Real-time RT-PCR Research Articles

The canonical Hippo pathway components modulate the differentiation of lamina propria regulatory T cells and T helper 17-like regulatory T cells in mouse colitis.

Regulatory T cells (Tregs) ameliorate inflammatory bowel diseases. However, their plasticity is not completely understood. In this study using a mouse colitis model, Tregs and T helper 17 (Th17)-like Tregs were detected and sorted using flow cytometry, followed by transcriptome sequencing, real-time RT-PCR, and flow cytometry to analyze the mRNA profiles of these cells. Treg plasticity was evaluated by in vitro differentiation assays. The immunosuppressive activities of Tregs and Th17-like Tregs were assessed in an adoptive transfer assay. We found Tregs-derived Th17-like Tregs in inflamed colonic lamina propria (LP). LP Th17-like Tregs expressed higher Th17-related cytokines and lower immunosuppressive cytokines compared with LP Tregs. Notably, Tregs expressed higher Yes-associated protein 1 (YAP1) but lower transcriptional coactivator with PDZ‑binding motif (TAZ) than Th17-like Tregs. Verteporfin-mediated inhibition of YAP1 activity enhanced Th17-like Treg generation, whereas IBS008739-induced TAZ activation did not affect Th17-like Treg generation. Besides, verteporfin enhanced while IBS008739 suppressed the differentiation of Th17-like Tregs into Th17 cells. Furthermore, YAP1 activated STAT5 signaling in Tregs, whereas YAP1 and TAZ activated STAT3 and STAT5 signaling in Th17-like Tregs. Compared with Tregs, Th17-like Tregs were less efficacious in ameliorating colitis. Therefore, YAP1 suppressed Treg differentiation into Th17-like Tregs. Both YAP1 and TAZ inhibited the differentiation of Th17-like Tregs into Th17 cells. Therefore, YAP1 and TAZ probably maintain the immunosuppressive activities of Tregs and Th17-like Tregs in colitis.

Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

Unveiling the emergence of SARS-CoV-2 JN.1 sub-variant: Insights from the first cases at Charles Nicolle Hospital, Tunisia.

The JN.1 sub-variant is a new variant of the SARS-CoV-2 Omicron strain, derived from the BA.2.86 sub-variant. It was first detected in late 2023 and has quickly spread to many countries, becoming the most prevalent variant in some regions. JN.1 exhibits a unique mutation (L455S) in the spike protein compared to the BA.2.86 lineage, which may affect its transmissibility and immune evasion capabilities. JN.1 has been designated as a "variant of interest" by the World Health Organization due to its rapidly increasing spread and is being closely monitored for its impact on the COVID-19 pandemic. This study describes the emergence of SARS-CoV-2 JN.1 sub-variant in Tunisia, and reports its mutation profiles.Nasopharyngeal samples collected over a four-month period (October 2023 to January 2024) were subjected to RNA extraction and real-time RT-PCR confirmation of SARS-CoV-2 infection. The whole-genome sequencing was performed by an iSeq 100 sequencer and COVIDSeq kit reagents (Illumina, USA).Mutation analysis, using the NextClade platform and GISAID database, revealed the presence of JN.1 in 15 out of 80 positive cases (18.75%) during the study period.The emergence of JN.1 highlights the ongoing evolution of SARS-CoV-2 and the need for continued surveillance and research to better understand the characteristics and impact of emerging variants.

Pregnancy and COVID—Complications and its Challenges: A Case Series from a Tertiary Care Center in the Northeastern Part of India

The objective was to describe the course of management of all the coronavirus disease 2019 (COVID-19)-positive pregnant women, admitted to a 150-bedded tertiary care center in Guwahati, Assam, from March 01, 2021, till June 15, 2021. We collected a case series of 13 COVID-19-positive pregnant women, mostly in their second and third trimesters, admitted to our center between March 01, 2021, and June 15, 2021. All of the cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were confirmed by a positive result on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests. We aimed to show the course of management and maternal outcomes in these cases. The Results show that among the 13 infected pregnant women, three presented with severe SARS-CoV-2 pneumonia and were treated in the intensive care unit (ICU) and the rest were treated in wards as the mild category and discharged successfully. Two among them required invasive mechanical ventilation (IMV), and one was managed on non-invasive ventilation (NIV). Of the two mechanically ventilated patients, one succumbed to the illness and the other patient was successfully weaned in 5 days and shifted out after 15 days of the ICU stay. The third patient was on NIV support for 7 days, weaned off to low-flow oxygen therapy via face mask/ nasal prongs, and shifted out after 17 days of the ICU stay. Data on pregnant women with coronavirus infection are scarce and contradictory as the effect of SARS-CoV-2 on pregnancy is not clear. It is highly important to systemically investigate the clinical characteristics and outcomes of pregnant women with COVID-19 to provide strong guidelines for prevention, treatment, and management.

