RT-PCR Research Articles

Evaluation of a commercial Immunochromatographic Strip Assay (ICT) for rapid detection of bovine, porcine and human Rotavirus A

The most frequent diarrhoeal pathogen “Rotavirus” is commonly detected by Ribo Nucleic Acid- Polyacrylamide Gel Electrophoresis (RNA-PAGE), Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Immunoassay (Enzyme Linked Immunosorbent Assay) in humans. However, use of these tests is limited in animals therefore, penside test like Immunochromatographic test/Lateral Flow Assay (ICT/LFA) is necessary. The present study was carried out to compare the diagnostic efficacy of rapid commercial ICT strip assay, RNA-PAGE and RT-PCR for specific detection of Rotavirus A (RVA) from stool samples of calves, piglets and children. A total of 313 faecal samples were collected between November 2022 to April 2023 from children below 5years of age (n=100), calves (n=100) and piglets (n=113) ≤ 3 months of age and were screened by RNA-PAGE, RT-PCR and commercial ICT kit for rotavirus A.The overall positivity of RVA from human, calves and piglets using RNA-PAGE, RT-PCR and LFA was found to be 35% (35/100), 8% (8/100) and 14.16% (16/113), respectively. Higher positivity of rotavirus was recorded in male children (44.26%, 27/61) than in female children (20.51%, 8/39). The kappa agreement between LFA and RT-PCR was 0.79 (substantial agreement); between LFA and RNA-PAGE was 0.61 (substantially low agreement) and between RNA-PAGE and RT-PCR was found to be 0.75 (substantial agreement). Relative diagnostic sensitivity and specificity of LFA in comparison with RT-PCR was 74.54% and 98.44%, respectively and in comparison with RNA-PAGE was 72.97% and 93.47%, respectively. RNA-PAGE in comparison with RT-PCR is having diagnostic sensitivity and specificity of 65.45% and 99.6%, respectively. In conclusion, the ICT strip assay is a rapid, convenient and effective method with satisfactory efficacy for detection of RVA from children, calves and piglets. ICT fulfilled the WHO ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Delivered) criteria for point-of-care testing. It can be useful in determining rotavirus outbreaks in resource-limited settings.

MiR-374 family is a key regulator of chronic primary pain onset.

Chronic primary pain conditions (CPPCs) are linked to catecholamine activation of peripheral adrenergic receptors. Yet, catecholamine-dependent epigenetic mechanisms, such as microRNA (miRNA) regulation of mRNA transcripts, remain largely unknown. We sought to identify RNA species correlated with case status in 3 pain cohorts, to validate RNAs found to be dysregulated in a mouse model of CPPC onset, and to directly test the role of adrenergic receptors in miRNA regulation. Furthermore, we tested antinociceptive effects of miR-374 overexpression. We used RNA-seq and quantitative reverse transcription polymerase chain reaction to measure RNA expression in 3 pain cohorts. Next, we validated identified RNAs with quantitative reverse transcription polymerase chain reaction in a mouse model of CPPC onset, measuring expression in plasma, peripheral (adipose, muscle, dorsal root ganglia [DRG]), and central (spinal cord) tissues. Then, we stimulated adrenergic receptors in primary adipocyte and DRG cultures to directly test regulation of microRNAs by adrenergic signaling. Furthermore, we used in vitro calcium imaging to measure the antinociceptive effects of miR-374 overexpression. We found that one miRNA family, miR-374, was downregulated in the plasma of individuals with temporomandibular disorder, fibromyalgia syndrome, or widespread pain following a motor vehicle collision. miR-374 was also downregulated in plasma, white adipose tissue, and spinal cord from mice with multisite mechanical sensitivity. miR-374 downregulation in plasma and spinal cord was female specific. Norepinephrine stimulation of primary adipocytes, but not DRG, led to decreased miR-374 expression. Furthermore, we identified tissue-specific and sex-specific changes in the expression of predicted miR-374 mRNA targets, including known (HIF1A, NUMB, TGFBR2) and new (ATXN7, CRK-II) pain targets. Finally, we demonstrated that miR-374 overexpression in DRG neurons reduced capsaicin-induced nociceptor activity. Downregulation of miR-374 occurs between adrenergic receptor activation and mechanical hypersensitivity, and its adipocyte source implicates adipose signaling in nociception. Further study of miR-374 may inform therapeutic strategies for the millions worldwide who experience CPPCs.

