Quantitative Reverse Transcriptase-polymerase Chain Research Articles

Circular RNA ACVR2A promotes the progression of hepatocellular carcinoma through mir-511-5p targeting PI3K-Akt signaling pathway

Circular RNA (circRNA) is thought to mediate the occurrence and development of human cancer and usually acts as a tiny RNA (miRNA) sponge to regulate downstream gene expression. However, it is not clear whether and how circACVR2A (hsa_circ_0001073) is involved in the progression of HCC. The purpose of this study is to clarify the potential role and molecular mechanism of circACVR2A in regulating the progression of hepatocellular carcinoma cells (HCC). The abundance of related proteins in circACVR2A, microRNA (miR511-5p) and PI3K-Akt signaling pathway was determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or Western blotting. Cell viability, invasion and apoptosis were analyzed by CCK-8, Transwell analysis and Tunel staining, respectively. The interaction between circACVR2A and microRNA was evaluated by double luciferase reporter gene assay. The results showed that circACVR2A was highly expressed in hepatocellular carcinoma cell lines. Our in vivo and in vitro data showed that circACVR2A promoted the proliferation, migration and invasion of HCC. In terms of mechanism, we found that circACVR2A can directly interact with miR511-5p and act as a miRNA sponge to regulate the expression of related proteins in PI3K-Akt signaling pathway.In HCC, circACVR2A can mediate miR-511-5p/mRNA network to activate PI3K signal pathway. This shows that the molecular regulatory network with circACVR2A as the core is a new potential target for diagnosis and treatment of hepatocellular carcinoma.

Role of SFRP5 in Non-Small Cell Lung Cancer and Its Correlation with SUV of 18F-FDG PET-CT

Aim: This study aimed to evaluate the relationship between secreted frizzled-related protein 5 (SFRP5) expression and fluorine 18-fluoro-deoxyglucose (18 F-FDG) uptake imaged with positron emission tomography/tomography (PET/CT) in patients with non-small cell lung cancer (NSCLC). In addition, we sought to elucidate the potential role and mechanism of action of SFRP5 in NSCLC.Materials and methods: The maximum standardized uptake value (SUVmax) of the lesions was calculated. SFRP5 expression was analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The correlation between SFRP5 expression and SUVmax was evaluated using Pearson’s correlation analysis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, wound healing, and transwell assays were used to analyze cell viability, apoptosis, migration, and invasion, respectively.Results and conclusion: The results indicated that the SUVmax was higher in patients with NSCLC than that in healthy volunteers. Moreover, SFRP5 expression was lower in tissues from the four types of NSCLC than that in the adjacent normal tissues. SUVmax negatively correlated with SFRP5 expression in the four types of NSCLC. In addition, up-regulation of SFRP5 decreased the viability, migration, and invasion abilities, and increased apoptosis of NSCLC cells. Furthermore, SFRP5 inhibited the Wnt/β-catenin pathway in NSCLC cells. In conclusion, SFRP5 modulates the biological behaviors of NSCLC through Wnt/β-catenin pathway.

Revealing New Prospects: Antipsychotic Drugs Exert Anti-Tumor Effects against Gastric Cancer through Inducing Apoptosis.

Globally, Gastric Cancer (GC) ranks as the fifth leading cause of cancer-related deaths. GC is a multifaceted malignancy with diverse etiologies; however, understanding the shared molecular mechanisms can aid in discovering novel targeted therapies for GC. This study has employed a drug repositioning approach to explore new drug candidates for treating GC. The human GC cell lines AGS, MKN-45, and KATO-III were treated with different concentrations of dopamine, cabergoline, thioridazine, and entacapone to determine effective doses and IC50 values. In vitro, cytotoxic activity on cancer cell lines was screened based on dose/time using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) was used to measure the mRNA expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Proliferating Cell Nuclear Antigen (PCNA) in each group. The percentage of apoptotic cells was evaluated using Annexin V/PI staining. Dopamine, cabergoline, thioridazine, and entacapone elicited cytotoxic effects on AGS and KATO-III cells in a dose-dependent manner and elevated the percentage of Annexin V-positive cells, suggesting the occurrence of apoptosis. The expression of Bcl-2 and PCNA was significantly decreased, whereas the expression of Bax was considerably increased in the AGS and KATO-III cells compared to that in the blank group (p < 0.05); however, no similar effect was observed in MKN-45 cells. Through in vitro experiments, this study provides evidence that the antipsychotic drugs cabergoline, dopamine, thioridazine, and entacapone can inhibit gastric cancer growth in AGS and KATO-III cells. These findings suggest that these drugs could be repurposed as novel therapeutic agents for the treatment of gastric cancer.

