RT-PCR Assay Research Articles

Sitagliptin Alleviates Obesity in Immature Mice by Inhibiting Oxidative Stress and Inflammation.

To investigate the impact of Sitagliptin against obesity and the underlying mechanism. Obese immature mice were treated with 10, 30, and 90mg/kg Sitagliptin, respectively. The body weights were recorded and the level of serum biochemical indexes were detected. The visceral fat ratio of each mouse was determined. The pathological change in adipose tissues was determined by HE staining, while F4/80 and CD206 levels in adipose tissues were determined by the immunohistochemical analysis. Lipid formation was evaluated by Oil red O staining assay. RAW264.7 cells were stimulated using oxLDL, followed by being incubated with different concentrations of Sitagliptin. The release of ADPN, IL-6, IL-1β, TNF-α, and the activity of SOD, was measured by ELISA assay. Western blotting was applied to determine adipsin, Nrf2, Keap1, and HO-1 protein levels. ROS level was checked using the DCFH-DA assay. RT-PCR assay was utilized to detect the mRNA levels of IL-6, IL-1β, TNF-α, Nrf2, Keap1, and HO-1. The body weight gain, infiltration of multinucleated cells, enlarged size of adipocytes, increased lipid accumulation, elevated visceral fat ratio, declined ADPN level, upregulated adipsin, and disordered serum biochemical indexes in obese immature mice were statistically significantly reversed by Sitagliptin. Excessive release of inflammatory factors and upregulated F4/80 and CD206 were observed in obese immature mice, which were statistically significantly repressed by Sitagliptin. Furthermore, the elevated MDA level, increased SOD activity, and inhibited Nrf2 pathway in obese immature mice were significantly reversed by Sitagliptin. In oxLDL stimulated RAW264.7 cells, increased release of inflammatory factors, ROS, and MDA, elevated SOD activity, and inactivated Nrf2 pathway were observed, which were statistically significantly abolished by the treatment of Sitagliptin. Sitagliptin alleviated obesity in immature mice by inhibiting inflammation and oxidative stress.

Potential role of peste des petits ruminants virus in small ruminant abortions

The aim of the present study was to investigate the frequency, genetic variability, and phylogeny of the peste des petits ruminants virus (PPRV) in ovine and caprine fetuses. During 2014 and 2017, a total of 1054 embryos/fetuses were collected in Turkey. A real-time RT-PCR assay was used for the detection of the PPRV RNA. Genetic characterization and phylogenetic analysis of the PPRV field isolates were conducted by sequencing fusion (F) protein and nucleoprotein (N) gene segments. Samples were also collected from ewes (n = 83) and nanny goats (n = 3) that had aborted and whose embryos/fetuses were found to be PPRV positive. PPRV positive embryos/fetuses were also tested for the presence of Listeria monocytogenes, Campylobacter spp., Coxiella burnetii, Chlamydophila abortus, Brucella spp., akabane virus, aino virus, bluetongue virus, border disease virus, bovine viral diarrhea virus, Cache Valley virus, and Schmallenberg virus. PPRV RNA was detected in 123 (11.7 %) of the 1054 embryos/fetuses, 78 of the 83 (94 %) ewes and 3 (100 %) nanny goats. Border disease virus RNA and Chlamydophila abortus DNA were detected in 7 and 12 PPRV positive sheep fetuses, respectively, while other bacterial and viral agents were not detected. Phylogenetically, the field isolates in this study belong to lineage IV, and compared to other strains of lineage IV considered in this study, they showed 1 and 5 new amino acid substitutions in the F and N gene sequences, respectively. The results of the study suggest that PPRV plays an important role in abortion. Therefore, PPRV needs to be taken into consideration in sheep and goats abortions.

