Transcription-polymerase Chain Reaction Assay Research Articles

Comparative Analysis of Viral Load and Cytokines during SARS-CoV-2 Infection between Pregnant and Non-Pregnant Women

To better understand the vulnerabilities of pregnant women during the COVID-19 pandemic, we conducted a comprehensive, retrospective cohort study to assess differences in immune responses to SARS-CoV-2 infection between pregnant and non-pregnant women. Nasopharyngeal swabs and serum specimens from 90 pregnant and 278 age-matched non-pregnant women were collected from 15 March 2020 to 23 July 2021 at NewYork-Presbyterian Queens Hospital in New York City. Multiplex reverse transcription polymerase chain reaction, neutralizing antibody, and cytokine array assays were used to assess the incidence, viral load, antibody titers and profiles, and examine cytokine expression patterns. Our results show a lower incidence of SARS-CoV-2 infection in pregnant women compared with non-pregnant women. Pregnant women infected with SARS-CoV-2 exhibited a substantially lower viral load. In addition, the levels of both anti-spike protein receptor-binding domain IgG neutralizing antibodies and anti-N Protein IgG were elevated in pregnant women. Finally, cytokine profiling revealed differential expression of leptin across cohorts. These findings suggest that pregnancy is associated with distinct immune and virological responses to SARS-CoV-2 infection, characterized by lower infection rates, substantially lower viral loads, and enhanced antibody production. Differential cytokine expression indicates unique immune modulation in pregnant women.

Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism

Objective: To investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation (EMT) of pancreatic cancer cells. Methods: The pancreatic cancer cells were divided into siControl, siSIRT7, over-expression SIRT7, siSIRT7+siCOL4A1, and siSIRT7+siSLUG groups using siRNA or plasmid transfection. The proliferation, migration and invasion of pancreatic cancer cells were detected by EdU, wound healing assay and Transwell experiments, respectively. The expression of EMT and cancer stem cell (CSC) markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) and western blot. RNA sequencing (RNA-seq) in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7. Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment (q-ChIP) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). The expressions of SIRT7 and target genes were detected by immunohistochemical (IHC) in pancreatic cancer tissues, and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset. The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website. Finally, GeneMANIA, STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs. Results: EdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1 [(19.33±0.35)%] and BxPC-3 cells [(17.00±1.89)%] were lower than those in the control group [(31.60±1.37)% and (24.33±0.78)%, respectively, P＜0.05]. The proliferation rates of SIRT7-knockdown PANC-1 [(23.94±1.00)% and (27.08±0.97)%] and BxPC-3 cells [(22.00±1.86)% and (25.96±1.61)%] were higher than those of the siControl group [(11.80±1.86)% and (13.42±1.39)%, respectively, P＜0.05]. In PANC-1 cells, the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells [(76.67±2.74)%] was lower than that of control cells [(100.00±2.13)%, P＜0.05]; the relative migration rate of cells with SIRT7 knockdown [(134.22±4.08)% and (199.82±9.20)%, respectively] was higher than that of siControl group [(102.24±3.13)%, P＜0.05]. Compared with the control group, SIRT7 overexpression decreased the number of migrated BxPC-3 cells (45.66±1.69 vs 28.33±2.62, P＜0.05); while SIRT7 knockdown increased these numbers (65.66±2.86 and 82.00±2.94 versus 33.00±0.81, P＜0.01). Transwell experiment revealed that the number of invaded cells in SIRT7 overexpression groups (16.33±2.05 and 34.66±1.69) was lower than that control groups (54.33±4.64 and 58.66±5.90, P＜0.05); with SIRT7 knockdown, the numbers of invaded PANC-1 (63.66±2.49 and 69.33±3.29) and BxPC-3 cells (134.33±3.09 and 181.66±4.02) were higher than those in control groups (35.33±2.49 and 42.00±0.81, P˂0.05). Also, SIRT7 knockdown decreased the expressions of epithelial markers and increased the expressions of mesenchymal and CSC markers. RNA-seq analysis showed that SIRT7 was involved in regulating a variety of cancer-related signaling pathways, including the pancreatic cancer pathway and the EMT pathway. Furthermore, SIRT7 could directly bind to the promoter regions of target genes, such as COL4A1 and SLUG. SIRT7 was negatively correlated with the expression and function of COL4A1 and SLUG in pancreatic cancer cells. The expressions of SIRT7, COL4A1, SLUG and SOX2 were verified in pancreatic cancer tissues by IHC. Finally, SIRT7 was predicted to be associated with many proteins and miRNAs based on GeneMANIA, STRING, and ENCORI online tools. Conclusions: SIRT7 can inhibit the EMT of pancreatic cancer cells through transcriptionally inhibiting the expression of target genes, such as COL4A1 and SLUG. Thus, SIRT7 may serve as a potential tumor suppressor gene in pancreatic cancer.

