Real-time Reverse Transcription Research Articles

Chemotoxic and phototoxic effects of liposomal co-delivery of green synthesized silver nanoparticles and ZnPcS4 for enhanced photodynamic therapy in MCF-7 breast cancer cells: An in vitro study.

Chemotoxic and phototoxic effects of liposomal co-delivery of green synthesized silver nanoparticles and ZnPcS4 for enhanced photodynamic therapy in MCF-7 breast cancer cells: An in vitro study.

MiR−33 as a novel diagnostic biomarker for distinguishing cholesterol from adenomatous polyps: a case-control study

Cholecystectomy is often excessively utilized in the management of gallbladder polyps. It is crucial to effectively differentiate between adenomatous and cholesterol polyps to reduce unnecessary cholecystectomies. This study aimed to investigate the potential of miR−33 as a novel diagnostic biomarker for distinguishing cholesterol from adenomatous polyps. Gallbladder specimens were retrospectively collected from gallbladder polyp patients who underwent laparoscopic cholecystectomy at the Second Department of General Surgery, Dongzhimen Hospital, Beijing University of Traditional Chinese Medicine, between June 2021 and December 2021. Pathological analysis categorized the specimens into two groups: the cholesterol polyp group (n = 13) and the adenomatous polyp group (n = 12). The expression levels of miR−33a and miR−33b in both groups were assessed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-33a level and the miR-33a/miR-33b ratio were significantly lower in cholesterol polyps than in adenomatous polyps (p < 0.05). Spearman correlation analysis showed a strong positive correlation between miR-33a and miR-33b (r = 0.956, p < 0.001). Stepwise logistic regression analysis revealed that decreased miR-33b and elevated miR-33a/miR-33b ratio are independent risk factors for cholesterol polyps (p < 0.05). A predictive model was constructed, with the model’s AUC for diagnosing adenomatous polyps being 0.885 (95% CI: 0.753−1.000, p = 0.001), exhibiting a notable specificity of 84.62% and a sensitivity of 83.33% at a cut-off of 0.424. MiR−33 could serve as a novel diagnostic biomarker for distinguishing cholesterol from adenomatous polyps to facilitate the diagnosis and treatment of clinicians.

Investigating the therapeutic potential of naringin in MK-801-induced schizophrenia Model: Focus on cognitive impairment and miR-25-3p-Regulated Pathways

Aim The aim of this study was to assess the ameliorative effects of naringin (NR) on cognitive impairment in schizophrenia(SZ) from multiple perspectives using behavioral, histopathological and molecular biological approaches. Materials and methods SZ models were established in rats via acute intraperitoneal injection of MK-801 in all groups except the control group, which received saline. Cognitive function was assessed using the Morris water maze test 21 days after prophylactic NR administration. Subsequently, Serum interleukin-6 (IL-6) and homocysteine (HCY) levels were quantified using enzyme-linked immunosorbent assay (ELISA), and hippocampal neuronal and synaptic structures were observed via microscopy. Molecular detection was performed using real-time reverse transcription polymerase chain reaction (RT-qPCR) and western blotting (WB) to assess the expression levels of molecules related to the microRNA-25-3p/salt inducible kinase 1/CREB regulated transcription coactivator 2/cAMP responsive element binding protein 1 (miR-25-3p/SIK1/CRTC2/CREB1) pathway, thereby elucidating the mechanism by which NR ameliorates cognitive impairment in SZ. Results NR was found to mitigate cognitive decline in learning and memory induced by MK-801. It lowered serum levels of IL-6 and HCY, reduced neuronal damage in the CA1 region of the hippocampus, increased the thickness of postsynaptic dense material, decreased the distance between synaptic gaps, decreased the expression of SIK1, and elevated the expression of miR-25-3p, CRTC2 and CREB1 in the hippocampus. Conclusion NR may protect neurons in the CA1 region of the hippocampus and enhance synaptic plasticity by regulating the miR-25-3p/SIK1/CRTC2/CREB1 signaling pathway, thereby promoting cognitive improvement.