Insights Into the Role of miR-877 and Histidine-Rich Glycoprotein in Preeclampsia

IntroductionThe clinical significance of miR-877 and histidine-rich glycoprotein (HRG) in preeclampsia (PE) remains unknown. Bioinformatics analyses have identified HRG as a target for numerous micro RNAs. Based on novelty and target score, miR-877 was selected for this study to assess the clinical significance of miR-877 and HRG in placental and serum samples from patients with PE, with the aim of elucidating their potential role in disease progression.Material and methodsThis study included 75 placental tissue samples and 75 serum samples obtained from patients with PE and normal controls. The PE group was subdivided into mild and severe cases. Relative quantification of miR-877 and HRG was performed using quantitative reverse transcriptase–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.ResultsPlacental and serum miR-877 levels were significantly elevated in pregnancies with PE, especially in severe cases, compared to normal controls. However, there were significant reductions in HRG serum levels along with significant increases in placental HRG levels among patients with PE. Moreover, significant differences in miR-877 and HRG levels were noted between the PE and control groups.ConclusionsmiR-877 and HRG may play a role in the pathogenesis and progression of PE. Moreover, miR-877 potentially plays a role in the dysregulation of HRG. In addition, these molecules are potential biomarkers for the detection of PE as well as differentiation of mild and severe cases.

Cyclooxygenase upregulation in cytomegalovirus-infected gingival fibroblasts: Implications for periodontal disease

ObjectiveTo assess the changes in the expression of COX-1 and COX-2 in primary gingival fibroblasts upon infection with two strains of human cytomegalovirus (CMV). MethodsPrimary human gingival fibroblasts (HGFs) were cultured and infected with two strains of human CMV the laboratory strain (Towne) and a clinical isolate (B52) at a multiplicity of infection of 0.5. The relative mRNA levels of COX-1 and COX-2 were evaluated using multiplex reverse transcriptase quantitative polymerase chain reaction with specific hydrolysis probes. Additionally, immunofluorescence was performed for COX-2, IE1, and IE2 viral proteins. ResultsAt 24 h post infection, CMV infection of HGFs resulted in a decrease in COX-1 transcripts in cells infected with both the Towne strain and B52 clinical isolate, and in non-infected cells. Conversely, COX-2 transcripts increased in infected cells (Towne and B52); they were significantly higher in B52 infected cells, similar to the immunofluorescence detection results for COX-2. ConclusionsInfection of HGFs with CMV increased in both mRNA and protein expression of COX-2. Furthermore, the B52 isolate induced higher COX-2 expression than the Towne laboratory strain. This study provides a basis for understanding the putative relationships between CMV infection and inflammatory responses in gingival diseases.

Effect of Quorum Sensing Systems on Biofilm Formation and Virulence in Salmonella

In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.

Fighting Emerging Caspofungin-Resistant Candida Species: Mitigating Fks1-Mediated Resistance and Enhancing Caspofungin Efficacy by Chitosan.