Silencing Exosomal circ102927 Inhibits Foot Melanoma Metastasis via Regulating Invasiveness, Epithelial-Mesenchymal Transition and Apoptosis.

Exosomes contain abundant circular RNAs (circRNAs), playing an important role in intercellular communication. However, the function and underlying molecular mechanism of exosomal circRNAs in foot metastatic melanoma remain unclear. Twelve differentially expressed exosomal circRNAs between patients with metastatic and primary foot melanoma were screened through high-throughput sequencing, and their expression levels were detected by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). CircRNA102927 silencing and overexpression A2058 cell line was constructed, and the effects of circRNA102927 on cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) were assessed using cell counting kit-8 (CCK-8), flow cytometry, wound healing, Transwell, and Western blot assays, respectively. Twelve differentially expressed exosomal circRNAs were screened and ROC curve showed that six circRNAs could be used as the diagnostic biomarkers for metastatic melanoma. Melanoma-secreted exosomes induced the differentiation of CD4+ T cells into Treg cells. CircRNA102927 was highly expressed in metastatic melanomas. Functionally, circRNA102927 silencing inhibited proliferation, EMT, migration, and invasion in metastatic melanoma cells, while promoting apoptosis. Meanwhile, overexpression of circRNA102927 had the opposite effects. Our investigation suggests that silencing exosomal circRNA102927 may suppress foot melanoma metastasis by inhibiting invasiveness, EMT and promoting apoptosis.

WITHDRAWN: Circular RNA CircRNA-0000520 promotes non-small cell lung cancer progression through sponging microRNA-521

The non-small cell lung cancer (NSCLC) accounts for 85 % of all lung cancers. This study investigated how the circular RNA (circRNA)-0000520 affected NSCLC and the underlying mechanisms, providing potential target for developing effective treatments of NSCLC. Firstly, circRNA-0000520 expression in tissues and cells were explored by the GEO2R online tool analyzing the public dataset obtained from the Gene Expression Omnibus (GEO) database and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). CircRNA-0000520 was upregulated in NSCLC (P < 0.05). Knockdown of circRNA-0000520 decreased the proliferation, migration and invasion of NSCLC cells, while increased the apoptosis and G0/G1 arrest of NSCLC cells (P < 0.05). Next, the downstream miRNAs of circRNA-0000520 were detected. By using RT-qPCR, the miR-521 was found to be downregulated in both NSCLC tissues and cell lines. And circRNA-0000520 knockdown obviously led to an increase of miR-521 in NSCLC cells. Circinteractome database was used to predict the binding sites of miR-521 in circRNA-0000520. And the interaction between circRNA-0000520 and miR-521 were verified by dual-luciferase reporter assay and RNA immunoprecipitation verified the interaction between circRNA-0000520 and miR-521. Furthermore, miR-521 depletion reversed the decreased levels of proliferation, migration and invasion of NSCLC cells, and the increased levels of apoptosis and G0/G1 arrest of NSCLC cells (P < 0.05) induced by circRNA-0000520 knockdown. In general, this study further verified that circRNA-0000520 played oncogenic roles in NSCLC and promoted the malignant behavior of NSCLC, and firstly proved that circRNA-0000520 acted as a sponge of miR-521, suggesting the potential of circRNA-0000520/miR-521 axis as the promising therapeutic targets for patients with NSCLC.