Plasma Circulating lncRNAs: MALAT1 and NEAT1 as Biomarkers of Radiation-Induced Adverse Effects in Laryngeal Cancer Patients

Background: The majority of head and neck cancers (HNCs) occur in the larynx. In clinical practice, adverse effects are frequently observed in laryngeal cancer (LC) patients undergoing radiotherapy (RT). Therefore, investigating markers that can predict these unfavorable events is of interest. Long non-coding RNAs (lncRNAs) have emerged as potential biomarkers for the early identification of patients susceptible to post-RT toxicity. MALAT1 and NEAT1 regulate various cellular processes, the inflammatory response, and resistance to anti-cancer treatments; however, their impact on the portability of post-RT adverse effects remains unknown. The aim of this study was to evaluate the clinical value of two plasma-circulating lncRNAs, MALAT1 and NEAT1, as predictive biomarkers for post-RT adverse effects in LC patients. Methods: The expression levels of the studied lncRNAs were determined using real-time quantitative reverse transcription PCR (qRT-PCR) in plasma samples obtained from 70 LC patients before the initiation of RT. These levels were then correlated with patient outcomes. Results: A low expression of MALAT1 was associated with a significantly higher probability of anemia, liver failure, and severe malnutrition (OR = 5.36; p = 0.040, OR = 6.07; p = 0.037, OR = 9.75; p &lt; 0.001, respectively) after the completion of RT. Similarly, patients with low NEAT1 expression had a significantly higher risk of anemia, liver failure, and mild or severe malnutrition (OR = 5.26; p = 0.020, OR = 5.70; p = 0.016, OR = 13.09; p = 0.002, respectively). Simultaneous lower expression levels of both lncRNAs were significantly associated with shorter median overall survival (OS) in RT-treated LC patients (HR = 5.44; p = 0.001). Conclusions: The analysis of MALAT1 and NEAT1 expression indicates clinical utility in predicting toxic events induced by RT-based therapy.

Validation and comparison of EvaGreen- and TaqMan-based real-time qRT-PCR for diagnosis of the black queen cell virus in the honey bees.

The black queen cell virus (BQCV) is a common agent that causes covert infection in hives and has a global distribution. In this study, TaqMan probe and EvaGreen (EG) dye-based real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays based on the amplification of coding senquences for the BQCV structural protein (SP) and the non-structural protein (NSP) were developed and validated for the detection of BQCV in adult bee and larvae/pupae. Additionally, the performances of commonly used EG and TaqMan chemicals for real-time qRT-PCR analyses were compared. The operating range for BQCV-SP TaqMan and EG real-time qRT-PCRs ranged 2.61-7.61 log10 RNA copies/reaction and 2.61-7.79 log10 RNA copies/reaction, respectively. The operating range for BQCV-NSP TaqMan and EG real-time qRT-PCRs ranged 6.83-11.83 log10 RNA copies/reaction and 6.98-11.98 log10 RNA copies/reaction, respectively. Based on these novel assays, the prevalence of BQCV in adult bees and larvae/pupae was 84% (88/105) and 44% (46/105), respectively. BQCV viral load was also within the operating range determined during assay validation. Comparable results were obtained in analytical and diagnostic performance analyses of the EG dye and TaqMan probe chemicals. Highly sensitive and analytically specific for the detection of BQCV, these real-time qRT-PCR assays will benefit the etiological and epidemiological studies of BQCV.

The Histone Demethylase Inhibitor GSK-J4 Attenuates Periodontal Bone Loss and Inflammation in a Rat Model of Periodontitis.

To investigate the treatment effect of the histone demethylase inhibitor GSK-J4, a small molecule that inhibits the demethylase activity of Jumonji domain-containing protein 3 (JMJD3), in the treatment of periodontitis. Gingival tissues from patients with moderate to severe chronic periodontitis and healthy controls were collected to evaluate JMJD3 expression via real-time quantitative reverse transcription PCR (RT-qPCR) and immunohistochemistry (IHC). Next, Sprague-Dawley (SD) rats were used to investigate the effect of GSK-J4 in vivo. The experimental periodontitis model was induced by upper first molar ligation and gingival sulcus injection of Porphyromonas gingivalis. The rats were divided into a healthy group, a periodontitis group, periodontitis plus GSK-J4 treatment groups (P + GSK-J415 mg/kg or 25mg/kg), and a periodontitis plus dimethyl sulfoxide (DMSO) group (P + DMSO). After 4weeks, maxillary molar segments were assessed via micro-computed tomography (CT) and hematoxylin and eosin (HE) staining. Serum tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assay (ELISA). Higher expression of the Jmjd3 gene and JMJD3 protein was detected in human inflamed gingiva than in healthy gingiva (P < 0.05). GSK-J4 administration reversed alveolar bone absorption [i.e., reduced alveolar bone crest (ABC)-cementoenamel junction (CEJ) distance], reduced inflammatory cell accumulation at the crest of the alveolar bone, and alleviated serum TNF-α levels in rats with periodontitis. Moreover, the number of H3K27me3-positive nuclei was greater in model rats treated with GSK J4 than in model rats. The histone demethylase inhibitor GSK-J4 attenuated periodontal bone loss and inflammation in a rat periodontitis model by targeting JMJD3.