HLA-DRB5 promotes immune thrombocytopenia via activating CD8+ T cells.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet (PLT) count and a high risk of bleeding, the clinical treatment for which still needs to be upgraded. Based on the critical role of human leukocyte antigen class II heterodimer β5 (HLA-DRB5) in immune system, we herein investigated its effect on ITP. ITP murine models were established by the injection of guinea pig anti-mouse platelet serum (GP-APS), and the PLT of mouse peripheral blood was counted during the modeling. Quantitative real-time reverse transcription polymerase chain reaction, western blot and immunofluorescence assay was performed to quantify expressions of HLA-DRB5, major histocompatibility complex II (MHC-II) and co-stimulatory molecules (CD80, CD86). Flow cytometry was conducted to analyze the percentage of CD8+ T cells. As a result, the PLT count was decreased in mouse peripheral blood. Expressions of HLA-DRB5, MHC-II and co-stimulatory molecules, as well as the percentage of CD8+ T cells were elevated in peripheral blood of ITP mice. HLA-DRB5 knockdown mitigated ITP by increasing peripheral PLT level, downregulating expressions of MHC-II and co-stimulatory molecules and inactivating CD8+ T cells. Collectively, the downregulation of HLA-DRB5 restores the peripheral PLT count in ITP mice by reducing MHC-II-mediated antigen presentation of macrophages to inhibit the activation of CD8+ T cells.

Development of a Risk Model and Genotyping Patterns Based on Disulfidptosis-Related lncRNAs to Predict Prognosis and Immune Landscape in Osteosarcoma.

Osteosarcoma (OS) is the most prevalent orthopedic malignancy with a dismal prognosis. Disulfidptosis-related lncRNAs (DRLncs) may be related to the progression of OS, but their potential molecular regulatory role is still unclear. Based on the data collected from The Cancer Genome Atlas (TCGA), we conducted correlation analysis and the univariate Cox analysis to screen prognosis-related DRLncs, followed by developing genotyping patterns and corresponding classifier. Subsequently, the survival analysis, enrichment analysis, drug sensitivity analysis and immune infiltration analysis were performed. Afterward, multivariate Cox regression was used to construct a risk model, which was further validated by the receiver operating characteristic (ROC) curve. The aberrant expression of hub DRLncs in OS was validated using the Reverse Transcription Polymerase Chain Reaction (RT-qPCR) assay. We identified 262 DRLncs and eleven prognosis-related DRLncs through filtering. We then constructed two distinct expression patterns of prognosis-related DRLncs and developed a classifier. We obtained 393 differentially expressed genes (DEGs) between different subtypes, which were significantly enriched in biological processes related to the extracellular matrix, integrin binding, focal adhesion, and Wnt signaling pathways. Through immune infiltration analysis, the activated CD4 memory T cells, resting natural killer (NK) cells, M1 macrophages, and resting dendritic cells (DC) were observed to exhibit different abundance in distinct subtypes. In the drug sensitivity analysis, tamoxifen showed a promising effect for drug-resistant OS. Furthermore, we identified five hub DRLncs and constructed a risk model. The RT-qPCR confirmed the aberrant expression of five hub DRLncs in OS. The present study identified DRLncs in OS, and conducted a comprehensive investigation of DRLncs-related expression patterns, survival status, immune landscape and drug sensitivity to reveal the difference in prognostic, pharmacological and immunological phenotype characteristics between distinct subtypes. Additionally, we developed a risk model to predict the prognosis, and constructed a genotyping classifier to predict the above phenotype characteristics in OS.