Analysis of real-time RT-PCR and rapid antigen test and its correlation with clinical characteristics, investigation profile, and imaging of SARI-suspected COVID-19 infections

Abstract Background: Coronavirus disease 2019 (COVID-19) continues to be a significant global public health issue. Aims and Objectives: This study aims to describe the demographic characteristics, comorbidities, clinical presentations, inflammatory biomarkers, and chest computed tomography (CT) findings of patients with severe acute respiratory infection (SARI) at a tertiary care hospital in Odisha, India. Additionally, the study aims to evaluate different COVID-19 diagnostic methods. Materials and Methods: A prospective observational study was conducted at a tertiary care hospital in Odisha, India. All admitted SARI patients underwent COVID-19 testing. Data on demographics, clinical presentations, laboratory investigations, and outcomes at 72 h were collected. COVID-19 diagnostic methods were also analyzed. Statistical Analysis: The statistical analysis was performed using GraphPad Prism 9.5. Results: Among the 830 patients studied, 305 tested positive for COVID-19. The rapid antigen test (RAT) demonstrated a sensitivity of 93.85% and a specificity of 97.43% compared to the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test. Discrepancies with RAT results were noted in cases with higher cycle threshold (Ct) values (&gt;25). A male predominance was observed, with a mean age of 43.30 ± 15.19 years in the COVID-19-positive group and 40.95 ± 17.15 years in the COVID-19-negative group. Patients with multiple comorbidities (15.68%) had a higher susceptibility to COVID-19 infection. The COVID-19-positive group showed a significantly elevated neutrophil-to-lymphocyte ratio (NLR) (P = 0.0001) and thrombocytopenia (P = 0.001). Inflammatory biomarkers, including CRP, LDH, ferritin, D-dimer, and IL-6, were markedly elevated in swab-positive cases. Bilateral chest involvement and ground glass opacities were common radiological findings in both groups. Conclusion: In resource-limited settings, the RAT is considered an alternative screening tool for COVID-19. Initial laboratory and radiological investigations may aid clinicians in effective management of such patients.

Epidemiological characteristics of human metapneumovirus among children in Nanjing, China.

The objective of this study was to examine the molecular epidemiology and clinical characteristics of HMPV infection among children with ARIs in Nanjing. The respiratory samples were collected from 2078 children (≤ 14 years) with acute respiratory infections and were tested for HMPV using real-time RT-PCR. Amplification and sequencing of the HMPV G gene were followed by phylogenetic analysis using MEGA 7.0. The detection rate of HMPV among children was 4.7% (97/2078), with a concentration in those under 5 years of age. Notably, the peak season for HMPV prevalence was observed in winter. Among the 97 HMPV-positive samples, 51.5% (50/97) were available for characterization of the HMPV G protein gene. Phylogenetic analysis indicated that the sequenced HMPV strains were classified into three sublineages: A2c111nt - dup (84.0%), B1 (2.0%), and B2 (14.0%). There was an incidence of HMPV among hospitalized children during 2021-2022 in Nanjing with A2c111nt - dup being the dominant strain. This study demonstrated the molecular epidemiological characteristics of HMPV among children with respiratory infections in Nanjing, China.

Effects of valproic acid on transcript levels in neurotrophin signaling pathway in mice hippocampus according to the implementation period

Abstract Background/Aim Valproic acid (VPA), is an antiepileptic drug and it has been known for a long time that exposure to VPA in the fetal period causes many behavioral, cognitive, and structural disorders. In this study, we aimed to evaluate the effects of the implementation period of VPA during pregnancy on both behavioral tests and the expression of 45 key genes belonging to the neurotrophin signaling pathway. Methods The study was designed to form four different groups of five female mice in each group. The experimental and control groups were divided into mature (n = 5) and immature (n = 5) subgroups, respectively. VPA (500 mg/kg dose) was subcutaneously administered to the experimental groups. All mice were first tested for behavioral changes, and then the hippo-campal tissues were used for RNA isolation. The expression levels of 45 transcripts associated with the neurotrophin signaling pathway were determined by real-time RT-PCR in the hippocampus. Results No differences were found between the groups in any of the behavioral tests. When gene expression was examined, there was a difference between the groups in the transcript levels of Ngf(neurotrophic growth factor), Ntrk2 (neurotrophic receptor tyrosine kinase 2), Shc1 (SHC adaptor protein 1), Chka (choline kinase alpha), and Mapk1 (mitogen-activated protein kinase 1) genes. Conclusions This study demonstrated the effects of VPA on transcript levels at different maturation stages. We believe that it is important to explain the structural and cognitive teratogenic side effects caused by VPA in human fetuses using genes whose expression changes.

Measles outbreak in 2019: a warning for the post-COVID-19 pandemic period.