Protective effects of vincamine against ethanol-induced gastric ulcer by attenuation of IL-6, IL-1β, and TNF-α mRNA expression levels in the gastric mucosa of BALB/c mice.

Vincamine, a monoterpenoid alkaloid, and an active constituent of plant Catharanthus roseus Linn, has been proclaimed for antioxidant and anti-inflammatory activities. This study was designed to evaluate the gastroprotective activity of Vincamine by ameliorating gastric ulcer in BALB/c mice. The study was also designed to find the possible mechanism of gastric protection by exploring the impact of Vincamine on gastric pH, acidic content, observing histopathology and molecular expression of inflammatory mediators like Interleukin- β (IL-1β), Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF- α) and oxidative stress markers in the gastric tissue. A total number of 36 BALB/c mice were divided into 6 groups mainly normal control (NC) treated with normal saline, disease control (DC) treated with high dose of absolute ethanol (5ml/kg) while treatment groups involved pretreatment with low- dose Vincamine (VLD) at 10mg/kg body weight, medium-dose vincamine (VMD) at 20mg/kg body weight and high- dose vincamine (VHD) at 40mg/kg body weight before ethanol high dose administration and reference drug control, omeprazole (OMT) at the dose of 20 mg/kg body weight. Molecular expression levels of mRNA expressions of inflammatory cytokines like IL-1β, IL-6 and TNF- α were evaluated by using reverse transcription real time polymerase chain reaction method (RT-PCR). Pre-treatment of DC group with low (VLD), medium (VMD) and high doses (VHD) of vincamine improved gastric ulcer score and ameliorated histopathological parameter such as, infiltration of inflammatory cells, edema, fibrinoid necrosis, hemorrhage, and erosion score when compared to DC group. Induction of gastric model significantly increased (all P < 0.05) the mRNA expression of IL-1β, IL-6 and TNF- α in the gastric tissue when same was compared to normal control group (NC). Pretreatment of DC group with different doses of vincamine (VLD, VMD and VHD) significantly downregulated (all P < 0.05) the mRNA expressions of IL-1β, IL-6 and TNF- α and ameliorated oxidative stress marker MDA and increased antioxidant markers like SOD and GSH in the gastric tissue when same was compared to the DC group. In a nutshell, vincamine provide gastric protection in the BALB/c mice of gastric ulcer group by increasing the gastric pH, attenuated total acidity of the stomach and modulated infiltration of inflammatory cells, edema, fibrinoid necrosis, hemorrhage, and erosion score when compared to the DC group. Furthermore, vincamine possesses antiulcer and gastroprotective activity which may be ascribed to down-regulation the mRNA expression of IL-1β, IL-6 and TNF- α in the gastric tissue of disease control group. Vincamine also provide gastroprotective role by increasing the concentration of SOD and GSH while decreasing the MDA in gastric tissue.

Chest HRCT versus RT-PCR for the Detection of COVID-19 in Bangladesh

The coronavirus disease 2019 (COVID-19) pandemic is a crisis of a completely different magnitude, and it is a challenge to life in the whole world, including Bangladesh. The real-time reverse transcription polymerase chain reaction (RT-PCR) tests are currently the gold standard diagnostic tool for COVID-19. This observational study was done to evaluate the diagnostic assessment of high-resolution computed tomography (HRCT) of the chest as compared to a RT-PCR test in COVID-19 patients attending the Radiology and Imaging department of Jalalabad Ragib-Rabeya Medical College Hospital, Sylhet, from 1st July 2020 to 31st December 2021. This study included 387 patients admitted to the ICU and suspected of having COVID-19 who went to the radiology and imaging department for HRCT of the chest for the diagnosis of COVID-19 pneumonia. Simultaneously, nasal swabs from the same number of patients were tested by RT-PCR in the government-approved centre to diagnose COVID-19. Demographic and clinical data were collected by a structured questionnaire, and analysis was done with the help of the Statistical Package for Social Science (SPSS) version 23. Abnormal HRCT was discovered in 317 patients, 309 of whom were positive and 8 of whom were negative by RT-PCR. The difference between the two groups was statistically significant (p&lt;0.05). These outcomes showed that the HRCT evaluation for prediction of COVID-19 sensitivity was 94.8%, specificity was 86.9%, accuracy was 93.5%, positive predictive value (PPV) was 97.5%, and negative predictive value (NPV) was 75.7%. RT-PCR is the gold-standard test for identifying COVID-19; HRCT can be useful in situations where RT-PCR is expected but COVID-19 isolation is a concern. HRCT evaluation for prediction of COVID-19 has more sensitivity and accuracy. Jalalabad Med J 2022; 19 (2): 54-59

Effects of oral administration of the probiotic Lactobacillus rhamnosus GG on the proteomic profiles of cerebrospinal fluid and immunoregulatory signaling in the hippocampus of adult male rats.