Invasive candidiasis poses a worldwide threat because of the rising prevalence of antifungal resistance, resulting in higher rates of morbidity and mortality. Additionally, Candida species, which are opportunistic infections, have significant medical and economic consequences for immunocompromised individuals. This study explores the antifungal potential of chitosan to mitigate caspofungin resistance in caspofungin-resistant Candida albicans, C. krusei, and C. tropicalis isolates originating from human and animal sources using agar well diffusion, broth microdilution tests, and transmission electron microscope (TEM) analysis of treated Candida cells. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression of SAGA complex genes (GCN5 and ADA2) and the caspofungin resistance gene (FKS) in Candida species isolates after chitosan treatment. The highest resistance rate was observed to ketoconazole (80%) followed by clotrimazole (62.7%), fluconazole (60%), terbinafine (58%), itraconazole (57%), miconazole (54.2%), amphotericin B (51.4%), voriconazole (34.28%), and caspofungin (25.7%). Nine unique FKS mutations were detected, including S645P (n = 3 isolates), S645F, L644F, S645Y, L688M, E663G, and F641S (one isolate in each). The caspofungin minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values before chitosan treatment ranged from 2 to 8 µg/mL and 4 to 16 µg/mL, respectively. However, the MIC and MFC values were decreased after chitosan treatment (0.0625-1 µg/mL) and (0.125-2 µg/mL), respectively. Caspofungin MIC was significantly decreased (p = 0.0007) threefold following chitosan treatment compared with the MIC values before treatment. TEM analysis revealed that 0.5% chitosan disrupted the integrity of the cell surface, causing irregular morphologies and obvious aberrant changes in cell wall thickness in caspofungin-resistant and sensitive Candida isolates. The cell wall thickness of untreated isolates was 0.145 μm in caspofungin-resistant isolate and 0.125 μm in sensitive isolate, while it was significantly lower in chitosan-treated isolates, ranging from 0.05 to 0.08 μm when compared with the cell wall thickness of sensitive isolate (0.03 to 0.06 μm). Moreover, RT-qPCR demonstrated a significant (p < 0.05) decrease in the expression levels of histone acetyltransferase genes (GCN5 and ADA2) and FKS gene of caspofungin-resistant Candida species isolates treated with 0.5% chitosan when compared with before treatment (fold change values ranged from 0.001 to 0.0473 for GCN5, 1.028 to 4.856 for ADA2, and 2.713 to 12.38 for FKS gene). A comparison of the expression levels of cell wall-related genes (ADA2 and GCN5) between caspofungin-resistant and -sensitive isolates demonstrated a significant decrease following chitosan treatment (p < 0.001). The antifungal potential of chitosan enhances the efficacy of caspofungin against various caspofungin-resistant Candida species isolates and prevents the development of further antifungal resistance. The results of this study contribute to the progress in repurposing caspofungin and inform a development strategy to enhance its efficacy, appropriate antifungal activity against Candida species, and mitigate resistance. Consequently, chitosan could be used in combination with caspofungin for the treatment of candidiasis.

Expression profile of long noncoding RNAs and comprehensive analysis of lncRNA-cisTF-DGE regulation in condyloma acuminatum

ObjectiveTo identify differentially expressed long noncoding RNAs (lncRNAs) in condyloma acuminatum (CA) and to explore their probable regulatory mechanisms by establishing coexpression networks.MethodsHigh-throughput RNA sequencing was performed to assess genome-wide lncRNA expression in CA and paired adjacent mucosal tissue. The expression of candidate lncRNAs and their target genes in larger CA specimens was validated using real-time quantitative reverse transcriptase polymerase chain reaction (RT‒qPCR). Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for the functional enrichment analysis of these candidate lncRNAs and differential mRNAs. The coexpressed mRNAs of the candidate lncRNAs, calculated by Pearson’s correlation coefficient, were also analysed using GO and KEGG analysis. In addition, the interactions among differentially expressed lncRNAs (DElncRNAs)-cis-regulatory transcription factors (cisTFs)-differentially expressed genes (DEGs) were analysed and their network was constructed.ResultsA total of 546 lncRNAs and 2553 mRNAs were found to be differentially expressed in CA compared to the paired control. Functional enrichment analysis revealed that the DEGs coexpressed with DElncRNAs were enriched in the terms of cell adhesion and keratinocyte differentiation, and the pathways of ECM-receptor interaction, local adhesion, PI3K/AKT and TGF-ß signaling. We further constructed the network among DElncRNAs-cisTFs-DEGs and found that these 95 DEGs were mainly enriched in GO terms of epithelial development, regulation of transcription or gene expression. Furthermore, the expression of 3 pairs of DElncRNAs and cisTFs, EVX1-AS and HOXA13, HOXA11-AS and EVX1, and DLX6-AS and DLX5, was validated with a larger number of specimens using RT‒qPCR.ConclusionCA has a specific lncRNA profile, and the differentially expressed lncRNAs play regulatory roles in mRNA expression through cis-acting TFs, which provides insight into their regulatory networks. It will be useful to understand the pathogenesis of CA to provide new directions for the prevention, clinical treatment and efficacy evaluation of CA.