Introduction. At the end of 2019 and the year before, there was a significant spread of measles in the World Health Organization (WHO) European Region.Gap statement. Among the countries that reported, a measles outbreak was Bosnia and Herzegovina (BiH).Aim. To describe the measles outbreak in BiH (an entity of the Federation of BiH, FBiH) in 2019.Methodology. Confirmatory IgM serology, measles nucleic acid detection by real-time RT-PCR and virus genotyping were done in the WHO-accredited laboratory for measles and rubella at the Clinical Center of the University of Sarajevo, Unit for Clinical Microbiology. Genotype was determined in all measles-RNA-positive cases by sequence analysis of the 450 nt fragment coding the C-terminal of measles virus nucleoprotein (N).Results. From 1 January to 31 December 2019, 1332 measles cases were reported, with the peak observed in April 2019 (413/1332, 31.01 %). Sarajevo Canton had the highest incidence, number of cases and percentage (206.4; 868/1332; 65.17 %) of measles cases. Around four-fifths of infected persons were unvaccinated (1086/1332, 81.53 %), while 4.58 % of the patients (61/1332) were immunized with one dose of measles-containing vaccine. The highest proportion of cases was found in children 0-6 years of age (738/1332, 55.41 %). Measles IgM positivity was determined in 75.88 % (346/456), while virus RNA was detected in 82.46 % (47/57) of the swab samples. All measles virus sequences belonged to genotype B3. SNP (position 216: C=>T) was detected in 1 of the 40 sequences obtained during this outbreak.Conclusion. Due to suboptimal immunization coverage, BiH belongs to countries at a high risk for measles outbreaks. Post-COVID-19 (coronavirus disease 2019) pandemic, targeted and tailored strategies are required to ensure routine vaccination demand and acceptance and broad partner and stakeholder group participation.

P7C3 suppresses astrocytic senescence to protect dopaminergic neurons: Implication in the mouse model of Parkinson's disease.

Astrocytic senescence is inextricably linked to aging and neurodegenerative disorders, including Parkinson's disease (PD). P7C3 is a small, neuroprotective aminopropyl carbazole compound that exhibits anti-inflammatory properties. However, the effects of P7C3 on astrocytic senescence in PD remain to be elucidated. An invitro, long culture-induced, replicative senescence cell model and a 1-methyl-4-phenylpyridinium (MPP+)/rotenone-induced premature senescence cell model were used to investigate the effects of P7C3 on astrocytic senescence. An invivo, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse PD model was used to study the role of P7C3 in astrocytic senescence. Immunoblotting, real-time quantitative RT-PCR (qPCR), immunofluorescence, subcellular fractionation assays, and immunohistochemistry were utilized to confirm the effects of P7C3 on astrocytic senescence and elucidate its underlying mechanisms. This study determined that P7C3 suppressed the senescence-associated secretory phenotype (SASP) in both cell models, as demonstrated by the reduction in the critical senescence marker p16 and proinflammatory factors (IL-6, IL-1β, CXCL10, and MMP9) and increased laminB1 levels, implying that P7C3 inhibited replicative astrocytic senescence and MPP+/rotenone-induced premature astrocytic senescence, Most importantly, we demonstrated that P7C3 prevented the death of dopamine (DA) neurons and reduced the behavioral deficits in the MPTP-induced mouse model of PD, which is accompanied by a decrease in senescent astrocytes in the substantia nigra compacta (SNc). Mechanistically, P7C3 promoted Nrf2/Sirt3-mediated mitophagy and reduced mitochondrial reactive oxygen species (mitoROS) generation, which contributed to the suppression of astrocytic senescence. Furthermore, Sirt3 deficiency obviously abolished the inhibitory effects of P7C3 on astrocytic senescence. This study revealed that P7C3 inhibited astrocytic senescence via increased Nrf2/Sirt3-mediated mitophagy and suppression of mitoROS, which further protected against DA neuronal loss. These observations provide a prospective theoretical basis for P7C3 in the treatment of age-associated neurodegenerative diseases, such as PD.