Dual pH/redox-responsive size-switchable polymeric nano-carrier system for tumor microenvironment DTX release

Innovation chemotherapeutic nano drug delivery systems (NDDSs) with various pharmacological achievement have become one of the hopeful therapeutic strategies in cancer therapy. This study focused on low pH, and high levels of glutathione (GSH) as two prominent characteristics of the tumor microenvironment (TME) to design a novel TME-targeted pH/redox dual-responsive P (AMA-co-DMAEMA)-b-PCL-SS-PCL-b-P (AMA-co-DMAEMA) nanoparticles (NPs) for deep tumor penetration and targeted anti-tumor therapy. The positively charged NPs exhibit strong electrostatic interactions with negatively charged cell membranes, significantly enhancing cellular uptake. Moreover, these NPs possess the unique size-shrinkable property, transitioning from 98.24 ± 27.78 to 45.56 ± 20.62 nm within the TME. This remarkable size change fosters an impressive uptake of approximately 100% by MDA-MB-231 cells within just 30 min, thereby greatly improving drug delivery efficiency. This size switchability enables passive targeting through the enhanced permeability and retention (EPR) effect, facilitating deep penetration into tumors. The NPs also demonstrate improved pH/redox-triggered drug release (∼70% at 24 h) within the TME and exhibit no toxicity in cell viability test. The cell cycle results of treated cells with docetaxel (DTX)-loaded NPs revealed G2/M (84.6 ± 1.16%) arrest. The DTX-loaded NPs showed more apoptosis (62.6 ± 3.7%) than the free DTX (51.8 ± 3.2%) in treated cells. The western blot and RT-PCR assays revealed that apoptotic genes and proteins expression of treated cells were significantly upregulated with the DTX-loaded NPs vs. the free DTX (P value<.001). In conclusion, these findings suggest that this novel-engineered NPs holds promise as a TME-targeted NDDS.

Elucidating the role of angiogenesis-related genes in colorectal cancer: a multi-omics analysis.

Angiogenesis plays a pivotal role in colorectal cancer (CRC), yet its underlying mechanisms demand further exploration. This study aimed to elucidate the significance of angiogenesis-related genes (ARGs) in CRC through comprehensive multi-omics analysis. CRC patients were categorized according to ARGs expression to form angiogenesis-related clusters (ARCs). We investigated the correlation between ARCs and patient survival, clinical features, consensus molecular subtypes (CMS), cancer stem cell (CSC) index, tumor microenvironment (TME), gene mutations, and response to immunotherapy. Utilizing three machine learning algorithms (LASSO, Xgboost, and Decision Tree), we screen key ARGs associated with ARCs, further validated in independent cohorts. A prognostic signature based on key ARGs was developed and analyzed at the scRNA-seq level. Validation of gene expression in external cohorts, clinical tissues, and blood samples was conducted via RT-PCR assay. Two distinct ARC subtypes were identified and were significantly associated with patient survival, clinical features, CMS, CSC index, and TME, but not with gene mutations. Four genes (S100A4, COL3A1, TIMP1, and APP) were identified as key ARCs, capable of distinguishing ARC subtypes. The prognostic signature based on these genes effectively stratified patients into high- or low-risk categories. scRNA-seq analysis showed that these genes were predominantly expressed in immune cells rather than in cancer cells. Validation in two external cohorts and through clinical samples confirmed significant expression differences between CRC and controls. This study identified two ARG subtypes in CRC and highlighted four key genes associated with these subtypes, offering new insights into personalized CRC treatment strategies.

Modulation of innate immunity related genes resulting in prophylactic antimicrobial and antiviral properties

BackgroundThe innate immunity acts during the early phases of infection and its failure in response to a multilayer network of co-infections is cause of immune system dysregulation. Epidemiological SARS-CoV-2 infections data, show that Influenza Virus (FLU-A-B-C) and Respiratory Syncytial Virus (RSV) are co-habiting those respiratory traits. These viruses, especially in children (mostly affected by ‘multi-system inflammatory syndrome in children’ [MIS-C] and the winter pandemic FLU), in the aged population, and in ‘fragile’ patients are causing alteration in immune response. Then, bacterial and fungal pathogens are also co-habiting the upper respiratory traits (e.g., Staphylococcus aureus and Candida albicans), thus contributing to morbidity in those COVID-19 affected patients.MethodsLiquid chromatography coupled with high-resolution mass spectrometry using the quadrupole orbital ion trap analyser (i.e., UHPLC-Q-Orbitrap HRMS) was adopted to measure the polyphenols content of a new nutraceutical formula (Solution-3). Viral infections with SARS-CoV-2 (EG.5), FLU-A and RSV-A viruses (as performed in BLS3 authorised laboratory) and real time RT-PCR (qPCR) assay were used to test the antiviral action of the nutraceutical formula. Dilution susceptibility tests have been used to estimate the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) of Solution-3 on a variety of microorganisms belonging to Gram positive/ negative bacteria and fungi. Transcriptomic data analyses and functional genomics (i.e., RNAseq and data mining), coupled to qPCR and ELISA assays have been used to investigate the mechanisms of action of the nutraceutical formula on those processes involved in innate immune response.ResultsHere, we have tested the combination of natural products containing higher amounts of polyphenols (i.e., propolis, Verbascum thapsus L., and Thymus vulgaris L.), together with the inorganic long chain polyphosphates ‘polyPs’ with antiviral, antibacterial, and antifungal behaviours, against SARS-CoV-2, FLU-A, RSV-A, Gram positive/ negative bacteria and fungi (i.e., Candida albicans). These components synergistically exert an immunomodulatory action by enhancing those processes involved in innate immune response (e.g., cytokines: IFNγ, TNFα, IL-10, IL-6/12; chemokines: CXCL1; antimicrobial peptides: HBD-2, LL-37; complement system: C3).ConclusionThe prophylactic antimicrobial success of this nutraceutical formula against SARS-CoV-2, FLU-A and RSV-A viruses, together with the common bacteria and fungi co-infections as present in human oral cavity, is expected to be valuable.