Identification of exosomal circSLC26A4 as a liquid biopsy marker for cervical cancer.

Circular RNA SLC26A4 (circSLC26A4) functions as an oncogene in the initiation and progression of cervical cancer (CC). However, the clinical role of plasma exosomal circSLC26A4 in CC is poorly known. This study aims to develop an accurate diagnostic method based on circulating exosomal circSLC26A4. In this study, exosomal circSLC26A4 derived from CC cell lines (CaSki, SiHa, and HeLa) and human cervical epithelial cells (HcerEpic) was measured and compared using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Additionally, 56 volunteers, including 18 CC patients, 18 cervical high-grade squamous intraepithelial lesion (HSIL) patients, and 20 healthy volunteers, were enrolled. qRT-PCR was also performed to measure the plasma exosomal circSLC26A4 levels in all participants. The exosomal circSLC26A4 expression level derived from CC cells was significantly elevated compared to it derived from HcerEpic cells. Plasma exosomal circSLC26A4 levels in CC patients were significantly higher than in healthy women and HSIL patients (P < 0.05). In addition, high plasma exosomal circSLC26A4 expression was positively associated with lymph node metastasis and FIGO stage (all P < 0.05). However, no significant correlation was found between plasma exosomal circSLC26A4 expression and age, intravascular cancerous embolus, and perineural invasion (P > 0.05). The high exosomal circSLC26A4 expression is closely related to the occurrence of CC. Plasma exosomal circSLC26A4 can be used as a diagnostic marker for CC.

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater

The comparison of decay rates of infectious SARS-CoV-2 and viral RNA in environmental waters and wastewater

Small non-coding RNA signatures in atrial appendages of patients with atrial fibrillation.

The development of high-throughput technologies has enhanced our understanding of small non-coding RNAs (sncRNAs) and their crucial roles in various diseases, including atrial fibrillation (AF). This study aimed to systematically delineate sncRNA profiles in AF patients. PANDORA-sequencing was used to examine the sncRNA profiles of atrial appendage tissues from AF and non-AF patients. Differentially expressed sncRNAs were identified using the R package DEGseq 2 with a fold change >2 and p < 0.05. The target genes of the differentially expressed sncRNAs were predicted using MiRanda and RNAhybrid. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. In AF patients, the most abundant sncRNAs were ribosomal RNA-derived small RNAs (rsRNAs), followed by transfer RNA-derived small RNAs (tsRNAs), and microRNAs (miRNAs). Compared with non-AF patients, 656 rsRNAs, 45 miRNAs, 191 tsRNAs and 51 small nucleolar RNAs (snoRNAs) were differentially expressed in AF patients, whereas no significantly differentially expressed piwi-interacting RNAs were identified. Two out of three tsRNAs were confirmed to be upregulated in AF patients by quantitative reverse transcriptase polymerase chain reaction, and higher plasma levels of tsRNA 5006c-LysCTT were associated with a 2.55-fold increased risk of all-cause death in AF patients (hazard ratio: 2.55; 95% confidence interval, 1.56-4.17; p < 0.001). Combined with our previous transcriptome sequencing results, 32 miRNA, 31 snoRNA, 110 nucleus-encoded tsRNA, and 33 mitochondria-encoded tsRNA target genes were dysregulated in AF patients. GO and KEGG analyses revealed enrichment of differentially expressed sncRNA target genes in AF-related pathways, including the 'calcium signaling pathway' and 'adrenergic signaling in cardiomyocytes.' The dysregulated sncRNA profiles in AF patients suggest their potential regulatory roles in AF pathogenesis. Further research is needed to investigate the specific mechanisms of sncRNAs in the development of AF and to explore potential biomarkers for AF treatment and prognosis.