The microbiome-gut-brain axis, by modulating bidirectional immune, metabolic, and neural signaling pathways in the host, has emerged as a target for the prevention and treatment of psychiatric and neurological disorders. Oral administration of the probiotic bacterium Lactobacillus rhamnosus GG (LGG; ATCC 53103) exhibits anti-inflammatory effects, although the precise mechanisms by which LGG benefits host physiology and behavior are not known. The goal of this study was to explore the general effects of LGG on the cerebrospinal fluid (CSF) proteome and a biological signature of anti-inflammatory signaling in the central nervous system (CNS) of undisturbed, adult male rats. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics were conducted using CSF samples collected after 21 days of oral treatment with live LGG (3.34 x 107 colony-forming units (CFU)/mL in the drinking water (resulting in an estimated delivery of ~1.17 x 109 CFU/day/rat) or water vehicle. Gene enrichment analysis (using DAVID, v. 6.8) and protein-protein interactions (using STRING, v. 11) were used to explore physiological network changes in CSF. Real time reverse transcription polymerase chain reaction (real time RT-PCR) was performed to assess gene expression changes of anti-inflammatory cytokines in the hippocampus. Genes associated with anti-inflammatory signaling that were analyzed included Il10, Tgfb1, Il4, and IL-4-responsive genes, Cd200, Cd200r1, and Mrc1 (Cd206). Oral LGG administration altered the abundance of CSF proteins, increasing the abundance of five proteins (cochlin, NPTXR, reelin, Sez6l, and VPS13C) and decreasing the abundance of two proteins (CPQ, IGFBP-7) in the CSF. Simultaneously, LGG increased the expression of Il10 mRNA, encoding the anti-inflammatory cytokine interleukin 10, in the hippocampus. Oral LGG altered the abundance of CSF proteins associated with extracellular scaffolding, synaptic plasticity, and glutamatergic signaling. These data are consistent with the hypothesis that oral administration of LGG may improve memory and cognition, and may promote a physiological resilience to neurodegenerative disease, by increasing glutamatergic signaling and promoting an anti-inflammatory environment in the brain.

Extracellular viral microRNAs as biomarkers of virus infection in human cells.

Extracellular viral microRNAs as biomarkers of virus infection in human cells.

Genetic and Phylogenetic Characterization of Dengue Virus among Clinically Suspected Patients in Eastern India

AIM: Dengue is an acute systemic viral disease well known globally in both endemic and epidemic transmission cycles. Main aim is to study serotypes/genotypes and lineages of dengue virus (DENV) are associated with more severe outbreaks. Many reports from India have shown an association between change in DENV genotype/lineage and magnitude of the outbreak and disease severity. Study Design. It is a hospital based cross sectional study. Place and Duration of Study: Molecular Biology laboratory in department of Microbiology, Indira Gandhi Institute of Medical Science and duration study from January2021 to January 2023. Material and Methods: This cross- sectional study was aimed to investigate the circulating DENV serotypes and genotypes in Bihar during year 2021-2023. For genetic characterization of prevalent serotypes, real-time reverse transcription polymerase chain reaction assay was used. Representative samples were sequenced for the envelope (E) gene. Results: All four prevalent serotypes, DENV-1, DENV-2, DENV-3 and DENV-4 were found to be circulating in Bihar with dominance of DENV-2. Mixed infection cases of DENV-2 with DENV-3 and DENV-1 with DENV-2 were also seen. Phylogenetic analysis based on C-prM gene revealed DENV-1 sequences to be 92% similar with Vietnam genotype I (2003) strain, DENV-2 isolates clustered with genotype IV of North India strains, Haryana (1996), Delhi (1996) and Jammu (1993) with 95%, 94% and 94% sequence similarity respectively. DENV-3 isolate was more closely related to genotype III of Indian origin (2003), Gwalior and Delhi with 98% and 99% sequence similarity respectively. Conclusion: With this background data, molecular monitoring of viruses circulating in the locality provides baseline data on the circulating serotypes/ genotypes of dengue virus (DENV). So this monitoring and early detection of changes in the circulation pattern may help in the prediction of DENV outbreaks and can augment disease control efforts.