Qualitative real-time RT-PCR assay for nOPV2 poliovirus detection

Based on the success of the Sabin2-based vaccine, a next-generation nOPV2 poliovirus vaccine has been developed. For epidemic monitoring and conducting epidemiological investigations, it is necessary to have a diagnostic assay with the ability to differentiate this variant from others. Here we describe such a real-time RT-PCR assay. The region with the cre insertion in the 5′-UTR was chosen as the target, and the limit of detection was 103 copies/mL (2.5×103 copies/mL using Probit analysis) determined using armored RNA particles. Sensitivity and specificity were 86.28 – 100 % and 76.84 – 100 %, respectively (with 95 % CI). Thus, this method can be effectively used when it is necessary to make a differential diagnosis of poliovirus strains.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL−1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL−1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

Headache and Dizziness as Prevailing Clinical Manifestations in Parkinson's Patients Following COVID-19

Background: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has posed significant global health challenges, particularly for vulnerable populations. Objectives: This retrospective report from the Iranian Network for Research in Viral Diseases (INRVD) investigates the common clinical symptoms of COVID-19 in individuals with Parkinson's disease (PD). Research suggests neurological complications can occur during SARS-CoV-2 infection, but there are limited studies on neurological symptoms in PD patients with COVID-19. Given the probable heightened vulnerability of PD patients, evaluating clinical symptoms, especially neurological manifestations, is crucial. Methods: Data from 54 patients were collected from the INRVD from March 2020 to May 2021. Nasal, oropharyngeal, and nasopharyngeal swabs were collected from patients presenting with respiratory symptoms, following Iranian CDC guidelines. Following sample collection, RT-PCR assays were performed for COVID-19 confirmation. Statistical analysis was conducted using the Chi-square test and Fisher's exact test. Results: Among the 54 PD patients included in the study, 27 tested positive for COVID-19. Of the 54 subjects analyzed, 38.9% were female and 61.1% were male. Comparison of COVID-19 symptoms revealed a higher rate of sore throat (74%) and shortness of breath (81%) in the COVID-19 positive group as common clinical symptoms of the disease. Interestingly, higher rates of dizziness (67%) and headache (74%) were observed in PD patients with COVID-19. Conclusions: This study enhances our understanding of the diverse clinical manifestations of COVID-19 and its impact on individuals with underlying health conditions such as PD. The increased prevalence of neurological symptoms among PD patients underscores the need for further investigation to clarify whether these symptoms directly result from COVID-19 infection or if they may also be influenced by PD itself or its treatment. It is important to consider that while neurological symptoms in Parkinson's patients can be attributed to the underlying disorder or linked to medication and medical interventions, respiratory symptoms are typically associated with COVID-19.

Temporin-GHaK Exhibits Antineoplastic Activity against Human Lung Adenocarcinoma by Inhibiting the Wnt Signaling Pathway through miRNA-4516.