Gastric juice piR-015551: A promising prognostic biomarker for gastric cancer.

e16074 Background: Emerging reports demonstrated that PIWI-interacting RNAs (piRNAs) played an indispensable role in tumorigenesis. However, it still remains elusive whether piR-015551 in gastric juice specific in stomach could be employed as a biomarker for gastric cancer (GC). The present work is aiming at exploring the possibility of piR-015551 in gastric juice as a potential marker to judge for diagnosis and prognosis of gastric cancer. Methods: Gastric juice was collected from 66 GC patients and 66 healthy individuals. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was employed to measure the levels of piR-015551 expression. Then, the pattern of piR-015551 expression in gastric juice was determined between GC patients and healthy individuals. A receiver operating characteristic (ROC) curve was constructed for distinguishing GC from healthy individuals. Results: Gastric juice piR-015551 levels in GC were higher than those of controls (P &lt; 0.0001). The value of area under ROC (AUC) was 0.885 (sensitivity, 90.9%; specificity, 74.2%; 95% confidence interval, 0.8286 to 0.9414). High gastric juice piR-015551 expression was signally correlated with tumor size (P = 0.013) and TNM stage (P = 0.001). GC patients with high piR-015551 expression in gastric juice exerted a poorer overall survival (OS) (P = 0.0152) and progression-free survival (PFS) (P = 0.013). COX regression analysis verified that gastric juice piR-015551 expression was an independent prognostic risk variable for OS (P &lt; 0.05). Conclusions: The current study suggested that piR-015551 in gastric juice had the potential to be a useful biomarker for GC detection and prognosis prediction.

Improving Intestinal Barrier Function in Sepsis by Partially Hydrolysed Guar Gum via the Suppression of the NF-κB/MLCK Pathway.

Partially hydrolyzed guar gum (PHGG) protects against intestinal barrier dysfunction and can ameliorate some intestinal diseases. However, whether PHGG has a role in protecting intestinal barrier function (IBF) during sepsis remains unclear. This study aimed to investigate the role and probable mechanism of PHGG in the intestinal mucosa in sepsis. A rat sepsis model was constructed using cecal ligation and puncture (CLP). FITC-dextran 4 (FD-4) flux, serum inflammatory mediator levels, tight junction (TJ) levels, jejunum mucosa pathology, and epithelial intercellular junction ultrastructure were monitored to evaluate the effect of PHGG on IBF. Caco-2 monolayers were used to study the impact and mechanism of PHGG on lipopolysaccharide (LPS)-induced barrier dysfunction in vitro. The expression of zonula occludens protein-1 and occludin and the location of P65 were studied by immunofluorescence. Nuclear factor kappa B (NF-κB) and myosin light chain kinase 3 (MLCK) pathway-related protein expression was verified by quantitative reverse transcriptase polymerase chain reaction or western blotting. The results indicated that the jejunal mucosa structure was destroyed, the villi were disrupted and shortened, and neutrophil infiltration was evident in the septic rats. Compared to Sham group, spetic rats had increased Chiu's score, serum inflammatory mediator levels, and FD-4 flux but decreased TJ and gap junction density. In addition, the expression of MLCK, p-MLC, and TJ proteins and the expression of P65 in the nucleus were increased in septic rats. Furthermore, compared to those in the Control group, LPS-treated Caco-2 cells showed lower cell viability and transepithelial electrical resistance, while had higher FD-4 flux and the expression of MLCK, p-MLC, TJ proteins and P65 in the nucleus. PHGG pretreatment reversed the above effects induced by CLP or LPS treatment. Moreover, SN50, an NF-κB inhibitor, attenuated the above effects of LPS on Caco-2 cells. Overall, PHGG reduced inflammation, increased TJ protein expression and localization, and relieved damage to the TJ structure and intestinal permeability through suppression of the NF-κB/MLCK pathway. This study provides new insights into the role of PHGG in sepsis therapy.

The Antihypertensive Effects and Mechanisms of Huotan Jiedu Tongluo Decoction in Rats with H-Type Hypertension.