WDR36 inhibits the osteogenic differentiation and migration of periodontal ligament stem cells

BACKGROUND Periodontitis is an inflammatory disease caused by the host’s immune response and various interactions between pathogens, which lead to the loss of connective tissue and bone. In recent years, mesenchymal stem cell (SC) transplantation technology has become a research hotspot, which can form periodontal ligament, cementum, and alveolar bone through proliferation and differentiation. AIM To elucidate the regulatory effects of WD repeat-containing protein 36 (WDR36) on the senescence, migration, and osteogenic differentiation of periodontal ligament SCs (PDLSCs). METHODS The migration and chemotaxis of PDLSCs were detected by the scratch-wound migration test and transwell chemotaxis test. Alkaline phosphatase (ALP) activity, Alizarin red staining, calcium content, and real-time reverse transcription polymerase chain reaction (RT-qPCR) of key transcription factors were used to detect the osteogenic differentiation function of PDLSCs. Cell senescence was determined by senescence-associated β-galactosidase staining. RESULTS The 24-hour and 48-hour scratch-wound migration test and 48-hour transwell chemotaxis test showed that overexpression of WDR36 inhibited the migration/chemotaxis of PDLSCs. Simultaneously, WDR36 depletion promoted the migration/chemotaxis of PDLSCs. The results of ALP activity, Alizarin red staining, calcium content, and RT-qPCR showed that overexpression of WDR36 inhibited the osteogenic differentiation of PDLSCs, and WDR36 depletion promoted the osteogenic differentiation of PDLSCs. Senescence-associated β-galactosidase staining showed that 0.1 μg/mL icariin (ICA) and overexpression of WDR36 inhibited the senescence of PDLSCs, and WDR36 depletion promoted the osteogenic differentiation of PDLSCs. CONCLUSION WDR36 inhibits the migration and chemotaxis, osteogenic differentiation, and senescence of PDLSCs; 0.1 μg/mL ICA inhibits the senescence of PDLSCs. Therefore, WDR36 might serve as a target for periodontal tissue regeneration and the treatment of periodontitis.

Anti-fibrotic effects of thrombin inhibition in systemic sclerosis-associated interstitial lung disease: Proof of concept.

Activation of the coagulation cascade leading to generation of thrombin is well documented in various forms of lung injury including systemic sclerosis-associated interstitial lung disease (SSc-ILD). We previously demonstrated that the direct thrombin inhibitor dabigatran inhibits thrombin-induced profibrotic signaling in lung fibroblasts isolated from scleroderma patients. The objective of this study was to characterize and compare lung fibroblasts from an SSc-ILD patient at baseline and after dabigatran treatment to ascertain this therapy's differential effects on fibrogenic gene expression. Lung fibroblasts isolated by bronchoalveolar lavage (BAL) from a SSc-ILD patient before and after receiving dabigatran (Pradaxa®) 75 mg twice daily for 6 months (ClinicalTrials.gov Identifier NCT02426229) were analyzed by RNA sequencing, real-time quantitative reverse transcription polymerase chain reaction (qRT)-PCR, and immunofluorescent staining. Thrombin inhibition for six-months by oral dabigatran resulted in significantly decreased expression of 708 lung fibroblast genes as compared to basal levels before dabigatran treatment. Using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, we determined that thrombin-inhibition by dabigatran primarily affected extracellular matrix (ECM) and ECM-related genes. Fibrosis-associated genes, including smooth muscle alpha-actin (SMA, ACTA2), tenascin C, collagen 1 alpha 1 (COL1A1), collagen 3 alpha1 (COL3A1), collagen 8 alpha 2 (COL8A2), collagen 10 alpha 1 (COL10A1), collagen 5 alpha 1 (COL5A1), fibronectin 1, connective tissue growth factor (CTGF), and procollagen-lysine-2-oxoglutarate-5-dioxygenase-2 (PLOD2) were all significantly down regulated following thrombin inhibition by dabigatran treatment. Real-time qRT-PCR and immunofluorescent staining confirmed significant downregulation of the selected genes at the mRNA and protein levels. Inhibition of thrombin in this SSc-ILD patient treated with low-dose dabigatran etexilate downregulated profibrotic proteins in lung fibroblasts, providing further support for the use of thrombin inhibitors as a therapeutic approach for the treatment of patients with SSc-ILD.

Reactivation of Oxidized Soluble Guanylate Cyclase as a Novel Treatment Strategy to Slow Progression of Calcific Aortic Valve Stenosis: Preclinical and Randomized Clinical Trials to Assess Safety and Efficacy.