(1) Background: GHaK is derived from the antimicrobial peptide temporin-GHa by substituting the amino acid H with K to enhance its bactericidal activity. The present research aims to broaden the pharmacological potential of GHaK by exploring its antineoplastic activity against human lung adenocarcinoma. (2) Methods: The cell viability, migration, invasion, apoptosis, and cell cycle of A549 and PC-9 cells were tested after GHaK treatment. miRNA sequencing, RT-PCR, Western blotting, and luciferase reporter gene assay were further performed to reveal the potential mechanism. (3) Results: GHaK significantly suppressed cell viability, migration, and invasion; induced apoptosis; and caused cell cycle arrest in the G2/M and S phase in PC-9 and A549 cells, respectively. The miRNA sequencing results show a total of 161 up-regulated and 115 down-regulated miRNAs. Furthermore, the study identified six up-regulated miRNAs (miR-4516, miR-4284, miR-204-5p, miR-12136, miR-4463, and miR-1296-3p) and their inhibitory effects on the expressions of target genes (Wnt 8B, FZD2, DVL3, and FOSL1) caused by miR-4516 directly interacting with Wnt 8B. Western blotting revealed the down-regulation of p-GSK-3β, along with a decreased expressions of cyclin A1 and CDK2 in A549 cells and cyclin B1 and CDK1 in PC-9 cells. (4) Conclusions: Temporin-GHaK exhibits antineoplastic activity against human lung adenocarcinoma by inhibiting the Wnt signaling pathway through miRNA-4516.

Molecular evolution and functional diversification of metal tolerance protein families in cereals plants and function of maize MTP protein

Plants employ metal tolerance proteins (MTPs) to confer tolerance by sequestering excess ions into vacuoles. MTPs belong to the cation diffusion facilitator (CDF) family, which facilitates the transport of divalent transition metal cations. In this study, we conducted a comprehensive analysis of the MTP gene families across 21 plant species, including maize (Zea mays). A total of 247 MTP genes were identified within these plant genomes and categorized into distinct subgroups, namely Zn-CDF, Mn-CDF, and Fe/Zn-CDF, based on phylogenetic analyses. This investigation encompassed the characterization of genomic distribution, gene structures, cis-regulatory elements, collinearity relationships, and gene ontology functions associated with MTPs. Transcriptomic analyses unveiled stress-specific expression patterns of MTP genes under various abiotic stresses. Moreover, quantitative RT-PCR assays were employed to assess maize MTP gene responses to diverse heavy metal stress conditions. Functional validation of metal tolerance roles was achieved through heterologous expression in yeast. This integrated evolutionary scrutiny of MTP families in cereals furnishes a valuable framework for the elucidation of MTP functions in subsequent studies. Notably, the prioritized MTP gene ZmMTP6 emerged as a positive regulator of plant Cd tolerance, thereby offering a pivotal genetic asset for the development of Cd-tolerant crops, particularly maize cultivars.

RT-PCR Testi ile doğrulanmış COVID-19 olgularında hızlı antikor testi uyumluluğunun değerlendirilmesi

INTRODUCTION: It was aimed to determine the reliability of rapid antibody detection test (RADT) results and compatibility with RT-PCR assay in the screening and control of COVID-19 infection. METHODS: The retrospective study was performed with the permission of the Ministry of Health and the local ethics committee of our hospital. Between May 2020 and August 2021, 624 health staffs laboratory results were recorded. Data processing hospital (HIS) and laboratory information system (LIS) records of our hospital were used as data collection method. The level of agreement between tests was estimated using Cohen's κ index. Statistical analyzes were performed using SPSS open source software. RESULTS: The mean age of the participants in the study was 28,46 ± 2,35. 54% were female (337), 46% (n=287) were male, and they had no additional disease. Both rapid antibody testing and RT-PCR tests were negative in 86% (n=540) of all tests. Two nasopharyngeal specimens were collected from each case to perform the RADT and RT-PCR. 13,6% (n=102) of the included patients had positive RT-PCR results. The RADT detected 84 of the 102 RT-PCR-positive cases and there were no false positive results. Overall sensitivity and specificity were 84,7% and 100%, respectively. Sensitivity was found &gt;95% in symptomatic cases. DISCUSSION AND CONCLUSION: The Weimi Bio-Tech COVID-19 Ag RADT can be useful in screening for COVID-19, especially in cases in the first or second weeks of symptomatic infection.