H-type hypertension, which is a specific form of hypertension characterized by elevated plasma homocysteine (Hcy) levels, has become a major public health challenge worldwide. This study investigated the hypotensive effects and underlying mechanisms of Huotan Jiedu Tongluo decoction (HTJDTLD), a highly effective traditional Chinese medicine formula commonly used to treat vascular stenosis. Methionine was used to induce H-type hypertension in rats, and HTJDTLD was administered intragastrically. Then, the systolic and diastolic blood pressures of the caudal artery of rats were measured by noninvasive rat caudal manometry. Histological assessment of the aorta was performed by hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure Hcy levels, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting were used to determine the mRNA and protein levels of Glucose regulatory protein 78 (GRP78), Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), c-Jun N-terminalkinases (JNK), and caspase-3. The results showed that HTJDTLD significantly lowered blood pressure, alleviated histopathological lesions, and decreased Hcy levels after methionine treatment. Moreover, HTJDTLD significantly inhibited the gene and protein expression of GRP78, JNK, TRAF2, and caspase 3, which are involved mainly in the endoplasmic reticulum (ER) stress-induced apoptosis pathway. Overall, the results indicated that HTJDTLD had effective antihypertensive effects in rats with H-type hypertension and revealed the antihypertensive mechanisms associated with inhibition of ER stress-induced apoptosis pathway activation.

Identification of cell-specific epigenetic patterns associated with chondroitin sulfate treatment response in an endemic arthritis, Kashin-Beck disease.

To assess the alterations in cell-specific DNA methylation associated with chondroitin sulphate response using peripheral blood collected from Kashin-Beck disease (KBD) patients before initiation of chondroitin sulphate treatment. Peripheral blood samples were collected from KBD patients at baseline of chondroitin sulphate treatment. Methylation profiles were generated using reduced representation bisulphite sequencing (RRBS) from peripheral blood. Differentially methylated regions (DMRs) were identified using MethylKit, while DMR-related genes were defined as those annotated to the gene body or 2.2-kilobase upstream regions of DMRs. Selected DMR-related genes were further validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to assess expression levels. Tensor composition analysis was performed to identify cell-specific differential DNA methylation from bulk tissue. This study revealed 21,060 hypermethylated and 44,472 hypomethylated DMRs, and 13,194 hypermethylated and 22,448 hypomethylated CpG islands for differential global methylation for chondroitin sulphate treatment response. A total of 12,666 DMR-related genes containing DMRs were identified in their promoter regions, such as CHL1 (false discovery rate (FDR) = 2.11 × 10-11), RIC8A (FDR = 7.05 × 10-4), and SOX12 (FDR = 1.43 × 10-3). Additionally, RIC8A and CHL1 were hypermethylated in responders, while SOX12 was hypomethylated in responders, all showing decreased gene expression. The patterns of cell-specific differential global methylation associated with chondroitin sulphate response were observed. Specifically, we found that DMRs located in TESPA1 and ATP11A exhibited differential DNA methylation between responders and non-responders in granulocytes, monocytes, and B cells. Our study identified cell-specific changes in DNA methylation associated with chondroitin sulphate response in KBD patients.

Serotonin 2C receptor in a rat model of temporal lobe epilepsy: From brainstem expression to pharmacological blockade in relation to ventilatory function.

Because of its involvement in breathing control and neuronal excitability, dysregulation of the serotonin (5-HT) 2C receptor (5-HT2C) might play a key role in sudden unexpected death in epilepsy. Seizure-induced respiratory arrest is thus prevented by a 5-HT2B/C agonist in different seizure model. However, the specific contribution of 5-HT2C in chronic epilepsy-related respiratory dysfunction remains unknown. In a rat model of temporal lobe epilepsy (EPI rats), in which we previously reported interictal respiratory dysfunctions and a reduction of brainstem 5-HT tone, quantitative reverse transcriptase polymerase chain reaction showed overexpression of TPH2 (5-HT synthesis enzyme), SERT (5-HT reuptake transporter), and 5-HT2C transcript levels in the brainstem of EPI rats, and of RNA-specific adenosine deaminase (ADAR1, ADAR2) involved in the production of 5-HT2C isoforms. Interictal ventilation was assessed with whole-body plethysmography before and 2 h after administration of SB242084 (2 mg/kg), a specific antagonist of 5-HT2C. As expected, SB242084 administration induced a progressive decrease in ventilatory parameters and an alteration of breathing stability in both control and EPI rats. However, the size of the SB242084 effect was lower in EPI rats than in controls. Increased 5-HT2C gene expression in the brainstem of EPI rats could be part of a compensatory mechanism against epilepsy-related low 5-HT tone and expression of 5-HT2C isoforms for which 5-HT affinity might be lower.

