qRT-PCR Method Research Articles

Exploring Competitive Relationship Between Haemophilus parainfluenzae and Mitis Streptococci via Co-Culture-Based Molecular Diagnosis and Metabolomic Assay

Various bacterial strains with nitrate-reducing capacity (NRC), such as Haemophilus, Actinomyces, and Neisseria, are known to promote NH3 production, control pH in the oral cavity, and inhibit the growth of aciduric bacteria. However, experimental evidence on various estimated bacterial networks within the salivary microbiome is insufficient. This study aims to explore potential bacterial compositional competition observed within saliva samples from dental caries patients through a co-culture assay of mitis Streptococci, which is a primary colonizer in the salivary microbiome, and nitrate-reducing bacteria Haemophilus parainfluenzae. We investigated bacterial growth efficiency change by co-culture time using the qRT-PCR method. In addition, we applied LC/Q-TOF-based metabolites screening to confirm metabolic interactions between oral bacterial species and their association with dental caries from a metabolomics perspective. As a result, we first found that the nitrate reduction ability of H. parainfluenzae is maintained even in a co-culture environment with the mitis Streptococci group through a nitrate reduction test. However, nitrate reduction efficiency was hindered when compared with monoculture-based nitrate reduction test results. Next, we designed species-specific primers, and we confirmed by qRT-PCR that there is an obvious competitive relationship in growth efficiency between H. parainfluenzae and two mitis Streptococci (S. australis and S. sanguinis). Furthermore, although direct effects of nitrate reduction on competition have not been identified, we have potentially confirmed through LC/Q-TOF-based metabolite screening analysis that the interaction of various metabolic compounds synthesized from mitis Streptococci is driving inter-strain competition. In particular, we constructed a basic reference core-metabolites list to understand the metabolic network between each target bacterial species (H. parainfluenzae and mitis Streptococci) within the salivary microbiome, which still lacks accumulated research data. Ultimately, we suggest that our data have potential value to be referenced in further metagenomics and metabolomics-based studies related to oral health care.

ALKBH5, an m6A demethylase, attenuates tumor growth and inhibits metastasis in papillary thyroid carcinoma

The significance of ALKBH5 in erasing mRNA methylation in mRNA biogenesis, decay, and translation control has emerged as a prominent research focus. Additionally, ALKBH5 is associated with the development of numerous human cancers. However, it remains unclear whether ALKBH5 regulates the growth and metastasis of papillary thyroid carcinoma (PTC). Here, we compared cancer tissues and paracancerous tissues from PTC patients, along with cultured cells expressing ALKBH5 (overexpression, silent gene expression, normal stable expression). Our primary objective was to investigate the impact of ALKBH5 on PTC. Selected 30 cases of PTC tissues and their adjacent noncancerous tissues to compare the protein expression levels of ALKBH5 between the two groups using immunohistochemical analysis. qRT-PCR and western blot were used to detect the expression of ALKBH5 in normal thyroid follicular epithelial cells (Nthy-ori3-1) and 4 PTC cell lines (human PTC cell lines K1, BCPAP, IHH4, and TPC1). Appropriate cell lines were screened for subsequent experiments. Immunofluorescence staining was used to localize the high accumulation of ALKBH5 in cells. Construct the ALKBH5 knockdown vector and ALKBH5 overexpression vector separately, and construct the overexpression ALKBH5-mut vector with m6A domain mutation. The impact of different levels of ALKBH5 in the three cell lines on RNA m6A methylation levels was compared using qRT-PCR and western blot methods. Furthermore, cell viability was assessed using the CCK-8 assay, while the impact on cell proliferation was examined using plate colony formation assay. Cell invasion was evaluated using the Transwell assay. Immunohistochemical staining results showed that the expression of ALKBH5 protein in PTC cancer tissue was significantly lower than in adjacent non-cancerous tissue (P < 0.05). Lymph node metastasis in PTC patients may have been linked to ALKBH5 protein levels in their cancerous tissues (P = 0.034). The expression of ALKBH5 in PTC cell lines BCPAP, IHH4, and TPC1 was significantly lower than Nthy-ori3-1 (P < 0.05). IHH4 and TPC1 cell lines were selected for subsequent experiments. Immunofluorescence single staining results showed a high accumulation of ALKBH5 protein in the cell nucleus. Cell viability results suggested that compared to the overexpression-negative control group, cell proliferation, and invasion were significantly decreased in the ALKBH5 overexpression group (P < 0.05) and the mut-ALKBH5 overexpression group (P < 0.05). Additionally, compared to the ALKBH5 overexpression group, cell proliferation and invasion were significantly more decreased in the mut-ALKBH5 overexpression group (P < 0.05). However, compared to the interference-negative control group, cell proliferation and invasion were significantly increased in the ALKBH5 interference group (P < 0.05). The presented findings suggested that m6A demethylase ALKBH5 inhibits tumor growth and metastasis in PTC. Moreover, effective inhibition of m6A modification of ALKBH5 might constitute a potential treatment strategy for PTC.