Long non-coding RNA AC105118.1 affects glycolysis to facilitate oxaliplatin resistance in colorectal cancer cells by modulating the miR-378a-3p/KIF26B axis

BackgroundOxaliplatin is a first-line chemotherapy drug for colorectal cancer (CRC), but many patients eventually lose treatment efficacy due to acquired resistance. AC105118.1 is a long non-coding RNA with unknown biological function. This research attempts to probe into the molecular regulatory mechanism of AC105118.1 in CRC oxaliplatin resistance. MethodsThe expression level of AC105118.1 in CRC tissues and cells was measured based on The Cancer Genome Atlas (TCGA) data and quantitative reverse transcription polymerase chain reaction (qRT-PCR). We utilized dual-luciferase assay and RNA immunoprecipitation to analyze the interaction between AC105118.1, miR-378a-3p, and their downstream target KIF26B. CCK-8, colony formation assay, and flow cytometry were employed to assess the half inhibitory concentration (IC50), cell proliferation, and apoptosis rate of HCT116/L-OHP cells treated with oxaliplatin. The glycolysis evaluation was completed by measuring the extracellular acidification rate (ECAR), glucose consumption, lactate production, and glycolysis-related proteins (HK2, GLUT1, and LDHA). TUNEL staining was used to detect the level of apoptosis. ResultsAC105118.1 was specifically upregulated in CRC tissues and cells. AC105118.1 indirectly facilitated the expression of miRNA target gene KIF26B by sequestering miR-378a-3p. In HCT116/L-OHP cells, transfection with si-AC105118.1 resulted in a decrease in glycolysis level, a lower maximum IC50 required for oxaliplatin-treated cells, inhibited cell proliferation, and an increase in apoptosis rate. All of these effects were alleviated when simultaneously transfecting miR-378a-3p inhibitor or oe-KIF26B. Knockdown of AC105118.1 significantly inhibited oxaliplatin resistance to CRC in mice. ConclusionAC105118.1 facilitates glycolysis and increases CRC cells’ resistance to oxaliplatin by targeting the miR-378a-3p/KIF26B axis. The present work shed new insights into the function and mechanism of AC105118.1 in molecular function and suggested that the AC105118.1/miR-378a-3p/KIF26B axis is a promising target for intervening CRC oxaliplatin resistance.

MiR-148b Caused Liver Injury in Rats with Traumatic Hemorrhagic Shock by Inhibiting SIRT6 Expression.

The purpose of this study was to investigate the role of miR- 148b in liver injury in rats with traumatic hemorrhagic shock (THS) and to elucidate its potential mechanism. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats were detected by enzyme-linked immune sorbent assay (ELISA), and the injury of rat liver was analyzed by hematoxylin-eosin (H&E) staining. Apoptosis of rat hepatocytes and normal rat liver cell line (BRL3A) was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry, respectively. MiR-148b and sirtuin 6 (SIRT6) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Lactate dehydrogenase (LDH) content and cell viability were measured by commercial kits and cell counting kit-8 (CCK-8) assay, respectively. The binding sites of miR-148b and SIRT6 were predicted by the Starbase database and verified by dual luciferase reporter assay. MiR-148b expression in THS rats or ischemia-reperfusion (I/R)-treated cells was higher than in the control group. Overexpression of miR-148b further promoted the effects of I/R, which enhanced the levels of ALT, AST and LDH, cell apoptosis of liver tissue or BRL3A cells and decreased the expression of SITR6. Besides, miR-148b negatively correlated with SIRT6, and upregulated the expression of SIRT6 could partly reverse the effect of miR-148b. Hepatocyte injury induced by I/R was achieved by regulating miR-148b /SIRT6 axis.

