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Background: Atrial fibrillation (AF) stands as a prevalent and detrimental arrhythmic disorder, characterized by intricate pathophysiological mechanisms. The availability of reliable and reproducible AF models is pivotal in unraveling the underlying mechanisms of this complex condition. Unfortunately, the researchers are still confronted with the absence of consistent in vitro AF models, hindering progress in this crucial area of research. Methods: Human induced pluripotent stem cells derived atrial myocytes (hiPSC-AMs) were generated based on the GiWi methods and were verified by whole-cell patch clamp, immunofluorescent staining, and flow cytometry. Then hiPSC-AMs were employed to establish the AF model by HS. Whole-cell patch clamp technique and calcium imaging were used to identify the AF model. The stability of 29 reference genes was evaluated using delta-Ct, GeNorm, NormFinder, and BestKeeper algorithms; Results: HiPSC-AMs displayed atrial myocyte action potentials and expressed the atrial-specific protein MLC-2 A and NR2F2, about 70% of the cardiomyocytes were MLC-2 A positive. After HS, hiPSC-AMs showed a significant increase in beating frequency, a shortened action potential duration, and increased calcium transient frequency. Of the 29 candidate genes, the top five most stably ranked genes were ABL1, RPL37A, POP4, RPL30, and EIF2B1. After normalization using ABL1, KCNJ2 was significantly upregulated in the AF model; Conclusions: In the hiPSC-AMs AF model established by HS, ABL1 provides greater normalization efficiency than commonly used GAPDH. [ABSTRACT FROM AUTHOR]

Abstract Background Atrial fibrillation (AF) stands as a prevalent and detrimental arrhythmic disorder, characterized by intricate pathophysiological mechanisms. The availability of reliable and reproducible AF models is pivotal in unraveling the underlying mechanisms of this complex condition. Unfortunately, the researchers are still confronted with the absence of consistent in vitro AF models, hindering progress in this crucial area of research. Methods Human induced pluripotent stem cells derived atrial myocytes (hiPSC-AMs) were generated based on the GiWi methods and were verified by whole-cell patch clamp, immunofluorescent staining, and flow cytometry. Then hiPSC-AMs were employed to establish the AF model by HS. Whole-cell patch clamp technique and calcium imaging were used to identify the AF model. The stability of 29 reference genes was evaluated using delta-Ct, GeNorm, NormFinder, and BestKeeper algorithms; Results HiPSC-AMs displayed atrial myocyte action potentials and expressed the atrial-specific protein MLC-2 A and NR2F2, about 70% of the cardiomyocytes were MLC-2 A positive. After HS, hiPSC-AMs showed a significant increase in beating frequency, a shortened action potential duration, and increased calcium transient frequency. Of the 29 candidate genes, the top five most stably ranked genes were ABL1, RPL37A, POP4, RPL30, and EIF2B1. After normalization using ABL1, KCNJ2 was significantly upregulated in the AF model; Conclusions: In the hiPSC-AMs AF model established by HS, ABL1 provides greater normalization efficiency than commonly used GAPDH.

Introduction: Tongue squamous cell carcinoma (TSCC) is a common malignant tumour type with aggressive invasion and a poor prognosis. To date, invasion-related gene expression signatures for the prognostic stratification of TSCC patients are unavailable in clinical practice. This study aimed to assess the impact of invasion-related genes on the prognosis of TSCC patients. Methods: We obtained mRNA profiles and clinical data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases (TCGA-TSCC and GSE41116, respectively). The TSCC samples from the TCGA-TSCC cohort were randomly divided into TCGA training and TCGA test datasets at a 7:3 ratio. Next, a disease-free survival (DFS) prognostic risk model was established on the basis of univariate and stepwise multivariate Cox regression analyses of the TCGA training cohort. Moreover, prognostic genes were screened. The model was subsequently evaluated and validated using the TCGA test and GSE41116 datasets. In addition, the prognostic genes were validated in the human TSCC cell line UM1 and the human oral keratinocyte (HOK) cell line using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Results: A total of 70 candidate genes related to invasion were identified in the TCGA-TSCC cohort. DFS data were subsequently constructed, and 6 prognostic genes, HMGN2, MYL12B, ACTB, PPP1CA, PSMB9, and IFITM3, were identified. The TSCC samples were divided into high- and low-risk groups in the TCGA training, TCGA test, and GSE41116 cohorts, respectively. In particular, patients with TSCC in the low-risk group had longer DFS than those in the high-risk group. Furthermore, qRT-PCR analysis confirmed that the expression levels of the 6 prognostic genes were significantly greater in the TSCC cell line UM1 than in the HOK cell line. Conclusion: This study identified new invasion-related target genes related to poor prognosis in TSCC patients, providing new insights into the underlying mechanisms of TSCC invasion. [ABSTRACT FROM AUTHOR]