Regulation of macrophage polarization by metformin through inhibition of TLR4/NF-κB pathway to improve pre-eclampsia.

Pre-eclampsia (PE) is a pregnancy complication featuring hypertension and proteinuria. Metformin exerts clinically preventive effects on PE with an unspecified mechanism. Placental tissues from PE patients and normal pregnant (NP) women were collected. Twenty-four pregnant mice were divided into control, PE (40μg/kg lipopolysaccharides (LPS)-induced modeling), aspirin, and metformin groups. After acquisition of bone marrow-derived macrophages (BMDM) and THP-1cells, cells were categorized into control, LPS (100ng/mL), metformin, and metformin+toll-like receptor 4 (TLR4) agonist RS 09 groups. Inflammatory factors and macrophage polarization were detected by ELISA, flow cytometry, immunofluorescence, immunohistochemistry, and qRT-PCR methods. TLR4/Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway protein expression was examined using Western blot. Both PE patients and PE-like mice enhanced inducible nitric oxide synthase (iNOS), TLR4, p-NF-κB/NF-κB, and p-inhibitor of NF-κB (IκBα)/IκBα expression, and lower arginase 1 (Arg-1) expression. Moreover, metformin treatment in PE-like mice increased fetal number and weight and reduced hypertension, proteinuria, insulin resistance, tumor necrosis factor-α (TNF-α), IL-6, IL-1β, chemokine ligand 4 (CCL4), C-X-C motif chemokine ligand 2 (CXCL2) expression and M1 macrophage polarization, with similar inhibition to aspirin. In LPS-induced cells, metformin had the same effects mentioned above. Decreased TLR4, p-NF-κB/NF-κB, and p-IκBα/IκBα protein expression was caused by metformin both in vivo and in vitro. In vitro, RS 09 intervention inhibited anti-inflammatory and pro-M2 polarizing effects of metformin, activating TLR4/NF-κB pathway. Metformin may ameliorate PE by promoting M2 macrophage polarization through up-regulating TLR4/NF-κB pathway, laying theoretical basis for metformin clinical application in PE.

Seroprevalence of Hepatitis C virus (HCV) infection and role of serological and molecular assay in HCV detection and management: A step towards achieving the aim of National Viral Hepatitis Control Program (NVHCP)

Purpose: Hepatitis C virus (HCV) is a small, enveloped, single stranded RNA virus of Flaviviridae family. HCV causes both acute and chronic infection. Diagnosis of HCV can be done by indirect serological tests that detect antibodies against HCV (anti-HCV) and direct test that detects and quantifies components of viral RNA particles. As per CDC recommendation; HCV testing should be initiated with anti-HCV test, those anti-HCV positive/reactive should have follow-up testing with Nucleic Acid Test. If the HCV RNA is detected, patient should be directed for the treatment with highly effective direct acting antiviral (DAA). This study aims to determine the seroprevalence of HCV infection, to estimate presumptive HCV infection by using serological assay by anti-HCV ELISA, to estimate the current HCV infection by detecting HCV-RNA using RT-PCR molecular assay and, to highlight the role of HCV RNA for initiation of treatment. Methods: This study is retrospective observational analysis of 36500 samples obtained for anti-HCV ELISA test from Jan to Dec 2022. The ELISA kit used was a 3rd generation sandwich assay. All ELISA positive samples were tested for HCV RNA, by automated qRT-PCR method. Results: Out of 36500 samples, 361 tested positive by anti-HCV ELISA with 0.9% seroprevalence. Out of 361, only 336 were tested for HCV RNA. Of 336, 240 samples were negative as target not detected, and 96 samples showed values as viral copies in IU/mL. Most common age group affected was 18-40 years, for both serology and molecular test. Conclusion: In current study, only 96 samples showed viral copies indicating current HCV infection and need to start treatment. Hepatitis C is curable; with the availability of highly effective DAA both in a brand and generic form can cure &gt; 95% of infected people, so the possibility of achieving the target of elimination HCV by 2030 can be predicted.