Stem cell secretome restore the adipo-osteo differentiation imbalance in diabetic dental pulp-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) from type 2 diabetes mellitus (T2DM) individuals exhibit increased adipogenesis and decreased osteogenesis. We investigated the potential of adipose tissue-derived MSCs (ADMSCs) secretome obtained from healthy individuals in restoring the tumor necrosis factor-α (TNF-α) mediated imbalance in the adipo/osteogenic differentiation in the dental pulp-derived MSCs obtained from T2DM individuals (dDPMSCs). dDPMSCs were differentiated into adipocytes and osteocytes using a standard cocktail in the presence of (a) induction cocktail, (b) induction cocktail + TNF-α, and (c) induction cocktail+ TNF-α + ADMSCs-secretome (50%) for 15 and 21 days resp. Differentiated adipocytes and osteocytes were stained by oil red O and alizarin red and analyzed by using ImageJ software. Molecular expression of the key genes involved was analyzed by using reverse-transcription polymerase chain reaction (RT-PCR). Treatment of TNF-α augmented the adipogenesis (9571 ± 765 vs. 19,815 ± 1585 pixel, p < 0.01) and decreased the osteogenesis (15,603 ± 1248 vs. 11,894 ± 951 pixel, p < 0.05) of dDPMSCs as evidenced by the oil red O and alizarin red staining respectively. Interestingly, dDPMSCs differentiated along with TNF-α and 50% ADMSCs secretome exhibited enhanced osteogenesis (11,894 ± 951 vs. 41,808 ± 3344 pixel, p < 0.01) and decreased adipogenesis (19,815 ± 1585 vs. 4480 ± 358 pixel, p < 0.01). Additionally, dDPMSCs differentiated along with ADMSCs secretome exhibited decreased expression of PPARg (p < 0.01), C/EBPa (p < 0.05), and FAS (p < 0.01) whereas mRNA expression of Runx2 (p < 0.05), Osterix (p < 0.01), and OCN (p < 0.05) was upregulated as revealed by the RT-PCR analysis. ADMSCs secretome from healthy individuals restore the TNF-α influenced differentiation fate of dDPMSCs and therefore can be explored for T2DM clinical management in the future.

Dietary sanguinarine supplementation improves the growth performance and intestinal immunity of broilers

Dietary sanguinarine (SAN) can enhance the growth performance of poultry and livestock, but the regulatory mechanism of the SAN monomer on intestinal homeostasis and how it promotes growth performance has not yet been clarified. In this study, 200 chickens were divided into four groups and fed different doses of SAN (0, 0.225, 0.75, 2.25mg/kg) for transcriptome and microbiota analysis. The data showed that different doses of SAN supplementation increased the feed conversion rate (FCR) of 22 to 42 d old and 1 to 42 d old broilers (P < 0.01), and 0.225 mg/kg SAN reduced the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA) and blood urea nitrogen (BUN) in serum (P < 0.01). Dietary SAN increased the villus height and the villus height/crypt depth (V/C) ratio in the ileum (P < 0.01). The levels of tight junction proteins (zonula occludens-1, occludin and claudin-1) were up-regulated in the ileum and cecum (P < 0.01) and the levels of immunoglobulin (Ig) A, IgM, IgG, interleukin (IL)-4, IL-10 and interferon (IFN)-γ were up-regulated in the serum and ileum (P < 0.01). RNA-seq analysis revealed 385 differentially expressed genes (DEGs) (|log2 fold change| ≥ 1, FDR < 0.05) between the SAN group and CON group. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed 15 pathways mostly associated with the immune system. Additionally, the reverse transcription-PCR results showed that the relative mRNA expression of β-defensin and mucin 2 were up-regulated (P < 0.01) and Toll-like receptor (TLR) 2 and TLR 4 mRNA were down-regulated by SAN (P < 0.01), which was consistent with the transcriptomic analysis. Western blot analysis also showed that SAN reduced the expression of inflammatory proteins such as TLR4, nuclear factor-kappa B and IL-1β in the ileum (P < 0.01). In addition, at the genus level, SAN significantly increased the relative abundance of bacteria (Bacteroides, unclassified_f__Lachnospiraceae, Lactobacillus and Romboutsia) involved in acetate and butyrate production in the cecum, which are associated with enhanced intestinal immune function and maintaining intestinal health. In conclusion, SAN ameliorates the growth performance of broilers, enhances intestinal immune function, regulates the structure of microbiota and maintains intestinal health.

Low Expression of Mitochondrial Ribosomal Protein S5 is Associated With Poor Prognosis in Patients With Clear Cell Renal Cell Carcinoma

