells in the breast. The malignant cells begin in the lining of the breast milk glands or ducts (ductal epithelium). BC is the most frequent cancer in women, but it may also occur in males. Long non-coding RNAs (lncRNA) have been demonstrated to control the development and incidence of cancer. However, some lncRNAs experience potential changes in BC, but their role has not been well studied. LINC01279 is known as a valuable biomarker in gastric cancer but has not yet been studied in BC. Changes in LINC01279 expression levels in BC samples were investigated by microarray. Q-PCR was also used to evaluate the expression of LINC01279 in the tumor and normal adjacent samples of 30 BC patients. The LINC01279 co-expressed gene module was discovered using weighted gene correlation network analysis (WGCNA) on the relevant dataset. The top ten hub genes were determined using gene ontology (GO) functional enrichments on the co-expressed gene module. The results of the bioinformatics study showed an increase in LINC01279 expression levels (log2FC = 3.228749561, adj.P.Val = 1.69E - 12) in tumor samples compared to normal marginal tissue. Q-PCR results also showed a significant increase in LINC01279 expression (P-value = 0.0005) in tumor samples. WGCNA analysis identified that the black module is the LINC01279 co-expressed module, and functional annotation analysis of black module genes enriched in significant cancer-related pathways and processes, including cell growth and/or maintenance, regulation of immune response, regulation of cell proliferation, and epithelial-to-mesenchymal transition (EMT). Regarding the real-time PCR results, the analysis of expression patterns has illuminated a distinct association between the heightened expression levels of LINC01279, and the stages of cancer progression as well as the metastatic potential of tumors. However, intriguingly, our observations have failed to reveal any statistically significant correlations between the relative expression of LINC01279 and tumor grade classification, or the presence of ER, PR, and HER2 biomarkers. The present study could provide a new perspective on the molecular regulatory. Processes associated with BC pathogenic mechanisms are linked to the LINC01279, although further research is needed on the possible role of this lncRNA in BC.

Comparison of triclosan and triclocarban in triggering immunotoxicity in larval zebrafish

As two efficient broad-spectrum sterilizing agents, triclosan (TCS) and triclocarban (TCC) are widely used, especially during the COVID-19 pandemic. The health risks caused by secondary pollution of TCS and TCC have aroused wide concern. Because of the similar mother nucleus structure and high lipophilicity, it remains unknown about the differences in the effect and mechanism of the toxicity (especially immunotoxicity) between TCS and TCC in organisms in the environment. In this study, we used zebrafish as a model to compare the immunotoxicity and mechanisms between the two pollutants at the same exposure concentration (0.6 µmol/L). The results showed that both TCS and TCC led to a hatching rate below 60% at the time point of 72 hours post fertilization (hpf) and the mortality rates of 40% and 50% at 120 hpf in larval zebrafish, respectively. The zebrafish exposed to TCS and TCC displayed malformations, such as shortened body, swimming sac closure, pericardial edema, yolk cyst deposition, and absorption disorder. Moreover, the developmental abnormalities caused by TCC were significantly severer than those caused by TCS. TCS exposure increased the proliferation rate of innate immune cells to 20% and decreased the number of mature T cells by 35%, while TCC exposure inhibited the differentiation of both innate immune cells and T cells, with the inhibition rates of 25% and 60%, respectively. The results of real-time quantitative PCR (RT-qPCR) and ELISA showed that TCS and TCC exposure up-regulated the expression levels of il-1β, il-6, and tnf-α, while il-10 and IgM exhibited opposite expression patterns. Additionally, both compounds slightly decreased C3 expression. The Pearson correlation analysis showed that the developmental toxicity induced by TCS and TCC had positive and negative correlations with the differentiation of immune cells, respectively. However, the toxicity induced by either TCS or TCC was positively correlated with the expression of pro-inflammatory cytokines. GO function and KEGG pathway enrichment analyses demonstrated that the target molecules of TCS and TCC were enriched in different signaling pathways, and the key network hub genes and the enriched regulatory pathways differed between TCS and TCC. The findings provide compelling evidence that TCS and TCC adopt different mechanisms in triggering immunotoxicity and offer a theoretical reference for the recognition, warning, and management of TCS and TCC-induced health risks.

Screening and expression validation of key proteins for secondary hair follicle growth in cashmere goats based on iTRAQ quantitative proteomics technology.