Botox-A Induced Apoptosis and Suppressed Cell Proliferation in Fibroblasts Pre-Treated with Breast Cancer Exosomes.

breast cancer-associated fibroblast (CAF) is linked to metastasis and is poor for breast cancer prognosis. Since Clostridium Toxin A (Botox-A) had represented a cytotoxic effect on fibroblasts, this study aims to assess Botox-A cytotoxicity in both normal fibroblasts and exosome-induced CAFs. the serum exosomes of 40 BC patients and 30 healthy individuals were isolated and lncRNA H19 (lnch19) levels were assessed by qRT-PCR method. After that, Breast Cancer (BC) exosomes co-cultured with Human foreskin fibroblasts (HFF) and qRT-PCR were applied to evaluate α-SMA, Vimentin, BCL-2, and BAX expression. Both Normal and malignant HFFs co-cultured with Botox-A, and Botox-A loaded exosome for 24 and 48 hours and their apoptosis, Cell proliferation, and viability were monitored by MTT assay, Annexin V-FITC and PI staining and qRT-PCR for BCL-2, BAX, and cyclin D1 mRNAs. Serum exosomes of BC patients had significantly higher levels of lncRNA H19 than healthy individuals. MTT assay results showed Botox-A decreased vital Human foreskin fibroblasts in a dose-dependent manner. BC exosomes significantly increased α-SMA, Vimentin, and BCL-2 mRNA levels in Human foreskin fibroblasts, on the other hand, BAX decreased meaningfully. Co-culture of exosome-treated HFF cells with both Botox-A and Botox-A loaded exosomes significantly boosted BCL-2 mRNA levels, completely contrary to BAX and cyclid d1 expression. Meanwhile, flow cytometry results confirmed a high rate of apoptosis in malignant Human foreskin fibroblasts treated with Botox-A loaded exosome. The findings of this study indicate that exosomal lncRNA H19 could be a diagnostic marker for Breast Cancer and these Breast cancer exosomes can induce malignant phenotype in fibroblasts and turn them into CAFs. Botox-A could be toxic for both normal fibroblasts and CAFs, inducing apoptosis and suppressing cell proliferation among them.

The Diagnostic Value of circFBXW7, circABCB10, and circ0103552 Levels in Breast Cancer.

Despite advances in cancer treatment, breast cancer (BC) remains one of the most common cancers affecting women worldwide. This study aimed to determine serum circFBXW7, circABCB10, and circ0103552 levels and compare BC patients and healthy controls to investigate their roles in the molecular mechanism of BC and the significance of these circRNAs in BC diagnosis. The study group consisted of 92 patients with BC and 31 healthy controls. Total RNA was isolated from serum samples. Following total RNA, complementary DNA was synthesized from this material. Following complementary DNA analysis, the circRNA levels were analyzed by the qRT-PCR method. Expression levels were evaluated in ΔCt values. High ΔCt values of circFBXW7 and circ0103552 and low ΔCt values of circABCB10 were correlated with BC diagnosis (circFBXW7, p = 0.043, r = 0.183, circ0103552, p < 0.001, r = 0.321, circABCB10, p = 0.001, r = -0.291). According to Fold Change (FC) values, circFBXW7 (FC = 0.30) and circ0103552 (FC = 0.26) showed low expression in the patient group compared to the control group, while circABCB10 (FC = 11.09) showed high expression (p < 0.05, for all comparisons). We think that our study is one of the rare studies investigating the relationship between BC and serum circRNA levels. This study concludes that the significant downregulation of circFBXW7 and circ0103552 and the upregulation of circABCB10 are directly related to the diagnosis of BC and can be used for diagnosis, but further studies are needed to elucidate the molecular mechanism of the relationship between circRNAs and BC.

A Novel circ_0075829/miR-326/GOT1 ceRNA Crosstalk Regulates the Malignant Phenotypes and Drug Sensitivity of Gemcitabine-Resistant Pancreatic Cancer Cells.