Mitochondrial ribosomal protein S5 (MRPS5) is abnormally expressed in various tumor tissues and may be a key molecule for the regulation of tumors. Our aim is to investigate the relationship between the expression of MRPS5 in clear cell renal cell carcinoma (ccRCC) and the prognosis of patients. MRPS5 expression in fresh tumoral tissues and peritumoral tissues of patients with ccRCC was examined by quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemical staining, respectively. MRPS5 expression level in paraffin-embedded tumoral tissue samples with ccRCC was evaluated by immunohistochemical scoring criteria. The relationship between the expression of MRPS5 and the clinicopathological parameters and prognosis of patients with ccRCC was analyzed statistically. The expression of MRPS5 mRNA and protein in fresh tumoral tissues was lower than that in peritumoral tissues. Among 160 paraffin-embedded tumoral tissue samples, 99 cases (61.9%) showed high expression and 61 cases (38.1%) showed low expression of MRPS5. The expression level of MRPS5 was significantly correlated with T classification, TNM stage, and Fuhrman grade. Kaplan-Meier method and log-rank test indicated that patients with low MRPS5 expression had significantly poorer overall survival and recurrence-free survival than high MRPS5 expression. Multivariate analysis revealed that MRPS5 expression was an independent predictor of overall survival and recurrence-free survival, respectively. MRPS5 low expression was a risk factor for the prognosis of patients. The expression level of MRPS5 is significantly correlated with the postoperative survival status, which has the potential to be used as a novel prognostic biomarker for patients with ccRCC.

Placental Histopathological Changes and the Level of Anti-Spike Antibody After Covid-19 Vaccination During Pregnancy: A Case Series

Background: COVID-19 infection during pregnancy could be associated with placental histopathological changes such as vascular diseases and malperfusion. There are studies showing that mRNA vaccines are not associated with significant placental pathological changes. Our objective was to evaluate the placental histopathology in pregnant women who received Sinopharm, an inactivated virus vaccine, during pregnancy. Case Presentation: The study included placental samples collected from mothers who gave birth of living singletons through elective cesarean sections performed between March 2022 and May 2022 at Imam Khomeini Hospital Complex. The study included women who had no history of positive reverse transcription polymerase chain reaction (RT-PCR) testing for COVID-19 during pregnancy, and had received at least one dose of COVID-19 vaccine during their pregnancy. Humoral levels of anti-SARS-CoV-2 spike IgG were measured in both the mothers and neonates. Results: The study included 20 mother-neonate pairs. The mean maternal age was 34±3.6 years, and all mothers received Sinopharm vaccine as their first and second doses. The last vaccine dose was administered during pregnancy, with 3 mothers receiving it in the first trimester, 9 in the second trimester, and 8 in the third trimester. The histopathological findings in the placental samples included decidual vasculopathy, subchorionic thrombosis, and chronic histiocytic intervillositis. All mothers and neonates, except one pair, were positive for anti-spike antibody. Conclusion: Multiple abnormal histopathological findings were reported in placenta of vaccinated mothers. However, similar to previous studies, these placental findings are considered mild lesions and have been observed in both vaccinated and unvaccinated mothers.

Identification of Key Genes Involved in Seed Germination of Astragalus mongholicus

Seed germination is a fundamental process in plant reproduction, and it involves a series of complex physiological mechanisms. The germination rate of Astragalus mongholicus (AM) seeds is significantly lower under natural conditions. To investigate the key genes associated with AM seed germination, seeds from AM plants were collected at 0, 12, 24, and 48 h for a transcriptomic analysis, weighted gene co-expression network analysis (WGCNA), and machine learning (ML) analysis. The primary pathways involved in AM seed germination include plant-pathogen interactions and plant hormone signaling. Four key genes were identified through the WGCNA and ML: Cluster-28,554.0, FAS4, T10O24.10, and EPSIN2. These findings were validated using real-time quantitative reverse transcription PCR (qRT-PCR), and results from RNA sequencing demonstrated a high degree of concordance. This study reveals, for the first time, the key genes related to AM seed germination, providing potential gene targets for further research. The discovery of N4-acetylcysteine (ac4C) modification during seed germination not only enhances our understanding of plant ac4C but also offers valuable insights for future functional research and application exploration.

Herbacetin ameliorates lipopolysaccharide-elicited inflammatory response by suppressing NLRP-3/AIM-2 inflammasome activation, PI3K/Akt/MAPKs/NF-κB redox inflammatory signalling, modulating autophagy and macrophage polarization imbalance.