Exploring Melezitose as a Potential Therapeutic Agent in Lung Cancer: Inhibitory Effects on Cell Proliferation and EMT-Mediated Signaling in A549 Cells

Background Lung cancer is one of the most common cancers worldwide and a leading cause of cancer-related deaths. The study delves into melezitose, a naturally occurring compound known for its biocompatibility. Purpose This study aims to uncover its therapeutic potential and molecular mechanisms within lung cancer, particularly in A549 cells. Melezitose’s impact on inhibiting cell proliferation and influencing epithelial-mesenchymal transitions (EMT) was the primary focus. Material and Methods In a time-dependent manner, A549 cells, representative of lung adenocarcinoma (LUAD), underwent melezitose treatment. Analysis of cytotoxicity by MTT assay, cell migration assay, and its responsible genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) kit method. Results The MTT assay indicated a significant reduction in A549 cell growth after 48 hours of melezitose treatment. Additionally, melezitose induced G1 phase cell arrest and stimulated apoptosis in A549 cells. Subsequent determination of IC50 values represents the concentration at which melezitose inhibits 50% of cell growth. The study also investigated EMT-related gene expression like claudin 1 (CLD1), E-cadherin (ECADH), SNAIL1, SLUG, and vimentin (VIM) through RT-PCR. The findings revealed strong binding associations between melezitose and these EMT targets, suggesting a potential regulatory role of melezitose in impeding EMT processes. Conclusion Overall, this study illuminates the significant role of melezitose in lung cancer. Its observed inhibition of lung cancer cell proliferation and its influential impact on EMT-related gene expression highlights. Melezitose has potential as a therapeutic agent, particularly in the context of NSCC. The multifaceted effects of melezitose on A549 cells open promising avenues for advancing our understanding of this disease and developing innovative therapeutic strategies.

A highly divergent enteric calicivirus in a bovine calf in India.

A highly divergent bovine calicivirus was identified in an Indian calf with enteritis. The whole genome of this virus was sequenced, revealing distinct amino acid motifs in the polyprotein encoded by open reading frame 1 (ORF1) that are unique to caliciviruses. Phylogenetic analysis showed that it was related to members of the genus Nebovirus of the family Caliciviridae. Although it showed only 33.7-34.2% sequence identity in the VP1 protein to the nebovirus prototype strains, it showed 90.6% identity in VP1 to Kirklareli virus, a nebovirus detected in calves with enteritis in Turkey in 2012. An in-house-designed and optimized reverse transcription polymerase chain reaction (RT-PCR) assay was used to screen 120 archived bovine diarrhoeic fecal samples, 40 each from the Indian states of Uttar Pradesh, Haryana, and Himachal Pradesh, revealing frequent circulation of these divergent caliciviruses in the bovine population, with an overall positivity rate of 64.17% (77/120). This underscores the importance of conducting a comprehensive investigation of the prevalence of these divergent caliciviruses and assessing their associations with other pathogens responsible for enteritis in India.

MiR-21-5p Modulates Airway Inflammation and Epithelial-Mesenchymal Transition Processes in a Mouse Model of Combined Allergic Rhinitis and Asthma Syndrome

Introduction: Combined allergic rhinitis and asthma syndrome (CARAS) is a concurrent allergic symptom of diseases of allergic rhinitis and asthma. However, the mechanism of CARAS remains unclear. The study aimed to investigate the impact of microRNA-21 (miR-21) on CARAS via targeting poly (ADP-ribose) polymerase-1 (PARP-1) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Methods: The levels of miR-21-5p and PARP-1 in CARAS patients were detected by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). An ovalbumin-sensitized mouse model of CARAS was established. And knock down of miR-21-5p was constructed by intranasally administering with miR-21-5p shRNA-encoding adeno-associated virus vector. Airway resistance and airway inflammatory response were detected. ELISA was used to evaluate IL-4/IL-5/IL-13 levels in bronchoalveolar lavage fluid (BALF). Expression levels of E-cadherin, fibronectin, and α-SMA were determined using Western blotting. The levels of PARP-1 and the activation of PI3K/AKT were assayed. Results: Downregulation of miR-21-5p relieved pathophysiological symptoms of asthma including airway hyperreactivity and inflammatory cell infiltration. Downregulation of miR-21-5p significantly reduced the levels of IL4, IL-5, and IL-13 in BALF. Additionally, downregulation of miR-21-5p inhibited the epithelial-mesenchymal transition (EMT) process in CARAS mice. Furthermore, miR-21-5p regulated PARP-1 and was involved in PI3K/AKT activation in CARAS mice. Conclusion: Downregulation of miR-21-5p ameliorated CARAS-associated lung injury by alleviating airway inflammation, inhibiting the EMT process, and regulating PARP-1/PI3K/AKT in a mouse model of CARAS.