Although gemcitabine (GEM) is the cornerstone of the treatment of pancreatic cancer (PC), GEM resistance frequently arises. Circular RNA (circRNA) circ_0075829 is highly expressed in PC. However, whether circ_0075829 contributes to GEM resistance of PC is largely unknown. To generate GEM-resistant PC cells (BxPC-3/GR and SW1990/GR), we exposed GEM-sensitive PC cells to GEM. Circ_0075829, microRNA (miR)-326, and glutamic-oxaloacetic transaminase 1 (GOT1) were quantified by a qRT-PCR or western blot method. Cell survival and viability were gauged by MTS assay. Cell proliferation, apoptosis, invasion, and migration were assessed by EdU, flow cytometry, transwell, and wound-healing assays, respectively. Dual-luciferase reporter assays were used to verify the relationship between miR-326 and circ_0075829 or GOT1. Mouse xenografts were performed to evaluate the role of circ_0075829 in vivo. Our data showed that circ_0075829 was upregulated in GEM-resistant PC tissues and cells. Knockdown of circ_0075829 impeded the proliferation, invasion, migration, and glutamine metabolism, and promoted cell apoptosis and GEM sensitivity of GEM-resistant PC cells. Moreover, circ_0075829 silencing suppressed the tumorigenicity of SW1990/GR cells and sensitized them to the cytotoxic effect of GME in vivo. Mechanistically, circ_0075829 bound miR-326 and exerted regulatory effects by affecting miR-326 expression. GOT1 was a direct miR-326 target and a key downstream effector of miR-326. Furthermore, circ_0075829 modulated GOT1 expression via miR-326. Our findings establish a novel regulatory network, the circ_0075829/miR-326/GOT1 competing endogenous RNA (ceRNA) crosstalk, in the regulation of GEM resistance in PC.

Dysregulation of MiR-21, MiR-221 and MiR-451 During Neoadjuvant Treatment of Breast Cancer: A Prospective Study.

Breast cancer is highly heterogeneous disease in which different responses are observed to the same treatment for different subtypes and extents of similar diseases. Considering this scenario, the search for tumor biomarkers is indispensable, with current evidence suggesting that circulating microRNAs are viable biomarkers. This study evaluated the expression of miR-21, miR-221, miR-195, and miR-451 in patients with breast cancer undergoing neoadjuvant treatment at oncology outpatient facilities in Brazil. We conducted a prospective and observational study in which blood samples were collected for microRNA expression analysis, comparing control and breast cancer patients who were candidates for neoadjuvant treatment groups. The expression of microRNAs was investigated by qRT-PCR method. For parametric data analysis, one-way ANOVA with Tukey's post hoc test was used. Thirty-three participants (all female) were included in the control group and twenty-seven participants were included in the study group. The non-special subtype of breast cancer was found in 96% of the study group participants; 88.9% were locally advanced tumors (T3, T4), 40.7% were luminal tumors, 33.3% were HER-2-positive, and 26% were triple negative tumors. Expression analysis of microRNAs during neoadjuvant treatment, using miR-16 as a normalizer, showed higher expression levels of miR-21 and miR-221 at the end of treatment, and high expression levels for miR-451 were also observed at the beginning of treatment. This is the first study that evaluates the expression of microRNAs in the context of neoadjuvant treatment of breast cancer in the Brazilian population. Our results suggest that there is a deregulation of miR-21, miR-221, and miR-451 during neoadjuvant treatment in these patients.

LncRNA TUG1 mitigates chronic kidney disease through miR-542-3p/HIF-1α/VEGF axis.

Renal interstitial fibrosis (RIF) is a common pathway in chronic kidney disease (CKD) that ultimately leads to end-stage renal failure, worsening both glomerulosclerosis and interstitial fibrosis. Ten percent of the adult population in the world suffers from CKD, and as the ageing population continues to rise, it is increasingly regarded as a global threat-a silent epidemic. CKD has been discovered to be closely associated with both long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), while the precise molecular processes behind this relationship are still unclear. This study evaluated the impact of miR-542-3p and lncRNA TUG1 on renal fibrosis, along with the underlying regulatory mechanisms. Through in vitro tube formation assays, research demonstrated that knocking down lncRNA TUG1 may enhance angiogenesis and repair damaged endothelial cell-cell connections. We used Western blot and qRT-PCR methods in the unilateral ureteral obstruction (UUO) model to identify tissue hypoxia and fibrotic lesions. Additionally, a cutting-edge method known as fluorescence microangiography (FMA) was employed to detect damage to the peritubular capillaries (PTCs), with MATLAB software utilised for data evaluation. Furthermore, the coexpression of CD31 and α-SMA helped identify cells in the obstructed kidney that were transitioning from endothelium to myofibroblasts. On the contrary, lncRNA TUG1 downregulation showed a protective effect against the transition from endothelial cells to myofibroblasts. Additionally, knocking down lncRNA TUG1 has been shown to reduce the expression of fibrotic markers by alleviating tissue hypoxia. This effect was significantly counteracted by the inhibition of miR-542-3p. Collectively, our findings offer fresh perspectives on how lncRNA TUG1 and the miR-542-3p/HIF-1α/VEGF axis are regulated as renal fibrosis advances.