The inhibition mechanism of co-cultured probiotics on biofilm formation of Klebsiella pneumoniae.

Klebsiella pneumoniae, an important opportunistic pathogen of nosocomial inflection, is known for its ability to form biofilm. The purpose of the current study is to assess how co- or mono-cultured probiotics affect K. pneumoniae's ability to produce biofilms and investigate the potential mechanisms by using a polyester nonwoven chemostat and a Caco-2 cell line. Compared with pure cultures of Lactobacillus rhamnosus and Lactobacillus sake, the formation of K. pneumoniae biofilm was remarkably inhibited by the mixture of L. rhamnosus, L. sake, and Bacillus subtilis at a ratio of 5:5:1 by means of qPCR and FISH assays. In addition, Lactobacillus in combination with B. subtilis could considerably reduce the adherence of K. pneumoniae to Caco-2 cells by using inhibition, competition, and displacement assays. According to the RT-PCR assay, the adsorption of K. pneumoniae to Caco-2 cells was effectively inhibited by the co-cultured probiotics, leading to significant reduction in the expression of proinflammatory cytokines induced by K. pneumoniae. Furthermore, the HPLC and RT-PCR analyses showed that the co-cultured probiotics were able to successfully prevent the expression of the biofilm-related genes of K. pneumoniae by secreting plenty of organic acids as well as the second signal molecule (c-di-GMP), resulting in inhibition on biofilm formation. Co-culture of L. sake, L. rhamnosus, and B. subtilis at a ratio of 5:5:1 could exert an antagonistic effect on the colonization of pathogenic K. pneumoniae by down-regulating the expression of biofilm-related genes. At the same time, the co-cultured probiotics could effectively inhibit the adhesion of K. pneumoniae to Caco-2 cells and block the expression of proinflammatory cytokines induced by K. pneumoniae.

The development and initial evaluation of a multiplex RT-PCR and high-throughput liquid chip assay for the simultaneous detection of five salmonid virus diseases

Viral diseases pose a major threat to salmonids aquaculture, and mixed infections against salmonids are common. Early detection and identification of viral pathogens are crucial for effective prevention and control measures. This study used samples of infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmonid alphavirus (SAV), and viral hemorrhagic septicemia virus (VHSV) to evaluate a multiplex RT-PCR assay developed for the simultaneous detection of these viruses. A liquid chip technique combined with multiplex RT-PCR was developed. The RT-PCR amplification products were tested using a liquid chip probe. The sensitivity of the assay system was evaluated using 10-fold serial dilutions of RNA extracted from IHNV, IPNV, ISAV, SAV, and VHSV with concentrations of 0.1, 0.4, 2, 0.9, and 1.6 pg µL−1, respectively. The assay was highly specific for these different viruses owing to the use of two nested degenerate primers and one probe. No cross-reactions occurred among the viruses nor did any non-specific reactions occur with other pathogens. The proposed method can be used for rapid, accurate, sensitive, and simultaneous detection of five salmon and trout viruses, thereby contributing to the rapid and accurate quarantine of aquatic animal samples.

Evaluating the efficiency of ELISA, monoplex and multiplex probe-based real-time reverse-transcription PCR assays in the detection of SARS-CoV-2 (COVID-19) and influenza A and B viruses: A cross-sectional study.

The current study aimed to evaluate the efficiency of Enzyme-linked immunosorbent assay (ELISA) assay and monoplex and multiplex real-time reverse-transcription PCR (rRT-PCR) in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A and B viruses (Flu A and Flu B). The SARS-CoV-2 -specific IgG and IgM antibodies, as well as, Flu A (H1N1 and H3N2 serotypes) and Flu B virus antibodies were determined by ELISA assay. The one-step qRT-PCR method was used to detect the SARS-CoV-2 in nasopharyngeal swab samples. Furthermore, the presence of Flu A and B viruses was evaluated using probe-based RT-PCR. Simultaneous detection of SARS-CoV-2, Flu A and B viruses was performed by multiplex rRT-PCR assay. SARS CoV-2 IgM and IgG antibodies were detected in 33.3% and 58.3% of patients, respectively. In contrast, the SARS CoV-2 genome was detected in 50% of patients using the one-step monoplex RT-PCR assay. Flu A serotypes H1N1 and H3N2 were found in 16.7% and 8.3% of patients. Probe-based RT-PCR revealed that 39.3% of patients were positive for the Flu A virus. Multiplex rRT-PCR detect the SARS-CoV-2, Flu A, and Flu B in 50%, 39.3%, and 19% of samples, respectively. The sensitivity and specificity of multiplex rRT-PCR assay in comparison to monoplex RT-PCR were 100% and 55%, respectively. Coinfection with SARS-CoV-2, Flu A, and Flu B viruses was found in 9.5% of patients. Multiplex rRT-PCR can be used as a repaid, cost-effective and suitable tool for molecular surveillance of SARS-CoV-2 and Flu A/B viruses.