Pharmacological treatments for fibrocalcific aortic valve stenosis (FCAVS) have been elusive for >50 years. Here, we tested the hypothesis that reactivation of oxidized sGC (soluble guanylate cyclase), the primary receptor for nitric oxide, with ataciguat is a safe and efficacious strategy to slow progression of FCAVS. We used quantitative real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry to characterize sGC signaling and the biological effects of ataciguat on signaling cascades related to nitric oxide, calcification, and fibrosis in excised human aortic valve tissue, aortic valve interstitial cells, and mouse aortic valves. We then conducted randomized, placebo-controlled phase I (14-day safety/tolerance) and phase II (6-month efficacy) trials in patients with moderate aortic valve stenosis. In excised human tissue, we found robust losses in sGC signaling despite upregulation of sGC subunits. In vitro, ataciguat increased sGC signaling and reduced BMP2 (bone morphogenetic protein 2) signaling in aortic valve interstitial cells. In mice with established FCAVS, treatment with ataciguat attenuated BMP signaling and slowed progression of valve calcification and dysfunction. In a phase I, randomized, placebo-controlled trial, treatment with ataciguat for 2 weeks was safe and well tolerated in patients with moderate FCAVS (https://www.clinicaltrials.gov; Unique identifier: NCT02049203). In a separate phase II, randomized, placebo-controlled trial, treatment with ataciguat for 6 months slowed the progression of aortic valve calcification and tended to slow the progression of valvular and ventricular dysfunction in patients with moderate FCAVS (https://www.clinicaltrials.gov; Unique identifier: NCT02481258). Collectively, this study highlights the therapeutic potential of the targeted restoration of the diseased/inactive form of sGC for treatment of FCAVS. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02049203. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02481258.

Maize 4-coumarate coenzyme A ligase Zm4CL-like9 gene positively regulates drought stress response in Arabidopsis thaliana

ABSTRACT Maize is a major food crop in China, and drought is one of the major abiotic stresses that threaten the growth and development of the crop, seriously affecting the crop yield. 4-coumaric acid coenzyme A ligase (4CL) is a key enzyme in the phenylpropane metabolic pathway, which can regulate the lignin content of the plant and play an important role in the plant’s resistance to drought stress, plays an important role in plant resistance to drought stress. In the present study, we screened the differentially expressed up-regulated gene Zm4CL-like9 under drought stress by pre-transcriptome sequencing data (PRJNA793522) in the laboratory, and analyzed the significant up-regulation of Zm4CL-like9 gene in roots under drought stress by qRT-PCR(Real-Time Quantitative Reverse Transcription PCR). The results of prokaryotic expression experiments showed that the protein encoded by the Zm4CL-like9 gene was able to be expressed in prokaryotic cells and could effectively improve the drought tolerance of E. coli. Phenotypic analysis of transgenic Arabidopsis plants under drought stress revealed that seed germination rate, root length, and plant survival after drought rehydration were significantly higher in transgenic Zm4CL-like9 Arabidopsis compared with wild-type Arabidopsis; physiological and biochemical indexes revealed that peroxidase activity, proline (Pro) content, and chlorophyll content were significantly higher in transgenic Arabidopsis compared with wild-type Arabidopsis. Under drought stress, the expression of drought-related genes was significantly up-regulated in transgenic Arabidopsis compared with wild-type Arabidopsis. Taken together, the Zm4CL-like9 gene enhances plant resistance to drought stress by reducing reactive oxygen species accumulation in plants.

Study the Expression of Two Circular RNAs, hsa_circ_0003227 and hsa_circ_0001666, in the Primary Breast Cancer Cell Line BT-20 and the Metastatic Breast Cancer Cell Line MCF-7.

Breast cancer is one of the most prevalent cancers globally and remains a significant cause of cancer-related mortality among women despite advancements in early detection and treatment. The heterogeneity of breast cancer arises from a complex interplay of genetic and environmental factors. Early-stage breast cancer is often asymptomatic, with initial signs including subtle changes in breast morphology and localized swelling, emphasizing the need for reliable diagnostic tools for early detection. Recent research has highlighted the potential of molecular biomarkers, particularly non-coding RNAs such as circular RNAs (circRNAs), in cancer diagnosis. CircRNAs, a unique subset of non-coding RNAs, are characterized by their covalently closed-loop structure, which confers exceptional stability and resistance to exonuclease degradation. They are present in various body fluids and have demonstrated regulatory roles in transcription, translation, and as microRNA sponges, making them promising candidates for cancer diagnostics and prognostics. This study focuses on evaluating the diagnostic potential of two circRNAs, hsa_circ_0001666 and hsa_circ_0003227, by examining their expression in normal, tumor, and metastatic breast cancer cell lines. Breast cancer cell lines representing normal (MCF-10A), tumor (MCF-7), and metastatic (BT-20) stages were cultured for analysis. Total RNA was extracted using a column-based RNA extraction kit, and RNA quality was assessed through NanoDrop spectrophotometry and agarose gel electrophoresis. Complementary DNA (cDNA) synthesis was performed using random hexamers, and the expression levels of hsa_circ_0001666 and hsa_circ_0003227 were quantified using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR), with beta-actin serving as the internal control. Statistical analyses were conducted using SPSS software to evaluate differences in expression levels across cell lines. A significant downregulation of hsa_circ_0001666 and hsa_circ_0003227 was observed in tumor and metastatic cell lines compared to normal breast cell lines (P < 0.05). These results suggest that the expression of these circRNAs correlates with the progression of breast cancer, with decreased levels observed as cells transition from normal to tumorigenic and metastatic stages. The findings of this study indicate that hsa_circ_0001666 and hsa_circ_0003227 have potential utility as diagnostic biomarkers for breast cancer. Their significant expression changes across different stages of breast cancer highlight their relevance in early detection and disease monitoring. This study reinforces the potential of RNA-based biomarkers, particularly circRNAs, in cancer diagnosis and treatment. However, in vitro findings require validation in clinical samples and larger cohorts. Future research should explore their roles in breast cancer progression and integration into non-invasive diagnostics.