Herbacetin, a flavonol abundant in traditional medicines, is documented as an anti-inflammatory agent. However, information regarding its attributes on lipopolysaccharide (LPS)-induced inflammatory immunopathies has not been delineated yet. The present study aimed to comprehend herbacetin effects on LPS-induced aspects of unwarranted, non-resolving inflammation, particularly via targeting the vicious circle of oxi-inflammatory stress, autophagy-apoptosis, macrophages polarization, impaired inflammasome activation, and inflammatory cascades. In-vitro model of LPS-stimulated RAW 264.7 macrophage was recapitulated to investigate different inflammatory anomalies using enzyme-linked immunosorbent assay, qRT-PCR (Real-Time Quantitative Reverse Transcription PCR), immunoblotting. Concanavalin A challenged splenocytes and in silico studies were performed to measure Tregs population and binding affinity, respectively. Herbacetin administration caused remarkable reduction in nitric oxide, reactive oxygen species, mitochondrial membrane potential hyperpolarization, tumor necrosis factor-α, interferon-γ, interleukin-6, inducible nitric oxide synthase and ratio of M1/M2 markers (inducible nitric oxide synthase/arginase-1/macrophage scavenger receptor-1/mannose receptor C type-1) in in vitro model of persistent inflammation. Suppression of interleukins-5,17 and matrix metalloproteinases-2,3,9,13 and proliferating cell nuclear antigen, signifies its anti-inflammatory attributes. Noticeable decline in monodansylcadaverine-Lysotracker staining, caspase-6, and enhanced p62, B-cell lymphoma-2 expression indicates apoptosis-autophagosome accumulation inhibition and lysosomal destabilization. These were accompanied by reduced NLRP3 activation, caspase-1, AIM-2 expression, and interleukin-1β release. Subsequently, up-regulated activation of TLR-4, NF-κB, PI3K, Akt, ERK1/2, and JNK was decisively thwarted by herbacetin. In silico investigation signified the interaction of herbacetin with these targets. Decreased cytokines and enhanced Tregs conferred its role in extenuating inflammation facilitated by T-cells depletion. Collectively, these findings comprehend attributes of herbacetin as an alternative therapeutic strategy in relieving LPS-associated chronic inflammatory disorders.

CKS2 induces autophagy-mediated glutathione metabolic reprogramming to facilitate ferroptosis resistance in colon cancer.

Ferroptosis, a form of cell death characterized by lipid peroxidation, plays a crucial role in tumor suppression, offering novel avenues for cancer therapy. Previous studies have indicated that high levels of cyclin-dependent kinase subunit 2 (CKS2) promote the progression of various cancers. However, the potential interplay between CKS2 and ferroptosis in colon cancer (CC) remains unclear. Bioinformatics and RNA-seq analyses were employed to study genes associated with the ferroptosis signaling pathway. CKS2 expression was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB). The in vitro and in vivo effects of CKS2 on CC cells were assessed through the CCK-8 assay, colony formation assay, propidium iodide (PI) staining, BODIPY staining, DCFH-DA staining, and animal experiments. Additionally, the impact of CKS2 on autophagy and glutathione (GSH) metabolism was investigated using a transmission electron microscope (TEM), immunofluorescence (IF) assays, WB experiments, and relevant assay kits. CKS2 expression was elevated in CC, indicating a poor clinical outcome. Knockdown of CKS2 significantly enhanced Erastin-induced ferroptosis in CC cells, leading to reduced GSH metabolism. Conversely, CKS2 overexpression produced opposite effects. Mechanistically, CKS2-induced autophagy reinforced GSH metabolism, thereby increasing resistance to ferroptosis in CC cells. Furthermore, inhibiting CKS2 promoted tumor ferroptosis by downregulating GPX4 expression. Additionally, CKS2 knockdown effectively increased sorafenib-induced ferroptosis both in vitro and in vivo. CKS2 suppresses ferroptosis in CC by modulating GSH metabolism in both in vitro and in vivo settings. These findings offer new insights into targeting CKS2 for CC treatment and shed light on the mechanism of ferroptosis in CC.

Succinylation modification-mediated upregulation of Sp1 promotes hepatocellular carcinoma cell proliferation.

Hepatocellular carcinoma (HCC) remains one of the most prevalent malignant tumors globally, characterized by high incidence and mortality rates. Despite ongoing research, the underlying molecular mechanisms of HCC development are not yet fully understood. Utilizing bioinformatic analysis, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blot assays, we identified that the expression of specificity protein 1 (Sp1) was significantly elevated in HCC cells compared to normal cells. Knockdown of the Sp1 gene led to a marked reduction in the viability and clonogenic potential of HCC cells. Further investigation revealed that the succinylation level of Sp1 was also increased in HCC cells. The upregulation of Sp1 expression was attributed to its succinylation, mediated by KAT2A, with lysine (K)562 identified as the succinylation site. Additionally, KAT2A and Sp1 were found to influence the upregulation of mTOR phosphorylation. Collectively, these findings suggest that KAT2A-promoted succinylation of Sp1 enhances the proliferative capacity of HCC cells by activating the mTOR pathway, providing a theoretical foundation for potential therapeutic strategies against HCC.