Flexible graphene textile-based sensor integrates programmable DNAzyme and logic gates for efficient small extracellular vesicle-miRNA detection

Nucleic acid signal amplification technologies and their cascade systems possess powerful ability to amplify signals. However, their application in biological computing, which necessitates a strict logical relationship, requires further investigation. To address this need, we developed a flexible sensing substrate consisting of carbon fiber cloth/graphene walls (CFC/GW), integrated with a 3D DNA tetrahedron (DNA-Te)-assisted bioplatform. This platform serves as the foundation for two types of Boolean logic gates: the “AND” and “OR” gate. The output system of the Cu2+ + DNAzyme logic gate module, activated by multiple miRNAs, was combined with the CFC/GW/Au-Te-based detection system to precisely identify small extracellular vesicle (sEV)-derived miRNAs in ultratrace quantities. Leveraging DNA Te-assisted DNAzyme molecular logic circuits, we designed higher-order Boolean logic operations to effectively identify and accurately read sEV-miRNAs in complex mixed samples. By integrating a material with low-background-signal and high-performance material with the original branch-strand migration logic gate system, we achieved an intelligent sensing platform that integrates capture, recognition, detection, and interpretation of sEV-miRNAs. We successfully demonstrated the response of the logic gate module of sEV-miRNAs in clinical samples, with detection results maintaining high consistency with conventional qRT-PCR methods. Our DNA logic gate platform boasts a simple design, adaptability, expandable complexity, and compatibility with various systems, enabling diverse possibilities for the nucleic acid-based development of intricate transduction networks in biomedical fields. This platform paves the way for more effective diagnosis, treatment, and monitoring of diseases, marking a significant advancement in nucleic acid signal amplification technologies and their utilization in biological computing.

One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses.

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

Development of RT-qPCR assay for assessing the expression of ACTB and SDHA housekeeping genes in the cell cultures of mammalian hosts of zoonotic infections

Background. The molecular mechanisms behind the maintenance of zoonotic pathogens in nature can be better understood by examining the gene expression in host cells in response to the infection. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study gene expression, especially with the use of endogenous reference genes (RG) to normalize the data. Therefore, it is critical to develop the reliable qRT-PCR assay and validate that selected RGs are stably expressed in the studied organism or cell culture.The aim. In this work, we aimed to develop the real-time qRT-PCR method for detecting mRNA of two candidate RGs, succinate dehydrogenase subunit A (SDHA) and beta-actin (ACTB), in the cell lines of mammalian hosts of zoonotic infections.Materials and methods. The SPEV (porcine embryonic kidney cell line) and ApnK (Korean field mouse kidney cell line) cells were grown in 24-well culture plates. Total RNA/DNA was isolated from trypsin-detached cell monolayers. Genomic DNA in the samples was removed with RNase-free DNase I, and one-step RT-qPCR was performed using primers for SDHA and ACTB gene fragments and the corresponding TaqMan hydrolysis probes. The experiment was performed in 4 independent replicates.Results. In the Korean field mouse cells, the linearity, efficiency, repeatability, and reproducibility of the RT-qPCR for ACTB gene mRNA corresponded to the modern requirements. However, RT-qPCR for SDHA exhibited good linearity and efficiency of the reaction, but CV values for repeatability and reproducibility slightly exceeded the recommended standards. In porcine cells, both assays had acceptable parameters. Thus, to use the SDHA as RG for ApnK cells, a detailed study of the stability of its expression in this particular model is required.Conclusions. New qRT-PCR assay was developed to assess the expression of housekeeping genes ACTB and SDHA in the cells of the Korean field mouse and domestic pig. Further research is necessary to validate these genes as references for quantitative assessment of gene expression in the cells of mammalian hosts of zoonotic infections.