β-hydroxybutyrate inhibits malignant phenotypes of prostate cancer cells through β-hydroxybutyrylation of indoleacetamide-N-methyltransferase

BackgroundProstate cancer (PCa) is one of the most prevalent cancers in men and is associated with high mortality and disability rates. β-hydroxybutyrate (BHB), a ketone body, has received increasing attention for its role in cancer. However, its role in PCa remains unclear. This study aimed to explore the mechanism and feasibility of BHB as a treatment alternative for PCa.MethodsColony formation assay, flow cytometry, western blot assay, and transwell assays were performed to determine the effect of BHB on the proliferation and metastasis of PCa cells. Tumor sphere formation and aldehyde dehydrogenase assays were used to identify the impact of BHB or indoleacetamide-N-methyltransferase (INMT) on the stemness of PCa cells. N6-methyladenosine (m6A)–meRIP real-time reverse transcription polymerase chain reaction and dual luciferase assays were conducted to confirm INMT upregulation via the METTL3–m6A pathway. Co-IP assay was used to detect the epigenetic modification of INMT by BHB-mediated β-hydroxybutyrylation (kbhb) and screen enzymes that regulate INMT kbhb. Mouse xenograft experiments demonstrated the antitumor effects of BHB in vivo.ResultsBHB can inhibit the proliferation, migration, and invasion of PCa cells by suppressing their stemness. Mechanistically, INMT, whose expression is upregulated by the METTL3–m6A pathway, was demonstrated to be an oncogenic gene that promotes the stem-like characteristics of PCa cells. BHB can suppress the malignant phenotypes of PCa by kbhb of INMT, which in turn inhibits INMT expression.ConclusionsOur findings indicate a role of BHB in PCa metabolic therapy, thereby suggesting an epigenetic therapeutic strategy to target INMT in aggressive PCa.Trial registrationNot applicable.

Curcuma longa L. extract increased immune responses in RAW 264.7 cells and cyclophosphamide-induced BALB/c mice

Curcuma longa L. extract (CLE) exerts various biological functions including antioxidant, anti-inflammation, anticancer, and antiallergenic effects. However, its immune-enhancing capacity remains unclear. Therefore, the immune-enhancing effect of CLE was investigated in RAW 264.7 cells and cyclophosphamide (CPP)-induced immunosuppression model. CLE upregulated nitric oxide (NO) and reactive oxygen species production and increased inducible nitric oxide synthase and cyclooxygenase-2 expression without affecting the RAW 264.7 cells viability. The results of quantitative real-time reverse transcription polymerase chain reaction and sandwich enzyme-linked immunosorbent assay showed that CLE increased the gene expression and protein levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β in RAW 264.7 cells. Moreover, CLE upregulated p65, I kappa B kinase α/β, and I kappa B α (IκBα) phosphorylation and downregulated IκBα expression in RAW 264.7 cells. CLE also increased p65 translocation from the cytoplasmic to the nucleus in RAW 264.7 cells. The oral administration of CLE increased organ indexes (including the spleen and thymus) and NO production in peritoneal macrophages and improved natural killer cell activity in CPP-induced immunosuppression BALB/c mice. Overall, CLE could be a useful health functional food material that can improve innate immunity via macrophage activation.

Inhibitory Effects of Eicosapentaenoic Acid on Vascular Endothelial Growth Factor-Induced Monocyte Chemoattractant Protein-1, Interleukin-6, and Interleukin-8 in Human Vascular Endothelial Cells.

Vascular endothelial growth factor (VEGF) induces monocyte chemoattractant protein-1 (MCP-1) and plays an important role in vascular inflammation and atherosclerosis. We investigated the mechanisms of VEGF-induced MCP-1 expression and the effects of eicosapentaenoic acid (EPA) in human umbilical vein endothelial cells (HUVECs). Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that VEGF enhanced MCP-1 gene expression and protein secretion in HUVECs. Western immunoblot analysis revealed that VEGF induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor (NF)-κB (IκB). Treatment with pharmacological inhibitors of p38 MAPK (SB203580) or NF-κB (BAY11-7085) significantly suppressed VEGF-induced MCP-1 in HUVECs. EPA inhibited VEGF-induced MCP-1 mRNA, protein secretion, phosphorylation of p38 MAPK, and the translocation of phospho-p65 to the nucleus. Additionally, VEGF also stimulated gene expressions of interleukin (IL)-6 and IL-8, which were suppressed by SB203580, BAY11-7085, and EPA. The present study has demonstrated that VEGF-induced activation of MCP-1, IL-6, and IL-8 involves the p38 MAPK and NF-κB signaling pathways and that EPA inhibits VEGF-induced MCP-1, IL-6, and IL-8 via suppressing these signaling pathways. This study supports EPA as a beneficial anti-inflammatory and anti-atherogenic drug to reduce the VEGF-induced activation of proinflammatory cytokine and chemokines.