Prognostic model development using novel genetic signature associated with adenosine metabolism and immune status for patients with hepatocellular carcinoma.

The high mortality rate of hepatocellular carcinoma (HCC) is partly due to advanced diagnosis, emphasizing the need for effective predictive tools in HCC treatment. The aim of this study is to propose a novel prognostic model for HCC based on adenosine metabolizing genes and explore the potential relationship between them. Regression analysis was performed to identify differentially expressed genes associated with adenosine metabolism in HCC patients using RNA sequencing data obtained from a public database. Adenosine metabolism-related risk score (AMrisk) was derived using the least absolute shrinkage and selection operator (LASSO) Cox regression and verified using another database. Changes in adenosine metabolism in HCC were analyzed using functional enrichment analysis and multiple immune scores. The gene expression levels in patient samples were validated using quantitative reverse transcription polymerase chain reaction. Thirty adenosine metabolism-related differentially expressed genes were identified in HCC, and six genes (ADA, P2RY4, P2RY6, RPIA, SLC6A3, and VEGFA) were used to calculate the AMrisk score; the higher the risk scores, the lower the overall survival. Moreover, immune infiltration activation and immune checkpoints were considerably higher in the high-risk group. Additional in vitro experiments validated the enhanced expression of these six genes in HCC. The established predictive model demonstrated that adenosine metabolism-related genes was significantly associated with prognosis in HCC patients.

Dengue Fever—Diagnosis, Risk Stratification, and Treatment.

Dengue fever is a common infectious disease in the tropical and subtropical zones, with more than 100 million symptomatic cases per year. Mosquitoes of the genus Aedes (Aedes aegypti, Aedes albopticus) are vectors of the disease, and their spread has led to rising case numbers around the world. Physicians in Europe, too, are increasingly being confronted by this challenge. This review is based on the findings of a selective search in international publication databases, as well as on the WHO guideline of 2009 and the current recommendations of the Robert Koch Institute. Dengue fever takes a mild course in more than 90% of cases. Severe dengue fever, up to and including shock and/or mucosal hemorrhages, is rare and carries a mortality of 1-5%. The disease characteristically takes a triphasic course (febrile phase, critical phase, recovery phase). It is diagnosed by the direct demonstration of the pathogen (e.g., with the reverse transcriptase polymerase chain reaction [RT-PCR] up to day 5 of the illness) or by serology. Patients are classified into one of three risk groups depending on their findings and comorbidities and are then treated either as outpatients or in the hospital. The treatment is symptomatic, as no treatment directed against the cause of the disease is available. The key measures are adequate volume replacement and, in patients with hemorrhage, the transfusion of blood products. Preventive steps include vaccination after a documented initial infection and the meticulous avoidance of mosquito bites. Climate change and global mobility have led to a worldwide increase in dengue fever. The disease only rarely takes a severe course. In such cases, rapid symptomatic treatment as needed is the key to the avoidance of severe complications.

Regulatory Effects of Copper on Ghrelin Secretion in Rat Fundic Glands.

Copper (Cu) is an effective additive in feed for promoting growth. Growth dan axis comprising growth hormone (GH), somatostatin (SS) and GH-releasing hormone (GHRH), with ghrelin regulating their release. The growth-promoting effects of Cu are closely related to ghrelin, but the specific mechanism behind the relationship remains unknown. We investigated the adjustment of ghrelin synthesis and secretion by Cu. Sprague-Dawley rats were fed basal diets with an addition of 0, 120 or 240 mg/kg Cu sulfate for 28 day to establish a growth-promoting model. Signalling molecules relevant to ghrelin synthesis and secretion were detected and mechanistically explored using enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction and Western blot analysis. The 120 mg/kg supplement improved growth performance; significantly increased the serum levels of ghrelin, ghrelin O-acyltransferase (GOAT), acylated ghrelin (AG), GH, and reactive oxygen species (ROS) and decreased those of SS; significantly increased the mRNA and protein expression of ghrelin, GOAT, ghrelin receptor (GHS-R1α), and activator protein 1 (AP-1); increased the phosphorylation ratio of JNK and p38 MAPK; and inhibited the mRNA and protein expression of SS and SS receptor subtype 2 (SSTR2) in gastric fundic gland tissues. Thus, Cu may affect gastric ghrelin synthesis at the transcriptional level by activating the JNK/p38 MAPK pathway through increased ROS levels and regulating the activation of the downstream redox-sensitive transcription factor AP-1. SS plays a crucial determinant role in ghrelin regulation via intragastric Cu. Cu promotes GOAT activity and ghrelin secretion by inhibiting SS secretion, affecting AG levels, and promoting ghrelin acylation through ghrelin/GOAT/GHS-R1α system, modulating ghrelin secretion.