Pleurotus sajor-caju (Fr.) Singer β-1,3-Glucanoligosaccharide (Ps-GOS) Suppresses RANKL-Induced Osteoclast Differentiation and Function in Pre-Osteoclastic RAW 264.7 Cells by Inhibiting the RANK/NFκB/cFOS/NFATc1 Signalling Pathway.

Edible grey oyster mushroom, Pleurotus sajor-caju, β (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti-microorganism and antioxidant. However, its biological activity is limited by low water solubility resulting from its high molecular weight. Our previous study demonstrated that enzymatic hydrolysis of grey oyster mushroom β-glucan using Hevea β-1,3-glucanase isozymes obtains a lower molecular weight and higher water solubility, Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS). Additionally, Ps-GOS potentially reduces osteoporosis by enhancing osteoblast-bone formation, whereas its effect on osteoclast-bone resorption remains unknown. Therefore, our study investigated the modulatory activities and underlying mechanism of Ps-GOS on Receptor activator of nuclear factor kappa-Β ligand (RANKL) -induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and its effect on osteoclast differentiation was determined by tartrate-resistant acid phosphatase (TRAP) staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by pit formation assay. The osteoclastogenic-related factors were assessed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), Western blot and immunofluorescence. The results revealed that Ps-GOS was non-toxic and significantly suppressed the formation of mature osteoclast multinucleated cells and their resorption activity by reducing the number of TRAP-positive cells and pit formation areas in a dose-dependent manner. Additionally, Ps-GOS attenuated the nuclear factor kappa light chain-enhancer of activated B cells' P65 (NFκB-P65) expression and their subsequent master osteoclast modulators, including nuclear factor of activated T cell c1 (NFATc1) and Fos proto-oncogene (cFOS) via the NF-κB pathway. Furthermore, Ps-GOS markedly inhibited RANK expression, which serves as an initial transmitter of many osteoclastogenesis-related cascades and inhibited proteolytic enzymes, including TRAP, matrix metallopeptidase 9 (MMP-9) and cathepsin K (CTK). These findings indicate that Ps-GOS could potentially be beneficial as an effective natural agent for bone metabolic disease.

Hypoxia and Giardia duodenalis: The Role of Hypoxia-Inducible Factor in Altered Epithelial Glucose Metabolism

The gastrointestinal system experiences frequent oxygenic fluctuations and must be able to maintain barrier integrity during periods of suboptimal oxygen concentration, or hypoxia. Epithelial cells rely on the Hypoxia-Inducible Factor (HIF) complex to activate genes that combat cellular stress and adapt cellular metabolism during hypoxia. Importantly, protozoan parasites can modulate host tissue oxygen tension and HIF activation, yet little is known regarding the relationship between enteric protozoa and hypoxia. This research aims to uncover the role of HIF upon Giardia duodenalis infection, a top cause of global diarrheal disease and an excellent infection model for the study of GI physiology. We hypothesize that HIF-target genes are activated upon Giardia infection, promoting alternative cellular glucose metabolism to sustain bioenergetic homeostasis. Caco-2 colonic epithelial cells were infected with Giardia isolate GS/M (MOI 10) for 1.5 or 4.5 hours under normoxic (21% O2) or hypoxic (~1%O2, STEMCELL hypoxia incubator) conditions to capture the early or peak activation of the oxygen-dependent HIF subunit (HIF-1α). RNA was extracted for assessment of transcriptional alterations of HIF-target genes via reverse transcriptase quantitative polymerase chain reaction. Investigation of HIF-mediated intracellular metabolic changes was carried out via liquid-chromatography mass-spectrometry analysis (hydrophilic-interaction chromatography method) on cocultures supplemented with a hypoxia mimetic (DMOG) or a HIF inhibitor (PX-478). Metabolomics experiments were repeated using Caco-2 cells transfected with sodium-dependent glucose cotransporter 1 (SGLT1), a key glucose transporter in the small intestine where Giardia localizes which is not reliably expressed in Caco-2 cells. Under normoxic conditions, genes that aid in cell stress responses ( VEGFA, ANKRD37, GADD45A) and glycolysis ( HK2, LDHA) are upregulated in Giardia-infected cells in a time-dependent manner (p&lt;0.05). Fewer HIF-target genes are upregulated under hypoxic conditions (e.g., VEGFA, GADD45A, PGK1; p&lt;0.05), indicating Giardia-infected cells exhibit a transcriptional profile similar to hypoxic uninfected cells. Interestingly, HIF1α was upregulated in Giardia-infected cells under hypoxic conditions at 1.5 hours (p&lt;0.05). Analysis of the Caco-2 intracellular metabolome indicated HIF-dependent changes to nucleic acid (e.g., guanosine, inosine, uracil) and amino acid (e.g., glycine, threonine) metabolism. DMOG treatment increased the abundance of glycolytic intermediates (e.g., PEP, DHAP) in both cell lines and inhibited the depletion of the Kreb’s Cycle intermediate aconitate in the Caco-2-SGLT1 transfected cells, confirming the promotion of glycolytic flux by HIF. Taken together, our findings indicate Giardia-infected cells illustrate a hypoxic signature, a novel metabolic cell rescue mechanism in response to enteropathogens. Elucidating the role of HIF during enteric parasitic infections will aid in our understanding of the cellular adaptations underpinning GI pathophysiology, while also shedding light on the potential of HIF as a therapeutic target. Natural Science and Engineering Research Council of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Whole-Transcriptome Sequencing of Knee Joint Cartilage from Kashin-Beck Disease and Osteoarthritis Patients.