INVESTIGATION OF ANTIMICROBIAL AND ANTIVIRAL EFFECTS OF TÜRKIYE PROPOLIS WATER EXTRACTS: AN IN VITRO STUDY

Propolis is a natural bee product used as a therapeutic agent for centuries. Propolis extracts are natural resources that attract the attention of scientists looking for new components due to the insufficiency of existing drugs. In current study, antiviral and antimicrobial activity of propolis water extracts prepared from three different raw propolis samples collected from Northeast of Türkiye (Ardahan, Rize, and Trabzon) were investigated. The total flavonoid contents (TFC) and total phenolic content (TPC) of the extracts were measured. It was determined that TPC and TFC ranged from 5.87±0.36 to 20.47±1.46 mg GAE g-1, and 0.48±0.04 to 2.10±0.22 mg QUE g-1, respectively. The antimicrobial activity of the extracts against 14 microorganisms (Bacillus cereus ATCC14579, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Acinetobacter haemolyticus ATCC 19002, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Enterobacter aerogenes ATCC 13048, Mycobacterium smegmatis ATCC 607, Klebsiella pneumoniae ATCC 13883, Chromobacterium violaceum ATCC 12472, Candida parapsilosis ATCC 22019, and Candida albicans ATCC 10231) and their effect against the Pseudomonas aeruginosa biofilm were investigated. Additionally, anti-quorum sensing and anti-swarming activities of the extracts were tested. The antiviral activity of the extracts was examined against Herpes simplex virus type 1 (HSV-1) by MTT and qRT-PCR methods. The water extracts of propolis samples did not show antimicrobial, anti-swarming, anti-quorum sensing, and anti-viral activities. However, extracts were found to have strong anti-biofilm activities. The results show that aquatic propolis extracts can be evaluated in the treatment of biofilms.

Network pharmacology and in vitro experiments to investigate the anti-gastric cancer effects of paeoniflorin through the RAS/MAPK signaling pathway

The aim of this study was to investigate the key targets and signaling pathways of paeoniflorin (PF) for the treatment of gastric cancer (GC). First, the differentially expressed genes (DEGs) of gastric cancer were obtained by analyzing GSE118916 Gene Chip, and then the active components of paeoniflorin and their targets of action were found. And the intersection genes of the two were analyzed for target and pathway analysis. In addition, cell viability after PF intervention was detected by CCK-8. Clone formation assay, wound scratch assay, transwell assay were used to detect cell migration and invasion. The qRT-PCR and Western blot methods were used to verify the mechanism of action. The results showed that a total of 286 paeoniflorin targets and 1799 DEGs were obtained. Secondly, we found that PF could treat gastric cancer through RAS/MAPK signaling pathway. In addition, through in vitro cellular experiments, we also found that PF had a significant therapeutic effect on gastric cancer. Therefore, we believe that PF inhibits the proliferation and metastasis of gastric cancer, and its effect may be exerted by regulating the RAS/MAPK signaling pathway. PF is a promising drug for the treatment of gastric cancer. Combined with the in vitro experiments, we found that the therapeutic effect of PF is related to the regulation of the RAS/MAPK signaling pathway, and the results of the present study preliminarily revealed its complex mechanism, which will lay the foundation for future clinical treatment.

Antiaging properties of chlorogenic acid through protein and gene biomarkers in human skin fibroblast cells as photoaging model

Background and purpose: Chlorogenic acid (CA) is a natural chemical that promises antiaging activity against photoaging skin damage. This research examined CA activities in mitigating skin photoaging. Experimental approach: UV-exposed human skin fibroblast cells were subjected to CA at 6.25, 12.5, and 25 μg/mL. The protein levels of cell secretion, such as cyclooxygenase (COX)-2, nitric oxide (NO), and interleukin (IL)-6 were measured using ELISA and colorimetry methods. Meanwhile, the mRNA expressions of glutathione peroxidase (GPX)-1, tissue inhibitor metalloproteinase (TIMP)-1, matrix metalloproteinase (MMP)-1, caspase (CASP)-3, CASP-8, and fibroblast growth factor (FGF)-2 were quantified using the qRT-PCR method. Findings/Results: CA treatment reduced inflammatory and aging biomarkers. CA at 6.25 μg/mL lowered NO, COX-2, and IL-6 levels to 89.44 μmol/L, 8.10 ng/mL, and 62.75 pg/mL, respectively. CA at 25 μg/mL resulted in the most significant down-regulation of MMP-1, CASP-3, and CASP-8 genes’ expression (3.27, 1.25, and 3.59, respectively). Furthermore, treatment with CA at 25 µg/mL demonstrated the most notable activity in up-regulating antioxidant markers, specifically GPX-1, and extracellular matrix (ECM) integrity markers, including TIMP-1 and FGF-2 genes’ expression. Conclusion: CA imposes its anti-aging activity by decreasing inflammatory and aging biomarkers, and increasing cellular antioxidant and ECM integrity.