The Anti-psoriatic Effect of Gallic Acid is Associated with the Suppression of Keratin 6 and Nrf2

Background: Psoriasis is recognized as an autoimmune dermatosis, and keratin 6 (KRT 6) is a hallmark of psoriasis. Gallic acid (GA) is a natural and small molecule with a series of biological activities. However, the effect of GA on psoriasis has not been clarified. Aims: This study aimed to investigate the anti-psoriatic activity of GA in psoriasis-like mice and in vitro. Methods: The transcriptions of the Homo sapiens KRT6 gene, and Mus musculus KRT6 gene, were identified using a quantitative real-time reverse transcriptase PCR (qRT-PCR) assay. Expressions of KRT 6, STAT3, pSTAT3, Nrf2, and pNrf2 in HaCaT cells and skin biopsies were determined with a western blotting assay. The immunofluorescence (IF) assay was used to examine the expression of KRT6, pSTAT3, and pNrf2 in HaCaT cells. The expression of KRT 6, PCNA, Ki67, and CD3 was evaluated on the skin of psoriasis-like mice and quantified with histochemical scores (H scores). Results: GA significantly inhibited KRT 6 gene transcription and expression in psoriasis-like disease both in vitro and in vivo. It significantly inhibited the expression of keratinocyte proliferation markers (PCNA and Ki67), suppressed the expression of CD3 (a marker of T cells), and decreased the thickness of the folded skin, as well as improved the splenomegaly in imiquimod-induced mice similar to psoriasis. Furthermore, the suppressing effect of GA on KRT 6 was abolished by the continuous activation of Nrf2 rather than STAT3, although GA significantly inhibited Nrf2 and STAT3 activation in IL-17A-induced HaCaT cells. Conclusions: KRT 6 acts as a potential target for GA against psoriasis, and the anti-psoriatic effect of GA could be related to Nrf2 signaling.

Astrocyte-mediated regulation of BLAWFS1 neurons alleviates risk-assessment deficits in DISC1-N mice.

Assessing and responding to threats is vital in everyday life. Unfortunately, many mental illnesses involve impaired risk assessment, affecting patients, families, and society. The brain processes behind these behaviors are not well understood. We developed a transgenic mouse model (disrupted-in-schizophrenia 1 [DISC1]-N) with a disrupted avoidance response in risky settings. Our study utilized single-nucleus RNA sequencing and path-clamp coupling with real-time RT-PCR to uncover a previously undescribed group of glutamatergic neurons in the basolateral amygdala (BLA) marked by Wolfram syndrome 1 (WFS1) expression, whose activity is modulated by adjacent astrocytes. These neurons in DISC1-N mice exhibited diminished firing ability and impaired communication with the astrocytes. Remarkably, optogenetic activation of these astrocytes reinstated neuronal excitability via D-serine acting on BLAWFS1 neurons' NMDA receptors, leading to improved risk-assessment behavior in the DISC1-N mice. Our findings point to BLA astrocytes as a promising target for treating risk-assessment dysfunctions in mental disorders.

Resurgence of influenza with increased genetic diversity of circulating viruses during the 2022-2023 season.

Introduction. After two seasons of absence and low circulation, influenza activity increased significantly in the winter of 2022-2023. This study aims to characterize virological and epidemiological aspects of influenza infection in Bulgaria during the 2022-2023 season and perform a phylogenetic/molecular analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of representative influenza strains.Hypothesis/Gap Statement. Influenza A and B viruses generate new genetic groups/clades each season, replacing previously circulating variants. This results in increased antigenic distances from current vaccine strains. Strengthening existing influenza surveillance is essential to meet the challenges posed by the co-circulation of influenza and SARS-CoV-2.Methodology. We tested 2713 clinical samples from patients with acute respiratory illnesses using a multiplex real-time RT-PCR kit (FluSC2) to detect influenza A/B and Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) simultaneously. Representative Bulgarian influenza strains were sequenced at the WHO Collaborating Centres in London, UK, and Atlanta, USA.Results. Influenza virus was detected in 694 (25.6 %) patients. Of these, 364 (52.4 %), 213 (30.7 %) and 117 (16.9 %) were positive for influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage virus, respectively. HA genes of the 47 influenza A(H1N1)pdm09 viruses fell into clades 5a.2. and 5a.2a.1 within the 6B.5A.1A.5a.2 group. Twenty-seven A(H3N2) viruses belonging to subclades 2b, 2a.1, 2a.1b and 2a.3a.1 within the 3C.2a1b.2a.2 group were analysed. All 23 sequenced B/Victoria lineage viruses were classified into the V1A.3a.2 group. We identified amino acid substitutions in HA and NA compared with the vaccine strains, including several substitutions in the HA antigenic sites.Conclusion. The study's findings showed genetic diversity among the influenza A viruses and, to a lesser extent, among B viruses, circulating in the first season after the lifting of anti-COVID-19 measures.