Antiviral and Virucidal Activities of Uncaria tomentosa (Cat's Claw) against the Chikungunya Virus.

Uncaria tomentosa (UT) is a medicinal plant popularly known as cat's claw belonging to the Rubiaceae family that has been reported to display antiviral and anti-inflammatory activities. The chikungunya virus (CHIKV) outbreaks constitute a Brazilian public health concern. CHIKV infection develops an abrupt onset of fever, usually accompanied by a skin rash, besides incapacitating polyarthralgia. There is no vaccine available or treatment for CHIKV infection. The present study evaluates the hydroalcoholic extract of UT bark as a potential antiviral against CHIKV. The in vitro antiviral activity of the UT extract against the Brazilian CHIKV strain was assessed using quantitative reverse transcription polymerase chain reaction, flow cytometry, and plaque assay. Results obtained demonstrated that UT inhibits CHIKV infection in a dose-dependent manner. At the non-cytotoxic concentration of 100 µg/mL, UT exhibited antiviral activity above 90% as determined by plaque reduction assay, and it reduced the viral cytopathic effect. Similarly, a significant virucidal effect of 100 µg/mL UT was observed after 24 and 48 h post-infection. This is the first report on the antiviral activity of UT against CHIKV infection, and the data presented here suggests UT as a potential antiviral to treat CHIKV infection.

Development and evaluation of a quadruple real-time fluorescence-based quantitative reverse transcription polymerase chain reaction assay for detecting Langya, Mojiang, Nipah, and Cedar viruses

The emerging viruses within the genus Henipavirus in the family Paramyxoviridae pose a great threat to public biosafety. To develop a quadruple real-time fluorescence-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay is pivotal for the early warning of the potential of zoonotic infectious diseases. Specific primers and probes were designed for the relatively conserved regions based on whole genome sequences of Langya virus (LayV), Mojiang virus (MojV), Nipah virus (NiV), and Cedar virus (CedV), followed by the establishment of a quadruple real-time fluorescence-based qRT-PCR detection method. No cross-reactivity was observed with other viral nucleic acids. The optimal linear detection range for LayV, MojV, NiV, and CedV was 101-108 copies/μL, and the lower limit of detection was 10 copies/μL. Three different DNA concentrations of LayV, MojV, NiV, and CedV (104, 105, and 106 copies/μL) were tested 14 times, achieving good repeatability. The standard deviation of the cycle threshold values for each concentration was <0.5 and the coefficient of variation was <3 %. Furthermore, the amplification efficiency of quadruple real-time fluorescence-based qRT-PCR was >90 %, and the correlation coefficient was >0.99. The established quadruple real-time fluorescence-based qRT-PCR assay for the detection of LayV, MojV, NiV, and CedV exhibits good sensitivity, specificity, and repeatability. Therefore, it can be used to detect Henipavirus and other related clinical specimens.

KLF4 interacts with TXNIP to modulate the pyroptosis in ulcerative colitis via regulating NLRP3 signaling.