The Effect of Circulating Exosomes Obtained from Young and Old Individuals on the Aging related hTERT and P16 Expression in Hematopoietic Stem Cells

Introduction: Functional reduction of telomeres can induce DNA damage response through cell cycle checkpoints and contribute to the senescence of stem cells. The effect of exosomes on the aging and rejuvenation of hematopoietic stem cells (HSCs) is not well known. Therefore, the present study is designed to examine the impact of plasma exosomes derived from young and old individuals on hTERT and P16 expression involved in the cellular aging process. Methods: Exosomes isolated from four young (Y-Exo) and four old (O-Exo) men were evaluated for CD63 protein expression, morphology, size and zeta potential. HSCs were treated with exosomes, and then, the cell viability and the mRNA expression (hTERT and P16) were evaluated using MTT and qRT-PCR methods, respectively. To measure the hTERT protein level, a western blot technique was performed. Results: The gene expression of hTERT was significantly decreased in HSCs treated with 5 μg/ml (O5-Exo) and 10 μg/ml (O10-Exo) doses of exosomes obtained from elderly individuals compared to the cells treated with young exosomes and the untreated HSCs (p &lt; 0.05). In addition, there was a profound elevation of hTERT protein in the HSCs treated with both doses of young exosomes in comparison with the cells treated with both doses of old exosomes (p &lt; 0.05). Moreover, P16 expression was markedly upregulated in the O5-Exo and O10-Exo groups compared to the untreated group (p &lt; 0.05). Conclusion: Our findings reinforce the concept that depending on the age of individuals, circulating exosomes may acquire properties that affect the pathways involved in the aging process in HSCs.

AP001885.4 promotes the proliferation of esophageal squamous cell carcinoma cells by histone lactylation- and NF-κB (p65)-dependent transcription activation and METTL3-mediated mRNA stability of c-myc

ABSTRACT Esophageal squamous cell carcinoma (ESCC) is an aggressive malignant neoplasm, and up to now, the role of long non-coding RNA (lncRNA) AP001885.4 in cancer, including ESCC, is absolutely unclear. The GEPIA database was applied to identify differentially expressed and prognosis-associated genes in esophageal cancer (ESCA). CCK-8, colony formation, Western blot, and qRT-PCR methods were harnessed to investigate the role and mechanism of AP001885.4 in esophageal carcinogenesis. By analyzing TCGA data in the GEPIA database, two lncRNAs were selected. AP001885.4 was overexpressed and positively associated with the unfavorable outcome of ESCC patients, and LINC001786 was under-expressed and negatively linked with the poor prognosis. Knockdown of AP001885.4 suppressed the proliferation and colony formation of ESCC cells. Importantly, the silence of AP001885.4 downregulated c-myc. Mechanically, the knockdown of AP001885.4 reduced METTL3 expression and m6A modification in c-myc mRNA, and METTL3 positively regulated c-myc. Furthermore, the knockdown of AP001885.4 diminished histone lactylation and NF-κB (p65) expression, and the protein lactylation inhibitors (2-DG, 2-deoxy-D-glucose and oxamate) and the NF-κB inhibitor (JSH-23) also lessened c-myc expression. Consequently, our findings suggested that AP001885.4 promoted the proliferation of esophageal squamous cell carcinoma cells by histone lactylation- and NF-κB (p65)-dependent transcription activation and METTL3-mediated mRNA stability of c-myc.

Evaluation of UCA1/miR-138/CDK6 Network in the Patients with Laryngeal Squamous Cell Carcinoma

Objective: Noncoding RNAs (ncRNAs) have the potential to be diagnostic and therapeutic targets, in many cancer types. miRNA ‐ mRNA ceRNA (competing endogenous RNA) network in cancer has been an interesting topic that has been studied in recent years. We aimed to investigate the relationship of UCA1/miR-138/CDK6 network with laryngeal squamous cell carcinoma (LSCC). Material and Methods: We studied with the cancerous tissue and as the control; neighboring normal tissue samples of the patients with diagnosis of LSCC. UCA1, miR-138 and CDK6 expression analysis was made by quantitative real-time PCR (qRT-PCR) method after RNA isolation of the samples. Results: Expressions of cancerous tissue samples and normal adjacent tissue samples as controls were compared. CDK6 and UCA1 levels were increased and miR-138 levels were found to be decreased in cancer tissues but not statistically significant. Conclusions: To the best of our knowledge there is no study to provide an expression profile of the UCA1/CDK6/miR-138 network for LSCC patients. Although we could not obtain statistically significant results, our results were similar with the UCA1, miR‐138, and CDK6 axis literature. Further studies with larger patient samples and LSCC cell lines may confirm the function of this axis, which might be a diagnostic and therapeutic target for LSCC.