Circulating CCRR serves as potential novel biomarker for predicting acute myocardial infarction

Abstract Objective Cold regions exhibit a high prevalence of cardiovascular disease, particularly acute myocardial infarction (AMI), which is one of the leading causes of death associated with cardiovascular conditions. Cardiovascular disease is closely linked to the abnormal expression of long non-coding RNA (lncRNA). This study investigates whether circulating levels of lncRNA cardiac conduction regulatory RNA (CCRR) could serve as a biomarker for AMI. Materials and methods We measured circulating CCRR from whole blood samples collected from 68 AMI patients and 69 non-AMI subjects. An AMI model was established using C57BL/6 mice. Quantitative reverse transcription PCR (qRT-PCR) was used to assess CCRR expression. Exosomes were isolated from cardiomyocytes, and their characteristics were evaluated using electron microscope and nanoparticle tracking analysis. The exosome inhibitor GW4869 was employed to examine the effect of exosomal CCRR on cardiac function using echocardiography. Protein expression was detected using Western blot and immunofluorescence staining. Results The circulating level of CCRR was significantly higher in AMI patients (1.93 ± 0.13) than in non-AMI subjects (1.00 ± 0.05, P &lt; 0.001). The area under the ROC curve (AUC) of circulating CCRR was 0.821. Similar changes in circulating CCRR levels were consistently observed in an AMI mouse model. Exosomal CCRR derived from hypoxia-induced cardiomyocytes and cardiac tissue after AMI were increased, a change that was reversed by GW4869. Additionally, CCRR-overexpressing exosomes improved cardiac function in AMI. Conclusion Circulating lncRNA CCRR is a potential predictor of AMI. Exosomal CCRR plays a role in the communication between the heart and other organs through circulation.

Expression Levels of PF4, ALOX12, ITGA2B, F131A in Pregnant COVID-19 Survivors.

COVID-19 is viral illness caused by SARS-CoV-2. The immediate complications of COVID-19 are well defined and associated with increased mortality. A global effort is required to determine its effects on implantation, fetal growth and labor. Post COVID-19 recovery period presents a further challenge regarding service provision, prevention, and management. To assess the expression of Platelet Factor 4 (PF4), Arachidonate 12-lipoxygenase (ALOX 12), Integrin alpha-IIb (ITGA2B) & Coagulation Factor XIII A Chain F13A1 in post-acute COVID-19 survivors pregnant women. Prospective case control study, conducted on 400 pregnant women. Case group consists of 200 singleton pregnancies who had recovered from COVID-19 since 4-6 weeks before conception. Control group consists of 200 singleton pregnancies with no history for COVID-19. Expression levels of ALOX12, PF4, ITGA2B, and F13A1genes were determined using quantitative reverse transcription polymerase chain reaction method (qRT-PCR). Expression levels of ALOX12, ITGA2B, and F13A1, were significantly higher in the patients group (3.82±9.6, 6.63±8.45, and 8.9±9.1, respectively) (p < 0.05) compared to those in the control group (1.0±6.0, 1.0±8.1, and 0.6±7.6, respectively). No significant difference in PF4 expression between patients and control group (p = 0.3). Results obtained from enrichment analysis have also supported the above findings. Relative expression levels of these candidate genes could be distinguished between post-acute COVID-19 survivors' pregnant women and control group, significant relative gene expression of ALOX12, ITGA2B, and F13A1 may be associated with an increased risk of placenta-mediated adverse pregnancy outcomes.

Mapping and quantification of potato virus A RNA genomes within viral particles and polysomes in infected plant cells.

Potato virus A belongs to the genus Potyvirus, a group of single-stranded positive sense RNA viruses infecting crops worldwide. To initiate infection in a host, its genome takes part in different activities, viz., translation, replication, encapsidation during the infection cycle. Extensive research has been carried out to scrutinize the stages of potyviral infection cycle and decipher the strategies it employs to cause disease. Nonetheless, the amount of viral RNA taking part in translation and virion formation, at a given time point, is missing. In this study, we quantified the percentage of viral RNA that exists as virions and those that associates with host polysome, relative to total viral RNA in infected plant tissue. We employed a revised version of immune-capture reverse transcription PCR and polysome profiling to address our queries. We tested three different coating antibody concentrations and further optimized the immuno-capture reverse transcription PCR protocol to address its limitation of binding and retaining viral particles. Our results indicate that most of the viral RNA (69%) exists as encapsidated genomes, while 3% of total viral RNA associates with host polysomes. These findings are crucial for correct interpretation of quantitative translational studies in which correlation must be made between the number of polysome-associated transcripts and the amount of protein synthesized.