Ulcerative colitis (UC) is one of the most common diseases in the gastrointestinal tract related to abnormal inflammation. Pyroptosis, which is characterized by the formation of inflammasome, activation of caspase-1, and separation of N- and C-terminus of gasdermin D (GSDMD), and may be involved in the pathogenesis of IBD. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in differentiated epithelial cells. KLF4 mediates proinflammatory signaling in macrophages. Here, we tested whether KLF4 is functional in pyroptosis of UC. In human UC tissues and/or lipopolysaccharide (LPS)/adenosine 5-triphosphate (ATP) stimulation human colon epithelial cells, KLF4, TXNIP, Cleave-Caspase-1, and GSDMD expression were detected through quantitative reverse transcription polymerase chain reaction (PCR), immunohistochemical and western blot assay. Interleukin (IL)-1β and IL-18 levels were quantified by enzyme-linked immunosorbent assay. We successfully constructed a KLF4-silenced colon epithelial cell line using an adenovirus vector. We apply the UCSC and JASPAR to predict the KLF4 binding sites in the promoter region of TXNIP. In human UC tissues and/or LPS/ATP stimulation human colon epithelial cells, KLF4, TXNIP, Caspase-1, and GSDMD expression level were significantly elevated via quantitative reverse transcription PCR, immunohistochemical and western blot assay. Moreover, We identified that there is an interaction between KLF4 and TXNIP through Yeast double hybrid assay and CO-IP assay. We successfully constructed a KLF4-silenced human intestinal epithelial cell line. In LPS/ATP stimulation KLF4-silenced human intestinal epithelial cells, KLF4, TXNIP, Cleave Caspase-1, ASC, and GSDMD expression level were significantly decreased via quantitative reverse transcription PCR. Our results confirm that KLF4 can positively regulate the expression of TXNIP and regulate the pyroptosis process of UC through the TXNIP/NLRP3 pathway.

Protective effects of an alcoholic extract of Kaempferia galanga L. rhizome on ethanol-induced gastric ulcer in mice

Ethnopharmacological relevanceThe rhizome of Kaempferia galanga L., a medicinal and edible Plant, was widely distributed in many Asian and African counties. It has been traditionally used to treat gastroenteritis, hypertension, rheumatism and asthma. However, there is a lack of modern pharmacology studies regarding its anti-gastric ulcer activity. Aim of the studyThe objective of this study is to investigate the protective effects of an extract from K. galanga L. rhizome (Kge) and its active components kaempferol and luteolin on ethanol-induced gastric ulcer. Materials and methodsThe kge was prepared by ultrasonic-assisted extraction, and the contents of kaempferol and luteolin were determined by HPLC. The mice were randomly divided into seven groups: blank control (0.5 % CMC-Na; 0.1 mL/10 g), untreatment (0.5 % CMC-Na; 0.1 mL/10 g), Kge (100, 200 and 400 mg/kg), kaempferol (100 mg/kg) and luteolin (100 mg/kg) groups. The mice were treated intragastrically once daily for 7 days. At 1 h post the last administration, the mice in all groups except the blank control group were intragastrically administrated with anhydrous alcohol (0.1 mL/10 g) once to induce gastric ulcer. Then, fasting was continued for 1 h, followed by sample collection for evaluation by enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction assay. ResultsThe contents of kaempferol and luteolin in Kge were determined as 3713 μg/g and 2510 μg/g, respectively. Alcohol induced severely damages with edema, inflammatory cell infiltration and bleeding, and the ulcer index was 17.63 %. After pre-treatment with Kge (100, 200 and 400 mg/kg), kaempferol and luteolin, the pathological lesions were obviously alleviated and ulcer indices were reduced to 13.42 %, 11.65 %, 6.54 %, 3.58 % and 3.85 %, respectively. In untreated group, the contents of Ca2+, myeloperoxidase, malondialdehyde, NO, cyclic adenosine monophosphate and histamine were significantly increased, while the contents of hexosamine, superoxide dismutase, glutathione peroxidase, and prostaglandin E2 were significantly decreased; the transcriptional levels of IL-1α, IL-1β, IL-6, calcitonin gene related peptide, substance P, M3 muscarinic acetylcholine receptor, histamine H2 receptor, cholecystokinin 2 receptor and H+/K+ ATPase were significantly increased when compared with the blank control group. After pre-treatment, all of these changes were alleviated, even returned to normal levels. Kge exhibited anti-gastric ulcer activity and the high dose of Kge (400 mg/kg) exhibited comparable activity to that of kaempferol and luteolin. ConclusionThe study showed that K. galanga L., kaempferol, and luteolin have protective effects against ethanol-induced gastric ulcers. This is achieved by regulating the mucosal barrier, oxidative stress, and gastric regulatory mediators, as well as inhibiting the TRPV1 signaling pathway and gastric acid secretion, ultimately reducing the gastric ulcer index.

Assessment of the yellow fever outbreak in Angola from December 2015 through December 2016: A retrospective study.