Methylene blue (MB) has been shown to reduce mortality and morbidity in vasoplegic patients after cardiac surgery. Though MB is considered to be safe, extravasation of MB leading to cutaneous toxicity has been reported. In this study, we sought to characterize MB-induced cutaneous toxicity and investigate the underlying mechanisms. To induce MB-induced cutaneous toxicity, we injected 64 adult male Sprague-Dawley rates with 200 µL saline (vehicle) or 1%, 0.1%, or 0.01% MB in the plantar hind paws. Paw swelling, skin histologic changes, and heat and mechanical hyperalgesia were measured. Injection of 1%, but not 0.1% or 0.01% MB, produced significant paw swelling compared to saline. Injection of 1% MB produced heat hyperalgesia but not mechanical hyperalgesia. Pain behaviors were unchanged following injections of 0.1% or 0.01% MB. Global transcriptomic analysis by RNAseq identified 117 differentially expressed genes (111 upregulated, 6 downregulated). Ingenuity Pathway Analysis showed an increased quantity of leukocytes, increased lipids, and decreased apoptosis of myeloid cells and phagocytes with activation of IL-1β and Fos as the two major regulatory hubs. qPCR showed a 16-fold increase in IL-6 mRNA. Thus, using a novel rat model of MB-induced cutaneous toxicity, we show that infiltration of 1% MB into cutaneous tissue causes a dose-dependent pro-inflammatory response, highlighting potential roles of IL-6, IL-1β, and Fos. Thus, anesthesiologists should administer dilute MB intravenously through peripheral venous catheters. Higher concentrations of MB (1%) should be administered through a central venous catheter to minimize the risk of cutaneous toxicity. [ABSTRACT FROM AUTHOR]

Introduction: During normal aging, telomeric DNA is gradually lost in dividing somatic cells, and critically short telomeres lead to replicative senescence, apoptosis, or chromosomal instability. We studied telomere length in bone marrow failure syndromes (BMFS) compared to normal healthy population. Methods: Peripheral blood was collected from the participants, and genomic DNA was extracted. Relative telomere length was measured using a quantitative polymerase chain reaction. Statistical analysis was performed using SPSS and GraphPad Prism 8.2 software. Results: The median age of normal Indian population was 31 (0–60) years. As expected, telomere length (TL) showed a decline with age and no difference in TL between males and females. The median age of 650 patients with aplastic anemia (AA) was 30 (1–60) years. TL was significantly shorter in patients with AA compared to healthy controls (p <.001). In FA and MDS patients, TL was significantly shorter than age‐matched healthy controls (p =.028; p <.001), respectively. There was no difference between the median TL in age‐matched AA and FA patients (p =.727). However, patients with MDS had shorter TL than age‐matched AA (p =.031). Conclusion: TL in BMF syndrome patients was significantly shorter than age‐matched healthy controls. [ABSTRACT FROM AUTHOR]

Background & Objective: This study aimed to determine the incidence of microRNA (miRNA; miR-1290) in the serum of oral squamous cell carcinoma (OSCC) patients compared to a control group using the qualitative real-time polymerase chain reaction (PCR) method. Methods: Blood serum samples were obtained from patients diagnosed with OSCC and confirmed through biopsy. The samples were collected from patients referred to the Mashhad Dental Faculty and Ghaem Hospital. The OSCC group consisted of 17 patients, while the healthy group included 15 individuals. RNA was extracted from the patient samples, and samples with an A260/280 ratio between 1.8 and 2.0 (indicating acceptable RNA quality) were immediately converted into complementary DNA (cDNA) using albumin and cDNA reference genes. The SYBR green real-time reverse transcriptase PCR method was used to measure the presence of miR-1290 in the blood samples. Results: A total of 32 patients were examined in this study, including 17 women (53.1%) and 15 men (46.9%). The mean age was 46.7 years in the healthy group and 54.6 years in the SCC group, indicating a significant difference (P<0.05). The expression level of the miR-1290 gene was higher in patients with SCC compared to the healthy group (P=0.000). While the expression level of miR-1290 was higher in grade 3 and advanced stage than in grades 2 and 1 and early stage, the differences were not statistically significant (P=0.173 and P=0.564 for grade and stage, respectively). Conclusion: The expression level of miR-1290 may increase in SCC patients compared to healthy individuals, making it a potential circulating biomarker. Further investigations for diagnostic utility would be warranted. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan–Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma. [ABSTRACT FROM AUTHOR]