The growth of secondary hair follicles (SHFs) in cashmere goats has periodic changes, including telogen, anagen, and catagen, during which proteins play important roles as the executor of life activities. In this study, the skin tissues of cashmere goats at three different growth stages of SHFs were collected for proteome sequencing and validation experiments. Through protein differential expression analysis and time series analysis, FKBP prolyl isomerase 10 (FKBP10) and fibrillin 2 (FBN2) were screened as the key proteins for SHF cycle growth of cashmere goats, and albumin (ALB), collagen type I alpha 1 chain (COL1A1) and elastin (ELN) were predicted to be their interacting proteins. The results of quantitative real-time PCR (qRT-PCR), western blot, and immunohistochemistry experiments showed that the mRNA and protein expression levels of FKBP10, FBN2, COL1A1, ELN and ALB were higher in anagen and lower in telogen. They were all highly expressed in the outer root sheath of SHFs in anagen. FKBP10, FBN2, COL1A1, ELN, and ALB can promote the growth of SHFs in cashmere goats. This study lays the foundation for analyzing the growth cycle regulatory mechanism of SHFs in cashmere goats, and provides new ideas for further improving cashmere yield and quality.

From eradication to reemergence: the growing threat of malaria in Turkey.

According to WHO, between 2000 and 2021, there were approximately 247 million malaria cases and 627,000 deaths globally, with the majority of cases occurring in sub-Saharan Africa. In Turkey, indigenous P. vivax malaria was a major public health problem until its eradication was achieved in 2010. Although indigenous malaria transmission has been significantly reduced since 2010, the country is challenged with imported malaria due to increasing global travel and migration from endemic regions. In this study, all imported malaria cases admitted to Dr. Sadi Konuk Research and Training Hospital, Istanbul, between 2018 and 2023 were included. DNA extraction was performed using archived slides and EDTA blood samples. Real-time PCR was performed to identify samples at the species level using previously reported primers and probes. In addition, all available patient demographics are presented. During the six years between 2018 and 2023, 157 patients were diagnosed with imported malaria. According to the real-time PCR results, 149 cases were P. falciparum (94.9%), five cases were P. vivax (3.2%), two cases were P. ovale (1.3%), and one case was P. malariae (0.6%). The male/female ratio among diagnosed patients was 2.34 (110♂/47♀) among diagnosed patients. Plasmodium falciparum was detected in patients from all African regions, whereas P. vivax was detected only in patients from Liberia and Djibouti. Although malaria cases in Turkey have significantly decreased due to elimination efforts and effective public health interventions, the recent increase in both imported and indigenous cases, as well as the presence of suitable vector species in the country, indicates that malaria still remains a serious public health problem for Turkey.

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of staphylococcus aureus

ABSTRACT The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and β-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced haemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and haemolytic activity in S. aureus.

RAA-CRISPR/Cas12a-Mediated Rapid, Sensitive, and Onsite Detection of Newcastle Disease in Pigeons.

Pigeon Newcastle disease, caused by pigeon paramyxovirus type 1 (PPMV-1), is a significant infectious disease in pigeons that can result in substantial mortality and poses a severe threat to the pigeon industry. The rapid and accurate onsite diagnosis of pigeon disease is crucial for timely diagnosis and the implementation of effective prevention and control measures. In this study, we established a rapid detection method for PPMV-1 based on recombinase-aided amplification (RAA) and CRISPR/Cas12a. The RAA primers target the conserved regions of the L gene for preamplification in clinical nucleic acid samples, followed by CRISPR/Cas12a detection of the target gene. Visualization could be achieved by combination with a lateral flow dipstick (LFD). This method demonstrated high specificity, showing no cross-reactivity with non-PPMV-1 samples. The sensitivity of the method assessed by fluorescence analysis reached 100 copies/µL, and when it was combined with an LFD, the sensitivity was 103 copies/µL. The constructed RAA-CRISPR/Cas12a-LFD visual detection method was applied to clinical sample testing and was found to enable the rapid and accurate detection of swab samples and tissue specimens. Its sensitivity was consistent with the current gold standard, quantitative real-time PCR results. The RAA-CRISPR/Cas12a-LFD detection method we developed provides a novel approach for the rapid, simple, precise, and specific onsite diagnosis of pigeon Newcastle disease.

NADPH-cytochrome P450 reductase involved in the lambda-cyhalothrin susceptibility on the green mirid bug Apolygus lucorum.