Characterization of clustered bacteriocin-type signal domain protein genes in Treponema denticola.

Periodontitis is caused by the dysbiosis of subgingival plaque, and Treponema denticola is the pathogen associated with this disease. Bacteriocins are involved in interbacterial competition during dysbiosis. In our previous study, three potential bacteriocin ABC transporter genes (tepA1-B1, tepA2-B2, and tepA3-B3) of T. denticola were investigated. Upstream of tepA1-B1, three genes annotated as bacteriocin-type signal domain proteins are located. However, the role of these proteins in T. denticola remains unclear. In the present study, these bacteriocin-type signal domain proteins were characterized to elucidate their putative roles in T. denticola. Gene clusters surrounding bacteriocin-type signal domain protein genes were compared in silico. The expression of proteins and transporters was evaluated using real-time quantitative reverse transcription PCR (qRT-PCR). Bacteriocin-type signal domain proteins were detected using immunoblot analysis. The expression of bacteriocin-like proteins was investigated by co-culturing with Treponema vincentii. The DNA sequences of the bacteriocin-type signal domain protein genes and upstream lipoprotein genes were highly conserved. Expression of the bacteriocin-type signal domain protein and tepA1 was slightly higher in the mid-log phase than in the stationary phase and was reduced upon co-culture with T. vincentii. Bacteriocin ABC transporter gene tepA1 was expressed independently of tepA2 and tepA3. Immunoblot analysis detected bacteriocin-like proteins in culture supernatants. However, bactericidal activity was not detected in the culture supernatant of T. denticola. Three tandem lipoprotein-bacteriocin-type signal domain protein genes may have originated from duplication. Bacteriocin-type signal domain proteins are expressed under unstimulated conditions and are secreted by T. denticola cells.

MiR-519d-3p from Placenta-Derived Exosomes Induce Immune Intolerance Regulating Immune Cells, Contributing to the Pathogenesis of Preeclampsia

ABSTRACT Background MiR-519d-3p, also called specific placenta biomarkers, is a member of the Chromosome 19 miRNA Cluster (C19MC) with the highest concentrations of miRNAs in human placenta and maternal serum. These miRNAs are secreted by fetal trophoblast cells within extracellular vesicles (EVs) and interact with the mother’s immune cells, which has been proposed to be crucial for immunological tolerance at the placental-maternal interface. A key mechanism in preeclampsia, a multifactorial, multipath hypertensive pregnancy illness, is an immunological imbalance between the mother and the fetus. Methods Using Next Generation Sequencing, we determined that the placenta-derived Exosomes (pEXOs) of preeclamptic patients had elevated expression of miR-519. To further develop an in vitro model of trophoblast-immune cell communication, HTR-8/Svneo cells and Jurkat T cells were employed and we utilized experiments such as Western blot (WB), Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR), Cell-Counting-Kit-8 (CCK-8) cell proliferation analysis, cell apoptosis analysis, and other techniques to accomplish research. Results It was discovered that miR-519d-3p in pEXOs promoted Jurkat T cell proliferation, inhibited apoptosis, and induced Jurkat T cell differentiation toward Th17. Conclusion MiR-519d-3p in pEXOs disrupts immune tolerance at the maternal-placental interface by encouraging Jurkat T cell proliferation, preventing Jurkat T cell apoptosis, and creating an imbalance in Th17/Treg differentiation. This likely leads to SIRS and unfavorable pregnancy complications like preeclampsia.