The acute tropical infectious disease known as yellow fever (YF) is caused by an arbovirus and is characterized by fever, jaundice, hemorrhage, headache, muscle pain, nausea, vomiting, and fatigue. Angola experienced a yellow fever virus (YFV) outbreak that was documented in December 2015. However, little is known about the outcome of this outbreak. We aimed to demonstrate epidemic features and lessons learned during the YF epidemic in Angola. A total of 4618 blood samples from suspected YF cases were sent to the Instituto Nacional de Investigação em Saúde (INIS), a national referral and public health laboratory, between December 5, 2015, and December 23, 2016. Sample analyses were conducted using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assays. Blood samples were sent from 16 out of the 18 provinces of Angola. We detected 884 (19.1%) cases that were positive for ELISA, which were confirmed by RT-PCR assay. Considering the positive cases, the incidence among male patients was around three times higher (n = 223; 10.9%) than in female patients (n = 59; 2.6%) in the 20-29 age group, followed by the age group 10-19 with n = 211 (6.8%) in males versus n = 108 (3.3%) in females; and the age group 30-39 had n = 68 (4.8%) in males versus n = 28 (1.8%) in females. The other groups had an incidence below 3.0%. The case fatality ratio for YF was in young adults in the age group 20-29 with n = 39 cases, followed by the age group 10-19 with n = 16 cases, and finally the age group 0-9 with n = 13 cases. The other age groups had several deaths by YF below 10 cases. This study demonstrates features of the YF epidemic that occurred in Angola. Also, it demonstrates that YF causes deaths in young people but is preventable by high vaccine coverage. Thus, public health laboratory surveillance must be strengthened to reduce the possibility of emerging and re-emerging human infections.

Circ0060467 sponges miR-6805 to promote hepatocellular carcinoma progression through regulating AIFM2 and GPX4 expression.

Circular RNAs (circRNAs) represent a subset of non-coding RNAs implicated in the regulation of diverse biological processes, including tumorigenesis. However, the expression and functional implications of circ0060467 in hepatocellular carcinoma (HCC) remain elusive. In this study, we aimed to elucidate the role of circ0060467 in modulating the progression of HCC. Differentially expressed circRNAs in HCC tissues were identified through circRNA microarray assays. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays revealed the upregulation of circ0060467 in both HCC cell lines and tissues. Various assays were conducted to investigate the roles of circ0060467 in HCC progression. Additionally, RNA immunoprecipitation (RIP) assays and luciferase assays were carried out to assess the interactions between circ0060467, microRNA-6085 (miR-6085), apoptosis-inducing factor mitochondria-associated 2 (AIFM2), and glutathione peroxidase 4 (GPX4) in HCC. Microarray and qRT-PCR analyses demonstrated a marked elevation of circ0060467 in HCC tissues and cell lines. Knockdown of circ0060467 suppressed HCC cell proliferation. Luciferase reporter and RIP assays confirmed the binding of circ0060467, AIFM2, and GPX4 to miR-6805. Subsequent experiments revealed that circ0060467 competes with AIFM2 and GPX4, thereby inhibiting cancer cell ferroptosis by binding to miR-6085 and promoting hepatocellular carcinoma progression. Collectively, circ0060467 modulates the levels of AIFM2 and GPX4, crucial regulators of tumor cell ferroptosis, by acting as a sponge for miR-6085 in HCC. Thus, circ0060467 may represent a novel diagnostic marker and therapeutic target for HCC.

Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes.

Glucagon plays an essential role in hepatic glucogenesis by enhancing glycogen breakdown, inducing gluconeogenesis, and suppressing glycogenesis. Moreover, glucagon increases cyclic adenosine monophosphate (cAMP) levels, thereby activating protein kinase A (PKA) and cAMP guanine nucleotide exchange factor (also known as Epac). Although the function of PKA in the liver has been studied extensively, the function of hepatic Epac is poorly understood. The aim of this study was to elucidate the role of Epac in mediating the action of glucagon on the hepatocytes. Epac mRNA and protein expression, localization, and activity in the hepatocytes were analyzed by reverse transcription polymerase chain reaction, western blotting, immunofluorescence staining, and Rap1 activity assay, respectively. Additionally, we investigated the effects of an Epac-specific activator, 8-CPT, and an Epac-specific inhibitor, ESI-05, on glycogen metabolism in isolated rat hepatocytes. Further mechanisms of glycogen metabolism were evaluated by examining glucokinase (GK) translocation and mRNA expression of gluconeogenic enzymes. Epac2, but not Epac1, was predominantly expressed in the liver. Moreover, 8-CPT inhibited glycogen accumulation and GK translocation and enhanced the mRNA expression of gluconeogenic enzymes. ESI-05 failed to reverse glucagon-induced suppression of glycogen storage and partially inhibited glucagon-induced GK translocation and the mRNA expression of gluconeogenic enzymes. Epac signaling plays a role in mediating the glucogenic action of glucagon in the hepatocytes.