NADPH-cytochrome P450 reductase (CPR) is crucial for the detoxification process catalysed by cytochrome P450, which targets various exogenous xenobiotics, as well as pesticides. In our research, we successfully obtained the complete cDNA sequence of Apolygus lucorum's CPR (AlCPR) using reverse transcription PCR along with rapid amplification of cDNA ends technology. Bioinformatics analysis exhibited that the inferred amino acid sequence of AlCPR is characteristic of standard CPRs, featuring an N-terminal membrane anchor and three conserved FMN, FAD and NADP binding sites. Phylogenetic result revealed that AlCPR was positioned within the Hemiptera cluster, showing a close evolutionary relationship with the CPR of Cimex lectularius. The real-time quantitative PCR results demonstrated widespread expression of AlCPR across various life stages and tissues of A. lucorum, with the most prominent expression in adults and the abdominal region. Injecting double-stranded RNA of AlCPR only significantly increased the lambda-cyhalothrin susceptibility in lambda-cyhalothrin-resistant strain rather than the susceptible strain. These findings suggest a potential link between AlCPR and the P450-dependent defence mechanism against lambda-cyhalothrin in A. lucorum.

Elucidation of the mechanisms of fluconazole resistance and repurposing treatment options against urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt

BackgroundThe incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains.ResultsFluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman’s correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models.ConclusionsThe fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.

Crosstalk between MIR-96 and IRS/PI3K/AKT/VEGF cascade in hRPE cells; A potential target for preventing diabetic retinopathy.

Regulation of visual system function demands precise gene regulation. Dysregulation of miRNAs, as key regulators of gene expression in retinal cells, contributes to different eye disorders such as diabetic retinopathy (DR), macular edema, and glaucoma. MIR-96, a member of the MIR-183 cluster family, is widely expressed in the retina, and its alteration is associated with neovascular eye diseases. MIR-96 regulates protein cascades in inflammatory and insulin signaling pathways, but further investigation is required to understand its potential effects on related genes. For this purpose, we identified a series of key target genes for MIR-96 based on gene and protein interaction networks and utilized text-mining resources. To examine the MIR-96 impact on candidate gene expression, we overexpressed MIR-96 via adeno-associated virus (AAV)-based plasmids in human retinal pigment epithelial (RPE) cells. Based on Real-Time PCR results, the relative expression of the selected genes responded differently to overexpressed MIR-96. While the expression levels of IRS2, FOXO1, and ERK2 (MAPK1) were significantly decreased, the SERPINF1 gene exhibited high expression simultaneously. pAAV-delivered MIR-96 had no adverse effect on the viability of human RPE cells. The data showed that changes in insulin receptor substrate-2 (IRS2) expression play a role in disrupted retinal insulin signaling and contribute to the development of diabetic complications. Considered collectively, our findings suggest that altered MIR-96 and its impact on IRS/PI3K/AKT/VEGF axis regulation contribute to DR progression. Therefore, further investigation of the IRS/PI3K/AKT/VEGF axis is recommended as a potential target for DR treatment.

Staphyloxanthin and Meropenem Effects on Some Capsular Genes Expressions of Klebsiella pneumoniae Isolated from UTIs

Klebsiella pneumoniae has become one of the most common etiologic agents in the context of recurrent UTI. Capsule is the earliest recognized factor determining bacterial pathogenicity. This study aimed to test the effects of staphyloxanthin, meropenem and a combination of them on capsule genes of Klebsiella pneumoniae. Eighty-eight gram negative bacterial isolates were collected from 150 urine specimens between September and November of 2022.after cultivation on MacConkey agar medium. Oxidase (negative) and catalase (positive) tests were performed. In addition, all isolates were found to be non-motile when their motility was further examined. The results presented that 77 (51.3%) could ferment lactose (lactose fermenter), while 30 isolates (20%) of them were confirmed as K. pneumonia. The antibiotics sensitivity test of (30) K. pneumoniae revealed that 28 (93%) of isolates were sensitive to meropenem, and about 20 (67%), 16 (53%), 8 (27%), 7 (23%), 5 (17%) and 4 (13%) were sensitive toward amikacin, ciprofloxacin, trimethoprim, cefepime, tetracycline and Augmentin respectively. S. aureus (producers of staphyloxanthin) isolates were taken; the staphyloxanthin pigment was extracted, purified by Millipore filter (0.22 mm). Conventional PCR results revealed the presence of galF gene (141 bp) in all eight tested isolates (100%). In addition, the wzi gene (177bp) was found in seven isolates (87.5%). According to the quantitative real-time PCR results, most treatments resulted in downregulation of galF and wzi genes expression, whereas some treatments resulted in upregulation.