Peripheral blood microbiome signature and Mycobacterium tuberculosis-derived rsRNA as diagnostic biomarkers for tuberculosis in human

BackgroundTuberculosis (TB) is a major global health issue. Early diagnosis of TB is still a challenge. Studies are seeking non-sputum biomarker-based TB test. Emerging evidence indicates potential significance of blood microbiome signatures for diseases. However, blood microbiome RNA profiles are unknown in TB. We aimed to characterize the blood microbiome of TB patients and identify Mycobacterium tuberculosis (Mtb) genome-derived small RNA molecules to serve as diagnostic biomarkers for TB.MethodsRNA sequencing data of the blood from TB patients and healthy controls were retrieved from the NCBI-SRA database for analyzing the blood microbiome and identifying rRNA-derived small RNA (rsRNA) of Mtb. Small RNA-seq was performed on plasma exosomes from TB patients and healthy controls. The levels of the candidate Mtb rsRNAs were determined by real-time quantitative reverse transcription PCR (RT-qPCR) on plasma from a separate cohort of 73 TB patients and 62 healthy controls.ResultsThe blood microbiome of TB patients consisted of RNA signals from bacteria, fungi, archaea, and viruses, with bacteria accounting for more than 97% of the total. Reduced blood microbial diversity and abundance of 6 Mycobacterium-associated bacterial genera, including Mycobacterium, Priestia, Nocardioides, Agrobacterium, Bradyrhizobium, and Escherichia, were significantly altered in the blood of TB patients. A diagnostic model for TB based on the 6 genera achieved an area under the curve (AUC) of 0.8945. rsRNAs mapped to the Mtb genome were identified from blood and plasma exosomes of TB patients. RT-qPCR results showed that 2 Mtb-derived rsRNAs, 16 S-L1 and 16 S-L2, could be used as diagnostic biomarkers to differentiate TB patients from healthy controls, with a high co-diagnostic efficacy (AUC = 0.7197).ConclusionsA panel of blood microbiome signatures and Mtb-derived rsRNAs can serve as blood biomarkers for TB diagnosis.

Nuclear receptor 4A1 inhibits chondrocyte inflammation and cartilage degeneration in osteoarthritis by inhibiting NF-κB signal pathway.

The objective of this study is to explore the mechanism of nuclear receptor subfamily 4 group A member 1 (NR4A1) inhibiting the inflammatory response and cartilage degeneration of osteoarthritis (OA) chondrocytes by inhibiting the nuclear factor κB(NF-κB). A total of 45 Sprague-Dawley (SD) rats were randomly divided into three groups (n = 15 in each group): the blank control group (BCG), OA model group (OAG), and NR4A1 overexpression group (OE-NR4A1). The rat model of knee OA was established by medial meniscectomy. A total of 1 week after the operation, the rat model of NR4A1 overexpression was established by injecting NR4A1 overexpression lentivirus into the articular cavity of rats; 5 weeks after the establishment of the model, the rats were killed, and the morphological changes of knee cartilage were observed by hematoxylin and eosin (HE) staining under light microscope. The expression of NR4A1 and NF-κB protein in cartilage tissue was detected by western blot, and the relative expression of NR4A1 and NF-κB mRNA in cartilage tissue was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The levels of interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α in the supernatant of chondrocytes were detected by ELISA, and the apoptosis of chondrocytes was detected by TUNEL staining. The relative expression of NR4A1 mRNA and protein in knee cartilage of rats in OE-NR4A1 were raised compared with those of OAG and BCG (P < 0.05). The relative expression of NF-κB mRNA and protein expression in knee joint cartilage in OE-NR4A1 were reduced compared with those of OAG and BCG, while their expression in the OE-NR4A1 (200 μL) and OE-NR4A1 (300 μL) groups were lower than in the OE-NR4A1 (100 μL) group(P < 0.05). The knee cartilage Mankin's score and knee joint diameter in the OAG were raised compared with those in the BCG (P < 0.05), while those in the OE-NR4A1 were reduced compared with the OAG (P < 0.05), but higher than in the BCG; these measures in the OE-NR4A1 (200 μL) and OE-NR4A1 (300 μL) groups were lower than in the OE-NR4A1 (100 μL) group (P < 0.05). The levels of IL-6, TNF-α, and IL-1β in knee synovial fluid of rats in OE-NR4A1 were reduced compared with those in the OAG (P < 0.05), but raised compared with those in the BCG; and these in the OE-NR4A1 (200 μL) and OE-NR4A1 (300 μL) groups were lower than in the OE-NR4A1 (100 μL) group (P < 0.05). The scorch rate of chondrocytes in the OE-NR4A1 was reduced compared with that in the OAG and higher than that of the BCG, and this measure in the OE-NR4A1 (200 μL) and OE-NR4A1 (300 μL) groups was lower than in the OE-NR4A1 (100 μL) group (P < 0.05). R4A1 can improve the level of intraarticular inflammatory factors by inhibiting the NF-κB signal pathway, thereby reducing intraarticular inflammation and cartilage degeneration, and it plays a protective role in OA.