Detection and discovery of plant viruses in Disporopsis through high-throughput sequencing

BackgroundDisporopsis, a member of the Liliaceae family and a perennial herb, is predominantly cultivated in southwestern and southeastern China. Its rhizome, referred to as Zhugenqi, serves as a traditional Chinese medicinal herb for the treatment of bone injuries. However, viral diseases have emerged as a significant challenge in the cultivation of Disporopsis.ObjectiveThe aim of this study was to identify and characterize viruses present in diseased samples of Disporopsis spp. using high-throughput sequencing (HTS) and reverse transcription-polymerase chain reaction (RT-PCR) to enhance the understanding of the virome associated with Disporopsis and to inform diagnostic and control strategies for viral diseases in this plant.MethodsDiseased samples of Disporopsis spp. were subjected to HTS and RT-PCR for virus identification. A total of five viruses were detected, including three novel viruses and two known viruses. The novel viruses were provisionally named Disporopsis chlorotic stripe virus (DCSV), Disporopsis pernyi-associated partitivirus (DaPTV), and Disporopsis pernyi-associated lispi-like virus (DaLV). Sequence identity and phylogenetic analyses were performed to confirm the novelty and taxonomic placement of these viruses.ResultsDCSV exhibited polyprotein sequence identities ranging from 47.6% to 83.6% with other potyviruses, with the highest identity (83.6%) shared with Polygonatum kingianum virus 5 (PKgV5). DaLV shared an amino acid sequence identity of 34.59% with maize suscal virus (MSV), and DaPTV shared an identity of 76.18–85.10% with Paris alphapartitivirus (ParAPV). Phylogenetic analyses supported the potential classification of the three novel viruses as new members of their respective genera. Two isolates of polygonatum mosaic-associated virus 1 (PMaV1) were identified in Disporopsis for the first time, showing divergences of 96.33% and 98.86% from existing isolates. RT-PCR analysis of 67 Disporopsis field samples collected from four cities in China revealed that more than half of the samples tested positive for at least one of the five viruses. PMaV1 and DaLV were the most prevalent, detected in 22 and 34 out of the 67 samples, respectively. Other viruses were detected at low rates and/or had limited distribution.ConclusionThis study provides insights into the virome infecting Disporopsis and offers valuable information for the diagnosis and control of viral diseases in this plant. The identification of five viruses, including three potential new members of their respective genera, contributes to the understanding of the viral threats to Disporopsis cultivation.

A Comparison of Clinical and Laboratory Features in Neonatal Proven Sepsis and COVID-19

Background: The clinical manifestations of COVID-19 in neonates are generally mild and commonly require only supportive treatment. However, it is important to note that they can sometimes present with symptoms like bacterial sepsis, which can lead to confusion in diagnosis. In this study, our objective was to compare laboratory data and clinical manifestations between 2 groups to identify opportunities for reducing the unnecessary use of antibiotics. Methods: The study was conducted as a cross-sectional study between January 2020 and 2023 on neonates who were admitted to the neonatal intensive care unit or the neonatal ward of Children’s Medical Center in Tehran, Iran. We specifically compared the laboratory data and clinical characteristics of neonates who tested positive for either a blood culture or a reverse transcription polymerase chain reaction for COVID-19. Results: Sixty-seven neonates in COVID-19 group and 68 neonates in the bacterial sepsis group entered. Prominent symptoms in the bacterial sepsis group include vomiting, seizure, apnea, mottling, increased need for ventilation and laboratory findings showing elevated levels of C-reactive protein and thrombocytopenia. In the COVID-19 group, patient exhibit symptoms such as cough, diarrhea, fever and laboratory findings that indicate neutropenia and leukopenia. Symptoms of nervous involvement were rare in this group. Conclusion: It is crucial to carefully assess the symptoms, laboratory results and overall condition of the patient before deciding on antibiotic initiation. By combining rapid COVID-19 testing and clinical variables, it is possible to identify low-risk infants who are unlikely to have bacterial infections.