Genome-wide analysis of the CCT gene family and functional characterization of SlCCT6 in response to drought stress in tomato

CCT transcription factors are important for photoperiod and abiotic stress regulation in Arabidopsis and rice. However, the CCT gene family has not been reported in tomato. Here, we systematically analyzed this. Thirty-one SlCCT genes were identified and divided into five groups (CMF, TIFY, PRR, S8, and COL), with members unevenly distributed across 12 chromosomes and the third chromosome exhibiting the most distribution. SlCCT was found to interact with an interacting protein (SlGI), transcription factor (MYB), and non-coding RNA (sly-miR156-5p) to jointly regulate the tomato stress response. cis-Acting element analysis of the SlCCT promoter region indicated large stress- and hormone-response elements in this family. Real-time PCR results indicated that SlPRR subfamily genes respond to various abiotic stresses and hormones. Tissue expression analysis revealed that several PRR subfamily genes are highly expressed in flowers, and subcellular localization analysis indicated an SlCCT6 nuclear location. Notably, SlCCT6 expression was significantly induced by drought, and its silencing reduced drought stress tolerance. Moreover, SlCCT6 overexpression enhanced tomato drought resistance by increasing antioxidant enzyme activity and activating stress-related genes, whereas SlCCT6 knockout decreased drought resistance. In conclusion, this provides valuable insights for future research on SlCCT functions.

Huaiqihuang (HQH) protects podocytes from high glucose-induced apoptosis and inflammation response by regulating PI3K/AKT/mTOR pathway

Diabetic kidney disease (DKD) is one of the common microvascular complications of diabetes, and there are still lack of effective treatments. Huaiqihuang (HQH) is a kind of traditional Chinese medicine mixed preparation, which is mainly made of Trametes robiniophila Murr, Fructus Lycii, and Polygonatum sibiricumhas. It has been shown to be effective in the treatment of DKD, but the specific mechanism has not been fully elucidated. Our results showed that HQH increased the protein expressions of synaptopodin, podocin, WT-1, and Bcl-2, decreased the protein expressions of Bax and cleaved-casepase-3, and activated the NF-ĸB and PI3K/AKT/mTOR pathway in MPC5 cells exposed to high-glucose (HG). Real-time PCR results showed that HQH reduced the mRNA expression of TNF-α, IL-1β, MCP-1, and IL-6. In conclusion, our results showed that HQH may attenuate podocyte injury by inhibiting MPC5 cell apoptosis induced by HG and NF-κB-mediated inflammation response through activation of the PI3K/AKT/mTOR pathway.

Cloning and Expression Analysis of TGF-β Type I Receptor Gene in Hyriopsis cumingii.

The TGF-β signaling pathway plays an important role in wound healing and immune response. In this study, a TGF-β type I receptor (TGF-βRI) homolog was cloned and characterized from freshwater mussel Hyriopsis cumingii. The full-length cDNA of the TGF-β RI gene was 2017 bp, with a 1554 bp open reading frame (ORF), and encoded 517 amino acids. The predictive analysis further identified distinct regions within the TGF-βRI protein: a signal peptide, a membrane outer region, a transmembrane region, and an intracellular region. Real-time quantitative PCR results showed that the TGF-β RI gene was expressed in all tissues of healthy mussels. The transcripts of TGF-β RI in hemocytes and hepatopancreas were significantly up-regulated at different periods after stimulation with Aeromonas hydrophila and peptidoglycan (PGN) (P < 0.05). The mRNA expression of TGF-β RI progressively increased from day 1 to day 10 after trauma (P < 0.05), and it returned to the initial level by day 15. The expression levels of TGF-β , Smad5, MMP1/19, and TIMP1/2, but not Smad3/4, were significantly up-regulated at different time points after trauma. However, the expression levels of TGF-β , MMP1/19, and TIMP2 were decreased after treatment with the inhibitor SB431542. Furthermore, the recombinant TGF-βRI proteins were expressed in vitro and existed in the form of inclusion bodies. Western blotting results showed that TGF-βRI proteins were expressed constitutively in various tissues of mussels, and their expression was up-regulated after trauma, which was consistent with the mRNA expression trend. These results indicate that TGF-β RI is involved in the process of wound repair and immune response.

A Quadruple Gene-Deleted Live BoHV-1 Subunit RVFV Vaccine Vector Reactivates from Latency and Replicates in the TG Neurons of Calves but Is Not Transported to and Shed from Nasal Mucosa.