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin–eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD. [ABSTRACT FROM AUTHOR]

This study used a correlative multianalytical approach to investigate the bacteriologic conditions in the apical root canal system of treated teeth with or without apical periodontitis and their correlation with the technical quality of the previous root canal obturation and the presence and volume of apical periodontitis lesions. Root apexes were obtained from recently extracted root canal–treated teeth with (n = 23) and without (n = 22) apical periodontitis lesions as demonstrated by cone-beam computed tomographic examination. The root apexes were sectioned and subjected to micro–computed tomographic (micro-CT) scanning. The specimens were cryopulverized, and DNA extracted from the powder was used as a template in real-time polymerase chain reaction assays to quantify total bacteria and members of the Streptococcus genus and Actinobacteria phylum. The bacteriologic findings were compared between the 2 groups and also evaluated for associations with cone-beam computed tomographic and micro–computed tomographic data. Bacteria were detected in all apical canal samples except 1. The mean counts of total bacteria, streptococci, and actinobacteria did not differ significantly between teeth with or without apical periodontitis (P >.05). Streptococcus levels were significantly lower by 80% in the apical canals of teeth with small lesions compared with those without lesions (P <.05). The limit of filling >2 mm short was significantly associated with more total bacterial counts compared with canals filled 0–2 mm short (P <.05). An adequate coronal restoration was significantly associated with lesser counts of Streptococcus (P <.05). Comparable bacterial loads were observed in the apical canal system of treated teeth with and without apical periodontitis, suggesting that factors other than only the total bacterial levels may also influence the development and progression of apical periodontitis. Bacteria were found in the apical canal in virtually all cases with a high prevalence of streptococci and actinobacteria. Streptococci counts were significantly higher in the apical canal of teeth with inadequate restorations and teeth with no lesions. Underfilled canals showed higher bacterial counts. [ABSTRACT FROM AUTHOR]

Aims: Osteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis. Methods: Blood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action. Results: In the LPS-induced bone loss mouse model, the levels of IL-19 in peripheral blood serum and femoral bone marrow suspension were significantly increased. The in vivo results indicated that global IL-19 deletion had no significant effect on RANKL content in the serum and bone marrow, but could increase the content of OPG in serum and femoral bone marrow, suggesting that IL-19 inhibits OPG expression in bone marrow mesenchymal stem cells (BMSCs) and thus increases bone resorption. Conclusion: IL-19 promotes bone resorption by suppressing OPG expression in BMSCs in a LPS-induced bone loss mouse model, which highlights the potential benefits and side effects of IL-19 for future clinical applications. Cite this article: Bone Joint Res 2023;12(11):691–701.

Background Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. Methods A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. Results The parasite load at disease presentation was 13 461.00 (2560.00–37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1–1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1–1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. Conclusion In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment. [ABSTRACT FROM AUTHOR]

Abstract Environmental DNA (eDNA) is revolutionizing species monitoring in nature. At the heart of any eDNA approach is the reliance upon sufficient DNA sequence information to satisfy the demands of eDNA assay specificity and sensitivity. The most common source of this information has been restricted to short barcoding regions of the mitochondrial genome (mitogenome) and marker genes. The use of these limited regions for assay design has often resulted in substantial trade‐offs in assay performance. With increased accessibility of full mitogenome assemblies, the potential for designing more robust eDNA assays is considerably enhanced. However, this also poses a new challenge to effectively identify suitable regions for assay design using considerably larger sequences. We present unikseq, a utility that uses words of length k (k‐mers) to identify unique regions in a reference sequence relative to tolerated (ingroup) and not‐tolerated (outgroup or non‐target) sequence sets, quickly and with low memory that can yield highly specific assays. We illustrate its application within an assay development workflow through use‐case examples for the design and validation of four quantitative real‐time polymerase chain reaction (qPCR)‐based assays selective for American bullfrog (Rana [Lithobates] catesbeiana), Burbot (Lota lota), Lake trout (Salvelinus namaycush), and Quillback rockfish (Sebastes maliger). The chosen target species vary in range, habitat, and degree of relatedness to their sympatric species that, consequently, impact eDNA assay design difficulty. We demonstrate the effectiveness of unikseq through assay validation and characterization using DNA from voucher specimens, synthetic DNA, and, where possible, field samples, to verify the specificity and sensitivity of the newly designed assays. By facilitating whole mitogenome sequence comparison, the creation of high‐performing eDNA assays is substantially enhanced. Having several adjustable parameters for specifying user requirements within unikseq, this approach can facilitate the identification of suitable regions for a broad range of applications requiring nucleotide sequence comparisons.