Viruses of Apple Are Seedborne but Likely Not Vertically Transmitted.

Many viruses occur in apple (Malus domestica (Borkh.)), but no information is available on their seed transmissibility. Here, we report that six viruses infecting apple trees, namely, apple chlorotic leaf spot virus (ACLSV), apple green crinkle-associated virus (AGCaV), apple rubbery wood virus 2 (ARWV2), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), and citrus concave gum-associated virus (CCGaV) occur in seeds extracted from apple fruits produced by infected maternal trees. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) assays revealed the presence of these six viruses in untreated apple seeds with incidence rates ranging from 20% to 96%. Furthermore, ASPV was detected by RT-PCR in the flesh and peel of fruits produced by infected maternal trees, as well as from seeds extracted from apple fruits sold for fresh consumption. Finally, a large-scale seedling grow-out experiment failed to detect ACLSV, ASGV, or ASPV in over 1000 progeny derived from sodium hypochlorite surface sterilized seeds extracted from fruits produced by infected maternal trees, suggesting no detectable transmission via embryonic tissue. This is the first report on the seedborne nature of apple-infecting viruses.

MicroRNA-15a-5p suppresses hypoxia-induced tumor growth and chemoresistance in bladder cancer by binding to eIF5A2.

In various malignant tumors (including bladder cancer) poor prognosis is associated with hypoxia and therapeutic resistance. Evidence indicates that in bladder cancer, microRNAs (miRNAs) have vital functions in acquired drug resistance. However, the involvement of miRNAs in hypoxia-mediated bladder cancer doxorubicin (Dox) resistance is unknown. Herein, we showed that hypoxia and Dox treatment downregulated miR-15a-5p expression. Using UM-UC-3 and J82 bladder cancer cell lines and in vivo mouse models of bladder cancer, we confirmed that miR-15a-5p arrests tumor cell growth and Dox resistance in vitro and in vivo. Furthermore, we determined the interaction between miR-15a-5p and eukaryotic translation initiation factor 5A-2 (eIF5A2) using dual luciferase reporters and quantitative real-time reverse transcription polymerase chain reaction assays. We also showed that a miR-15a-5p agomir repressed EIF5A2 expression in bladder cancer cells, thereby inhibiting the epithelial-mesenchymal transition (EMT) induced by Dox or hypoxia. Moreover, ectopic expression of miR-15a-5p abrogated eIF5A2-mediated Dox resistance in bladder cancer cells. Collectively, these data indicated that hypoxia promotes tumor growth and chemoresistance through the HIF-1α/miR-15a-5p/eIFTA2/EMT pathway. This new finding not only has implications for improving our understanding of the Dox resistance process during bladder cancer progression but also indicates that the miR-15a-5p agomir is a promising tool to prevent Dox resistance in patients with bladder cancer.

D-dimer and HbA1c levels findings in COVID-19 Iraqi patients.

On March 11, 2020, the World Health Organization (WHO) declared a new coronavirus infection caused by the SARS-CoV-2 virus as a pandemic, making it the 11th pandemic of the 20th and 21st centuries. This study investigated the clinical and laboratory results (D-dimer, conventional coagulation, and HbA1c biomarker concentrations) of 150 patients (75 male and 75 female) with confirmed COVID-19 pneumonia and 50 controls (25 male and 25 female). For disease diagnosis, all COVID-19 patients were given a Real-Time Reverse Transcription Polymerase Chain Reaction Assay (RT-PCR). The findings revealed that D-dimer and HbA1c levels in COVID-19 patients were significantly higher (P 0.001) at the time of admission; In COVID-19 patients, there was also a strong correlation between D-dimer levels and HbA1c levels (P 0.001). In conclusion, COVID-19 patients are more likely to have a poor prognosis if their D-dimer and HbA1c levels remain uncontrolled over a lengthy period. To lower the likelihood of a bad prognosis in COVID-19, patients with higher levels of D-dimer and HbA1c should be continuously monitored.