Bovine herpesvirus type 1 (BoHV-1) establishes lifelong latency in trigeminal ganglionic (TG) neurons following intranasal and ocular infection in cattle. Periodically, the latent virus reactivates in the TG due to stress and is transported anterogradely to nerve endings in the nasal epithelium, where the virus replicates and sheds. Consequently, BoHV-1 is transmitted to susceptible animals and maintained in the cattle population. Modified live BoHV-1 vaccine strains (BoHV-1 MLV) also have a similar latency reactivation. Therefore, they circulate and are maintained in cattle herds. Additionally, they can regain virulence and cause vaccine outbreaks because they mutate and recombine with other circulating field wild-type (wt) strains. Recently, we constructed a BoHV-1 quadruple mutant virus (BoHV-1qmv) that lacks immune evasive properties due to UL49.5 and glycoprotein G (gG) deletions. In addition, it also lacks the gE cytoplasmic tail (gE CT) and Us9 gene sequences designed to make it safe, increase its vaccine efficacy against BoHV-1, and restrict its anterograde neuronal transport noted above. Further, we engineered the BoHV-1qmv-vector to serve as a subunit vaccine against the Rift Valley fever virus (BoHV-1qmv Sub-RVFV) (doi: 10.3390/v15112183). In this study, we determined the latency reactivation and nasal virus shedding properties of BoHV-1qmv (vector) and BoHV-1qmv-vectored subunit RVFV (BoHV-1qmv sub-RVFV) vaccine virus in calves in comparison to the BoHV-1 wild-type (wt) following intranasal inoculation. The real-time PCR results showed that BoHV-1 wt- but not the BoHV-1qmv vector- and BoHV-1qmv Sub-RVFV-inoculated calves shed virus in the nose following dexamethasone-induced latency reactivation; however, like the BoHV-1 wt, both the BoHV-1qmv vector and BoHV-1qmv Sub-RVFV viruses established latency, were reactivated, and replicated in the TG neurons. These results are consistent with the anterograde neurotransport function of the gE CT and Us9 sequences, which are deleted in the BoHV-1qmv and BoHV-1qmv Sub-RVFV.

Antimicrobial potential of carvacrol against Edwardsiella piscicida in vitro

With the alarming rise of antibiotic-resistant bacteria, novel antibacterial substances are urgently needed for controlling and treating multidrug-resistant bacterial infections. Edwardsiella piscicida is an important zoonotic enteric pathogen, that can cause systemic hemorrhagic septicemia in fish. Carvacrol, a major terpene of oregano essential oil, has a wide range of antibacterial activities. This study aimed to analyze the effect of carvacrol on the growth and virulence of E. piscicida in vitro. The minimum inhibitory concentration (MIC) of carvacrol against E. piscicida was 125 μg/mL. The sub-inhibitory concentrations of carvacrol significantly decreased the biofilm formation of E. piscicida in a dose dependent manner, whereas increased the hemolytic activity with a negative correlation. The quantitative real-time PCR results showed that carvacrol at sub-MICs downregulated the expression of related virulence genes, including flagellum (fimA, fliC, flgN), hemolysins (ethA, ethB), quorum sensing systems (luxR, qseB), T3SS (esrB, esrC) and T6SS (evpB, evpC). Moreover, carvacrol (≤1/8 MIC) reduced the cytotoxicity, adherence and internalization activities of E. piscicida to the EPC cells. In vivo trial, the diet mixed with carvacrol increased the survival of zebrafish infected with E. piscicida. Overall, these findings suggested that carvacrol might be a promising therapeutic agent against E. piscicida infection in aquaculture.

Abamectin at environmentally relevant concentrations impairs bone development in zebrafish larvae

Abamectin (ABM) is a widely used pesticide in agriculture and veterinary medicine, which primarily acts by disrupting the neurological physiology of pests, leading to their paralysis and death. Its extensive application has resulted in contamination of many natural water bodies. While the adverse effects of ABM on the growth and development of non-target organisms are well documented, its impact on bone development remains inadequately studied. The present study aimed to investigate the effects of environmentally relevant concentrations of ABM (1, 5, 25 μg/L) on early bone development in zebrafish. Our results indicated that ABM significantly affected both cartilage and bone development of zebrafish larvae, accompanied by dose-dependent increase in deformity and mortality rates, as well as exacerbated apoptosis. ABM exposure led to deformities in the ceratobranchial (cb) and hyosymplectic (hs), accompanied by significant increases in the length of the palatoquadrate (pq). Furthermore, significant decreases in the CH-CH angle, Meckel's-Meckel's angle, and Meckel's-PQ angle were noted. Even at the safe concentration of 5 μg/L (1/10 of the 96 h LC50), ABM delayed the process of bone mineralization in zebrafish larvae. Real-time fluorescent quantitative PCR results demonstrated that ABM induced differential gene expression associated with cartilage and bone development in zebrafish. Thus, this study provides preliminary insights into the effects and molecular mechanisms underlying ABM's impact on the bone development of zebrafish larvae and offers new evidence for a better understanding of its toxicity.