Diverse genotypes of norovirus genogroup I and II contamination in environmental water in Thailand during the COVID-19 outbreak from 2020 to 2022

Noroviruses (NoVs) are the most significant viral pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. This study aimed to investigate the prevalence and diversity of NoVs contaminated in the environmental water in Chiang Mai, Thailand. A total of 600 environmental water samples were collected from ten sampling sites in Chiang Mai from July 2020 to December 2022. The presence of NoV genogroups I (GI), GII, and GIV were examined using real-time RT-PCR assay. The genotype of the virus was determined by nucleotide sequencing and phylogenetic analysis. The results showed that NoV GI and GII were detected at 8.5% (51/600) and 11.7% (70/600) of the samples tested, respectively. However, NoV GIV was not detected in this study. NoV circulated throughout the year, with a higher detection rate during the winter season. Six NoV GI genotypes (GI.1-GI.6) and eight NoV GII genotypes (GII.2, GII.3, GII.7, GII.8, GII.10, GII.13, GII.17, and GII.21) were identified. Among 121 NoV strains detected, GII.17 was the most predominant genotype (24.8%, 30 strains), followed by GII.2 (21.5%, 26 strains), GI.3 (17.4%, 21 strains), and GI.4 (16.5%, 20 strains). Notably, NoV GII.3, GII.7, GII.8, and GII.10 were detected for the first time in water samples in this area. This study provides insight into the occurrence and seasonal pattern of NoV along with novel findings of NoV strains in environmental water in Thailand during the COVID-19 outbreak. Our findings emphasize the importance of further surveillance studies to monitor viral contamination in environmental water.

The full genome characterization of avian encephalomyelitis virus, Iran: a vertical transmission case.

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). The causative virus can be transmitted both horizontally and vertically. In the present study, an AEV suspected outbreak with typical neurological signs occurred in broilers. Histopathological examination, RT-PCR assay and full genome sequencing were applied to confirm the presence of AEV. Phylogenetic analysis of the full genome sequence showed that the detected AEV strain at 7055 nucleotide length is classified in cluster I and is closely related to vaccinal USA and China originated isolates. Although, the outbreaks of AE in progeny of vaccinated breeders have been reported previously, the source of infection was unknown. Based on the results obtained in this study, the outbreaks are vaccine-originated. This study provides the first whole genome analysis of AEV from Iran and reveals that the AEV possesses a hepatitis C virus-like internal ribosome entry site.

Study on the Mechanism of Action of the Traditional Chinese Medical Prescription Gushukang in Treating Osteoporosis Based on Network Pharmacology and Experimental Verification.

Gushukang (GSK), a traditional Chinese medical prescription, has made a great and extensive contribution to the treatment of different forms of osteoporosis, but polypharmacology studies of its mechanism of action are lacking. This study investigates the pharmacological mechanism of osteoporosis using network pharmacology and molecular docking. Experimental verification was carried out to confirm the efficacy of GSK on RANKLinduced osteoclast differentiation in RAW264.7 cells to verify the network pharmacology studies. The effective chemical components and corresponding targets of osteoporosis with oral bioavailability of more than 30% and drug-like properties greater than 0.18 were searched in the TCMSP and TCM-ID databases. DrugBank, GeneCards, OMIM, TTD, and other databases were examined for targets related to osteoporosis. Using Cytoscape software, a network of possible TCM-active ingredient-osteoporosis targets was created. STRING software was used to create the networks of protein-protein interactions. The DAVID program was carried out to conduct GO and KEGG pathway enrichment analyses of the targets. Molecular docking and pattern of action analysis were carried out using software like AutoDock Vina and Discovery Studio Visualizer. The growth media for RAW264.7 cells contained varying doses of GSK serum and 50 ng/mL RANKL. The activity of TRAP was altered. Additionally, genes related to osteoclasts were examined using an RT-PCR assay. Network pharmacological analysis revealed that the primary efficacy targets of osteoporosis were PTGS2, PTGS1, HSP90AA1, NCOA2, ADRB2, ESR1, NCOA1, and AR. The pharmacological targets of osteoporosis may be mediated by substances including quercetin, kaempferol, luteolin, naringenin, icariin, anthocyanin, tanshinone IIA, and cryptotanshinone. GSK markedly inhibited RANKL-induced TRAP activity. qRT-PCR results revealed decreased expression of the PTGS2 and ADRB2 genes upon GSK treatment. The findings of network pharmacology, molecular docking, as well as experimental verification provide a new further study for elucidating the pharmacodynamic substance basis and polypharmacology mechanism of GSK in treating osteoporosis.

Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Melting curve analysis reveals false-positive norovirus detection in a molecular syndromic panel

BackgroundMolecular syndromic panels can improve rapidity of results and ease clinical laboratory workflow, although caution has been raised for potential false-positive results. Upon implementation of a new panel for infectious diarrhea (BioFire® FilmArray® Gastrointestinal [GI] Panel, bioMérieux) in our clinical laboratory, a higher than expected number of stool samples with norovirus were detected. ObjectivesThe goal of this study was to investigate positive percent agreement and the false-positive rate of norovirus detected by the multiplex BioFire GI panel compared to a singleplex commercial assay. Study designFrom October 2023 to January 2024, all prospective stool samples with a positive norovirus result by BioFire had melting curves reviewed manually using the BioFire FilmArray Torch System. Stool samples further underwent testing by a supplementary real-time RT-PCR assay (Xpert® Norovirus, Cepheid) for comparative analysis. ResultsOf the 50 stool samples with norovirus detected by BioFire, 18 (36 %) tested negative by Xpert (deemed "false-positives"). Furthermore, melting curve analysis revealed nearly all of these samples had atypical melting curve morphologies for the "Noro-1" target on BioFire (16/18, 89 %), which was statistically significant (Odds Ratio 173.2, 95 % CI [22.2, 5326.9], p < 0.0001). Stool samples with multiple pathogens detected by BioFire including norovirus were not more likely to produce false-positive norovirus results (Odds Ratio 1, 95 % CI [0.3, 3.3], p = 1). ConclusionsAlthough not described in the manufacturer's Instructions for Use, we propose routine manual review of melting curves for the BioFire GI panel prior to reporting, to mitigate potential false-positive norovirus results.

Genetic characterization and evidence for multiple reassortments of rotavirus A G3P[3] in dogs and cats in Thailand

Rotavirus A (RVA) causes gastroenteritis in humans and animals. The zoonotic potential of RVA has been reported and raises major concerns, especially in animal-human interface settings. The study aimed to characterize and investigate the genetic diversity among RVAs in dogs and cats in Thailand. We collected 572 rectal swab samples from dogs and cats in Bangkok animal hospitals from January 2020 to June 2021. The one-step RT-PCR assay detected RVAs in 1.92% (11/572) of the samples, with 2.75% (8/290) in dogs and 1.06% (3/282) in cats. Two canine RVA and one feline RVA were subjected to whole genome sequencing. Our results showed that all three viruses were identified as RVA genotype G3P[3]. The genetic constellation of RVAs is unique for different species. For canine RVAs is G3-P [3]-I3-R3-C3-M3-A9-N2-T3-E3-H6, while Feline RVA is G3-P [3]-I8-R3-C3-M3-A9-N3-T3-E3-H6. Notably, both canine and feline RVAs contained the AU-1 genetic constellation with multiple reassortments. The results of phylogenetic, genetic, and bootscan analyses showed that canine RVAs may have reassorted from dog, human, and cat RVAs. While feline RVA was closely related to RVAs in humans, bats, and simians. This study provided genetic characteristics and diversity of RVAs in dogs and cats and suggested possible multiple reassortments, suggesting the zoonotic potential of the viruses. Thus, public health awareness should be raised regarding the zoonotic potential of RVAs in dogs and cats. Further studies on RVAs on a larger scale in dogs and cats in Thailand are needed.