First detection of multiple cases related to CV-A16 strain of B1c clade in Beijing in 2022.

Coxsackievirus A16 (CV-A16) is a significant etiologic agent of hand, foot, and mouth disease (HFMD) and herpangina (HA), with the capacity to progress to severe complications, including encephalitis, aseptic meningitis, acute flaccid paralysis, myocarditis, and other critical conditions. Beijing's epidemiological surveillance system, established in 2008, encompasses 29 hospitals and 16 district disease control centers. From 2019 to 2021, the circulation of CV-A16 was characterized by the co-circulation of B1a and B1b clades. Multiple cases of HFMD linked to clade B1c has not been reported in Beijing until 2022. This study enrolled 400 HFMD and 493 HA cases. Employing real-time RT-PCR, 368 enterovirus-positive cases were identified, with 180 selected for sequencing. CV-A16 was detected in 18.89% (34/180) of the cases, second only to CV-A6, identified in 63.33% (114/180). Full-length VP1 gene sequences were successfully amplified and sequenced in 22 cases, revealing the presence of clades B1a, B1b, and B1c in 14, 3, and 5 cases, respectively. A cluster of five B1c clade cases occurred between June 29 and July 17, 2022, within a 7-km diameter region in Shunyi District. Phylogenetic analysis of five complete VP1 gene sequences and two full-genome sequences revealed close clustering with the 2018 Indian strain (GenBank accession: MH780757.1) within the B1c India branch, with NCBI BLAST results showing over 98% similarity. Comparative sequence analysis identified three unique amino acid variations (P3S, V25A, and I235V). The 2022 Shunyi District HFMD cases represent the first instances of spatiotemporally correlated CV-A16 B1c clade infections in Beijing, underscoring the necessity for heightened surveillance of B1c clade CV-A16 in HFMD and HA in this region.

Clinical features of hand, foot and mouth disease caused by Coxsackievirus A6 in Xi'an, China, 2013-2019: A multicenter observational study

PurposeTo investigate the clinical features of hand, foot, and mouth disease (HFMD) caused by coxsackievirus A6 (CVA6) and this work may help early diagnose of atypical HFMD. MethodsFrom January 2013 to December 2019, a total of 7,208 patients with a clinical diagnosis of HFMD in Xi'an Children's Hospital, Xi'an Central Hospital, and Xi'an Jiaotong University Second Affiliated Hospital, were included in this observational study. The clinical data, specimens and follow-up results were collected. Real-time RT‒PCR was performed for the detection and typing of enterovirus nucleic acids. ResultsOf the 7,208 clinically diagnosed HFMD patients, 5,622 were positive for enterovirus nucleic acids, and the positive proportions of CVA6, enterovirus 71 (EV-A71), coxsackievirus A16 (CVA16), and other enteroviruses were 31.0% (1,742/5,622), 27.0% (1,518/5,622), 35.0% (1,968/5,622), and 7.0% (394/5,622), respectively. Based on the etiology, patients were divided into CVA6 group, EV-A71group, and CVA16 group. The mean age at onset was significantly higher in the CVA6 group (4.62±2.13 years) than in the EV-A71 group and CVA16 group (3.45±2.25 years and 3.35±2.13 years, respectively; both P < 0.05). The male/female ratio was 1.45 (1,031/711) in the CVA6 group and was not significantly different from the other two groups. The incidence of fever was significantly higher in the CVA6 group [82.5% (1,437/1,742)] than in the EV-A71 group [51.3% (779/1,518)] and the CVA16 group [45.9% (903/1,968)] (P < 0.05). In the CVA6 group, the rashes were more frequently on the trunk and elbows/knees and were significantly different from the other two groups (P < 0.05). The number of patients with two or more rash morphologies was significantly higher in the CVA6 group than in the other two groups (P < 0.05). The incidence of bullous rash in the CVA6 group [20.2%; n = 352] was higher than in the EV-A71 group [0.33%; n = 5] and CVA16 group [0.66%; n = 13] (P < 0.05). The incidence of neurological complications was significantly higher in the EV-A71 group [52.1% (791/1,518)] than in the CVA16 group [5.1% (100/1,968)] and the CVA6 group [0.8% (14/1,742)] (P < 0.05). In the follow-up period, 160 patients (9.2%) with CVA6 HFMD experienced onychomadesis, but no onychomadesis was observed in the EV-A71 and CVA16 groups. The average WBC count was significantly higher in the CVA6 group than in the CVA16 group (P < 0.05). The number of patients with increased CRP was significantly larger in the CVA6 group than in the CVA16 group but was significantly smaller than that in the EV-A71 group (P < 0.05). ConclusionsCVA6 has become one of the main pathogens of HFMD in the Xi'an area during 2013–2019. The main clinical manifestations were slightly different from those of HFMD caused by EV-A71 or CVA16, with a higher frequency of fever, diverse morphologies and diffuse distribution of rashes, fewer neurological complications and some onychomadesis.