Long noncoding RNAs (lncRNAs) plays important role in disease spread and its invasion. Overexpression of prostate cancer antigen 3 gene (PCA3gene) is reported in prostate cancer. To analyze the PCA3 lncRNA expression in chronic myeloid leukemia (CML) patients.

Objective: To establish a rapid detection method for viable Penicillium expansum by propidium monoazide (PMA) combined with quantitative real-time polymerase chain reaction (qPCR). Methods: PMA-qPCR detection method for viable P. expansum was established, including the optimization of the treatment concentration, dark incubation and exposure time of PMA, the screening of specific primers of P. expansum, and the conduction of qPCR. In addition, the standard curve was constructed, which was applied to the detection of artificially contaminated apples samples. The reliability of this method was also evaluated by comparing with the plate counting method. Results: The optimal PMA treatment conditions were: 10 µg/mL for PMA concentration, 5 min for the dark incubation and 10 min for the exposure time. Among the 4 pairs of primers, Pexp-patF showed strong specificity for P. expansum, which could be used as a optimal primer for PMA-qPCR detection. The correlation coefficient of the established quantitative standard curve was 0.9948, and the detection limit of the method was 102.6 CFU/mL. There was no obvious difference between the detection result of this method and the plate counting method, and the viable P. expansum could be detected in the non-rotted part of apples. Conclusion: The established PMA-qPCR technique could be applied to the detection of P. expansum in apples, which would provide technical support for the prevention and control of P. expansum.

Summary: Background: The Wnt pathway is aberrantly activated in chronic lymphocytic leukemia (CLL) and contributes to the antiapoptotic and mitogenic characteristics of CLL cells. Lymphoid enhancer-binding factor‑1 (LEF1) acts as a mediator and key transcription factor of the Wnt/β-catenin pathway. LEF1 helps to regulate important genes involved in tumor cell death mechanisms. Objective: To analyze the expression levels of Wnt signaling pathway member (WNT3) and its key mediator LEF1 in Egyptian CLL patients and to detect the potential use of these genes as markers of CLL outcome. Methods: We quantified the expression levels of Wnt3 and LEF1 in peripheral blood mononuclear cells of 30 untreated CLL and 19 healthy controls by qRT-PCR (quantitative real time polymerase chain reaction). Results: Our study demonstrated significant upregulation of both Wnt3 and LEF1 in CLL (P < 0.0001). WNT3 and LEF1 were significantly decreased in CLL patients with ECOG performance 2 and 3 than those with 0 and 1 (p = 0.023 and 0.007, respectively). CLL patients with 17p deletion express significantly low LEF1 (p = 0.033). Low levels of WNT3 and LEF1 expression indicated a shorter overall survival (P = 0.007, 0.005, respectively). The predictive power of WNT3 and LEF1 expression showed good discrimination of CLL patients from controls (AUC >0.9). Conclusion: Upregulation of WNT3 and LEF1 could be used as helpful and specific markers to distinguish our CLL patients. Low WNT3 and LEF1 expression is associated with shortened survival in CLL patients. These results indicate that WNT3 and LEF1 represent an attractive therapeutic target for future therapies in Egyptian CLL patients. [ABSTRACT FROM AUTHOR]