The aim of this study was to provide a comprehensive understanding of similarities and differences in mRNAs, lncRNAs, and circRNAs within cartilage for Kashin-Beck disease (KBD) compared to osteoarthritis (OA). We conducted a comparison of the expression profiles of mRNAs, lncRNAs, and circRNAs via whole-transcriptome sequencing in eight KBD and ten OA individuals. To facilitate functional annotation-enriched analysis for differentially expressed (DE) genes, DE lncRNAs, and DE circRNAs, we employed bioinformatic analysis utilizing Gene Ontology (GO) and KEGG. Additionally, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), we validated the expression levels of four cartilage-related genes in chondrocytes. We identified a total of 43 DE mRNAs, 1451 DE lncRNAs, and 305 DE circRNAs in KBD cartilage tissue compared to OA (q value < 0.05; |log2FC| > 1). We also performed competing endogenous RNA network analysis, which identified a total of 65 lncRNA-mRNA interactions and 4714 miRNA-circRNA interactions. In particular, we observed that circRNA12218 had binding sites for three miRNAs targeting ACAN, while circRNA12487 had binding sites for seven miRNAs targeting COL2A1. Our results add a novel set of genes and non-coding RNAs that could potentially serve as candidate diagnostic biomarkers or therapeutic targets for KBD patients.

Protective effect of quercetin on lipopolysaccharide‑induced miscarriage based on animal experiments and network pharmacology.

Spontaneous abortion (SA) occurs in woman of child‑bearing age, jeopardizing their physical and mental health. Quercetin is a natural flavonoid, which exhibits a variety of pharmacological activities. However, the role and mechanisms of quercetin in SA still need to be further explored. Animal experiments were performed to examine the effect of quercetin in treating SA. Institute of Cancer Research mice were injected with lipopolysaccharide into the tail vein on the 7th day of gestation to establish a SA model. Gavage was performed during days 3‑8 of gestation with high‑, medium‑ and low‑dose of quercetin. Then the effect of quercetin on embryos was evaluated. Animal experiment showed that quercetin could remarkably reduce the embryo loss rate and increase the mean weight of surviving embryos to some degree. Furthermore, network pharmacology was employed to explore the underlying mechanisms of quercetin in the treatment of SA. Several databases were used to collect the targets of SA and quercetin. Protein‑protein interaction network, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed to elucidate the interactions between SA and quercetin. The relative mRNA expressions of several targets in uterine were detected by quantitative reverse transcriptase polymerase chain reaction (RT‑qPCR). Network pharmacology indicated that the effects of quercetin in treating SA were mainly related to hormone response and the modulation of defense response and inflammatory response, involving signaling pathways such as PI3K‑Akt, VEGF, MAPK and core targets such as AKT1, albumin, caspase‑3. RT‑qPCR showed that quercetin could up‑regulate AKT1, MAPK1, PGR, SGK1 and down‑regulate ESR1, MAPK3. The results showed that quercetin may modulate multiple signaling pathways by targeting core targets to prevent and treat SA.