CircPDSS1 (hsa_circ_0017998) silencing induces ferroptosis in non-small-cell lung cancer cells by modulating the miR-137/SLC7A11/GPX4/GCLC axis

BackgroundCircular RNAs (circRNAs) regulate the tumorigenesis of non-small-cell lung cancer (NSCLC). CircPDSS1 (hsa_circ_0017998) has been newly discovered, and its role in NSCLC remains elusive. We aimed to investigate the functional roles and downstream targets of circPDSS1 in NSCLC cells. Materials and methodsCellular viabilities were measured through the Cell Counting Kit-8 (CCK-8) assay, whereas cell death was assessed through flow cytometry. The lactate dehydrogenase activity, malondialdehyde levels, ferrous iron, and reactive oxygen species were measured using commercial assay kits. The interaction between circPDSSA/ microRNA-137 (miR-137) and miR-137/solute carrier family 7 member 11 (SLC7A11) was assayed through a dual luciferase activity assay. Finally, the mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blots, respectively. ResultsCircPDSS1 expression was upregulated in NSCLC cells, compared with healthy lung cells. CircPDSS1 silencing suppressed the viability of NSCLC cells. Additionally, circPDSS1 knockdown induced ferroptosis rather than other types of cell death in NSCLC cells. Mechanically, circPDSS1 functions as a “sponge” to inversely control miR-137 expression, which directly targets SLC7A11. Moreover, circPDSS1 silencing causes the downregulation of glutathione peroxidase 4 (GPX4) and glutamate-cysteine ligase catalytic subunit (GCLC). ConclusionsTargeting the circPDSS1/miR-137/SLC7A11/GPX4/GCLC axis may be a promising strategy to kill NSCLC cells.

Rapid Differential Detection of Wild-Type Classical Swine Fever Virus and Hog Cholera Lapinized Virus Vaccines by TaqMan MGB-Based Dual One-Step Real-Time RT-PCR.

To establish a rapid real-time RT-PCR method for differentiating wild-type classical swine fever virus (CSFV) strains from vaccine strains (HCLV), we designed a universal primer targeting the NS3 gene to detect wild-type CSFV strains and vaccine strains simultaneously, and two TaqMan-MGB probes were designed to differentiate between wild-type and vaccine strains. After optimizing the RT-qPCR conditions, a rapid dual TaqMan-MGB RT-qPCR method for the detection and identification of CSFV and HCLV was developed. The results showed that method could specifically detect CSFV and HCLV with no cross-reactivity with other swine pathogens. The analytic sensitivity for the NS3 gene of CSFV and HCLV were 1.67 × 101 copies/μL, respectively. For precision testing, the repeatability and reproducibility of the test was less than 2%. This method was successfully used for the rapid detection of 193 biological samples collected from CSFV-vaccinated pigs. This fast and accurate detection technology can be used for the detection of CSFV and is suitable for differentiating between wild-type CSFV strains and vaccine strains.

Downregulation of B4GALNT1 inhibits proliferation and metastasis of osteosarcoma cells.