Effect of Moderate Intensity Interval Training on the Expression of Genes Related to Necroptosis of Heart Tissue in Male Heart Attack Model Rats

Introduction: Myocardial ischemia-reperfusion injury is a set of cellular and molecular patho-mechanisms that lead to the death of living cardiomyocytes and compromised cardiac function. The aim of this study was to determine the effect of moderate intensity interval training on the expression of RIPK-1, RIPK-3 and MLKL genes in the heart of male heart attack model rats. Methods: In this experimental study, thirty 16-week-old male Wistar rats weighing 220 to 280 gr were randomly divided into three groups: control (healthy), myocardial infarction (MI), and moderate intensity interval training. Myocardial infarction was performed by direct intervention with occlusion in the left anterior descending artery (LAD) for 30 minutes. Interval training protocol was performed three days a week, for 8 weeks, with moderate intensity of 60 minutes of interval running on a treadmill, each interval including 4 minutes of running with an intensity of 65-70% of VO2max and 2 minutes of active recovery with an intensity of 50-60% of VO2max. qRT-PCR method was used to check the gene expression of research variables. Data were compared using SPSS software version 16, one-way analysis of variance and Tukey's post hoc test at a significance level of p&lt;0.05. Results: The expression of cardiac RIPK-1, RIPK-3 and MLKL genes were higher in the MI group compared to the control group; however, moderate intensity interval training decreased the expression of RIPK-1, RIPK-3 and MLKL genes compared to the MI group (p=0.001 and p=0.006, respectively). Conclusion: The results of the research showed that myocardial infarction is associated with the activation of the necroptosis cell death pathway, likewise, interval training with moderate intensity has an effect on reducing the expression of necroptosis genes (RIPK1, RIPK3, and MLKL). However, due to the lack of evidences and limitations of research, it still needs more investigations.

Development of a Triplex Endpoint PCR Assay for the Detection of SARS-CoV-2: Insights on Cost-Efficiency and Method Design

Introduction: Lower respiratory tract infections, including COVID-19, have a substantial global impact, making the development of diagnostic tests crucial. The study aimed to develop a new, accurate, fast, and cost-effective PCR-based detection method for SARS-CoV-2, applicable in limited settings and capable of detecting all current variants and potential future pathogens. Methods: The study was conducted between 2020 and 2022 at the molecular biology department of Mures County Clinical Hospital (MCCH), Romania. Initially, pharyngeal and nasal secretions were collected and processed using the real-time qRT-PCR method for routine COVID-19 diagnosis. Ninety-two samples were randomly selected to develop the assay, including samples from different infection periods and negative controls. Complementary DNA was prepared from the selected samples, and the presence and integrity of the extracted RNA were evaluated by amplifying the GAPDH housekeeping gene. Primers for three specific viral genes (N, ORF1ab, and S) were designed, and their efficiency was evaluated using endpoint PCR and sequencing. Finally, the method was optimized and implemented as a one-step triplex PCR assay for routine diagnostic use. Results: The molecular laboratory at the MCCH analyzed a total of 41,818 samples between June 2020 and December 2022. Among these samples, 26.15% tested positive for SARS-CoV-2, while 70.9% were negative and 2.95% were inconclusive or invalid. Three peaks of positive tests were observed in November 2020, April 2021, and February 2022. The study selected 92 preserved RNA samples for triplex PCR assay development, validating the primers’ specificity and confirming the quality of the nucleic acids. The comparative analysis showed the efficiency and accuracy of the endpoint reverse transcription triplex PCR method (RT-PCR), indicating its potential as a cost-effective alternative to real-time reverse transcription PCR (qRT-PCR) in low-income countries with limited infrastructure for COVID-19 testing. Conclusion: This method has the potential to facilitate large-scale diagnosis of SARS-CoV-2 infections, allowing for rapid and appropriate therapeutic management and ongoing monitoring of patients. Additionally, the method can be easily adapted for the detection of other pathogens.