Osteosarcoma is the most common malignant bone tumor in children and adolescents, characterized by a high potential for proliferation and metastasis. Patients with osteosarcoma who have distant metastases generally have a poor prognosis. Challenges in treatment include incomplete resection of tumor and chemotherapy resistance, with no effective cure currently available. Recent studies suggest that β-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1) plays a role in the progression of various malignant tumors. However, the function of B4GALNT1 in osteosarcoma cells has not been reported. This study aims to investigate the expression of B4GALNT1 in osteosarcoma tissues compared to normal tissues and to explore its effects on the proliferation, migration, and invasion of osteosarcoma cells, thereby providing new theoretical foundations and directions for the treatment of osteosarcoma patients. Tumor tissues and corresponding normal tissue samples were collected from 16 osteosarcoma patients who underwent tumor resection at the Second Xiangya Hospital of Central South University. The patients' ages ranged from 8 to 17 years (median age 12 years). The expression of B4GALNT1 mRNA in osteosarcoma tissues, corresponding normal tissues, 3 osteosarcoma cell lines (MG63, Saos-2, and U2OS), and human fetal osteoblastic cells (hFOB) was detected using real-time reverse transcription PCR (real-time RT-PCR). The effects of B4GALNT1 knockdown on the proliferation of osteosarcoma cells Saos-2 and U2OS were analyzed using cell counting kit-8 (CCK-8) assays and colony formation assays. The effects of B4GALNT1 knockdown on the migration and invasion abilities of Saos-2 and U2OS cells were evaluated using Transwell migration and invasion assays. Western blotting analysis was performed to assess the impact of B4GALNT1 knockdown on the expression of epithelial-mesenchymal transition (EMT) and invasion-related proteins in Saos-2 and U2OS cells. Real-time RT-PCR results showed that B4GALNT1 mRNA expression levels were significantly higher in osteosarcoma tissues and the 3 osteosarcoma cell lines compared to normal tissues and hFOB cells (all P<0.01). CCK-8 and colony formation assays indicated that B4GALNT1 knockdown significantly reduced the proliferation rate of osteosarcoma cells compared to the control group (all P<0.05). Transwell migration and invasion assays demonstrated that B4GALNT1 knockdown significantly decreased the number of migrating and invading osteosarcoma cells (all P<0.01). Western blotting analysis revealed that B4GALNT1 knockdown inhibited the expression of N-cadherin, Snail, Vimentin, and matrix metalloproteinase 9 (MMP9) compared to the control group (all P<0.01). B4GALNT1 is upregulated in osteosarcoma tissues and cell lines, and its knockdown suppresses the malignant phenotype of osteosarcoma cells. B4GALNT1 may function as an oncogene in the proliferation and metastasis of osteosarcoma cells.

Involvement of multiple epigenetic mechanisms by altered DNA methylation from the early stage of renal carcinogenesis before proliferative lesion formation upon repeated administration of ochratoxin A

Ochratoxin A (OTA) is a rat renal carcinogen that induces karyomegaly and micronuclei in proximal tubular epithelial cells (PTECs). We previously performed comprehensive gene profiling of alterations in promoter-region methylation and gene expression in PTECs of rats treated with OTA for 13 weeks. The OTA-specific gene profile was obtained by excluding genes showing expression changes similar to those upon treatment with 3-chloro-1,2-propanediol, a renal carcinogen not inducing karyomegaly. In this study, we validated the candidate genes using methylated DNA enrichment PCR and real-time RT-PCR, and identified Gen1, Anxa3, Cdkn1a, and Osm as genes showing OTA-specific epigenetic changes. These genes and related molecules were subjected to gene expression and immunohistochemical analyses in the PTECs of rats treated with OTA, other renal carcinogens, or non-carcinogenic renal toxicants for 4 or 13 weeks. Cdkn1a upregulation and increase of p21WAF1/CIP1+ karyomegalic PTECs were observed with OTA, matching the findings associated with micronucleus-inducing carcinogens. This suggested that the increase of p21WAF1/CIP1+ karyomegalic PTECs is linked to micronucleus formation, which in turn accelerates chromosomal instability. The upregulation of Cdkn1a-related genes with OTA suggests the acquisition of a senescence-associated secretory phenotype, which promotes the establishment of a carcinogenic environment. Meanwhile, OTA specifically caused a decrease of GEN1+ PTECs reflecting Gen1 downregulation and an increase of ANXA3+ PTECs reflecting Anxa3 upregulation, as well as Osm upregulation. OTA may efficiently disrupt pathways for repairing the DNA double-strand breaks that it itself causes, via Gen1 downregulation, and enhance cell proliferation through the upregulation of Anxa3 and Osm. This may exacerbate the chromosomal instability from the early stage of OTA-induced renal carcinogenesis before proliferative lesions form. OTA may cause renal carcinogenesis involving multiple epigenetic mechanisms.