Dysbiosis plays an important role in the development of bacterial infections in the gastric mucosa, particularly Helicobacter pylori. The international guidelines for the treatment of H. pylori infections suggest standard triple therapy (STT). Nevertheless, because of the increasing resistance rates to clarithromycin, metronidazole has been widely considered in several countries. Unfortunately, the non-justified administration of antibiotics induces dysbiosis in the target organ. We characterized the gastric microbiota of patients diagnosed with follicular gastropathy and pangastropathy attributed to H. pylori infection, before and after the administration of STT with metronidazole. Dominant relative abundances of Cutibacterium were observed in pre-treatment patients, whereas H. pylori was observed at <11%, suggesting the multifactor property of the disease. The correlation of Cutibacterium acnes and H. pylori with gastric infectious diseases was also evaluated using quantitative real-time polymerase chain reaction. The dominance of C. acnes over H. pylori was observed in gastritis, gastropathies, and non-significant histological alterations. None of the microorganisms were detected in the intestinal metaplasia. Post-treatment alterations revealed an increase in the relative abundances of Staphylococcus, Pseudomonas, and Klebsiella. Non-H. pylori gastrointestinal bacteria can be associated with the initiation and development of gastric diseases, such as pathobiont C. acnes. [ABSTRACT FROM AUTHOR]

Aims: Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP. Methods: Using quantitative real-time polymerase chain reaction (qRT-PCR), osteogenesis-related genes (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2)), PCBP1-AS1, microRNA (miR)-126-5p, group I Pak family member p21-activated kinase 2 (PAK2), and their relative expression levels were determined. Western blotting was used to examine the expression of PAK2 protein. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. To examine the osteogenic differentiation, Alizarin red along with ALP staining was used. RNA immunoprecipitation assay and bioinformatics analysis, as well as a dual-luciferase reporter, were used to study the association between PCBP1-AS1, PAK2, and miR-126-5p. Results: The expression of PCBP1-AS1 was pre-eminent in OP tissues and decreased throughout the development of human bone marrow-derived mesenchymal stem cells (hBMSCs) into osteoblasts. PCBP1-AS1 knockdown and overexpression respectively promoted and suppressed hBMSC proliferation and osteogenic differentiation capacity. Mechanistically, PCBP1-AS1 sponged miR-126-5p and consequently targeted PAK2. Inhibiting miR-126-5p significantly counteracted the beneficial effects of PCBP1-AS1 or PAK2 knockdown on hBMSCs’ ability to differentiate into osteoblasts. Conclusion: PCBP1-AS1 is responsible for the development of OP and promotes its progression by inducing PAK2 expression via competitively binding to miR-126-5p. PCBP1-AS1 may therefore be a new therapeutic target for OP patients. Cite this article: Bone Joint Res 2023;12(6):375–386.

Abstract Background Streptococcus, Bifidobacteria, Lactobacillus and Actinomyces are acidogenic aciduria that may be associated with root caries (RC). The aim of the study was to analyze Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Bifidobacterium spp., Lactobacillus spp. and Actinomyces naeslundii (A. naeslundii) in the saliva of nursing home elderly, to assess the correlation between bacterial composition and RC for five putative catiogenic organisms. Methods In this study, we collected 43 saliva samples and divided into two groups: the root caries group (RCG, n = 21) and the caries-free group (CFG, n = 22). Bacterial DNA was extracted from the saliva samples. The presence and abundance of the five microorganisms were detected by Quantitative real-time PCR (qPCR). Spearman correlation test was performed to evaluate the relationship between the numbers of root decayed filled surfaces (RDFS) and root caries index (RCI) and salivary levels of the bacteria. Results The salivary levels of S. mutans, S. sobrinus, Bifidobacterium spp. and Lactobacillus spp. were significantly higher in RCG than in CFG (p 0.05). Conclusion S. mutans, S. sobrinus and Bifidobacterium spp. in saliva appear to be associated with RC in the elderly. Taken together, the findings indicate that specific salivary bacteria may be involved in the progression of RC.

Ferroptosis plays crucial roles in the pathology of spinal cord injury (SCI). The purpose of this study was to identify differentially expressed ferroptosis-related genes (DE-FRGs) in human acute SCI by bioinformatics analysis and validate the hub DE-FRGs in non-SCI and SCI patients. The GSE151371 dataset was downloaded from the Gene Expression Omnibus and difference analysis was performed. The differentially expressed genes (DEGs) in GSE151371 overlapped with the ferroptosis-related genes (FRGs) obtained from the Ferroptosis Database. A total of 41 DE-FRGs were detected in 38 SCI samples and 10 healthy samples in GSE151371. Then, enrichment analyses of these DE-FRGs were performed for functional annotation. The GO enrichment results showed that upregulated DE-FRGs were mainly associated with reactive oxygen species and redox reactions, and the KEGG enrichment analysis indicated involvement in some diseases and ferroptosis pathways. Protein-protein interaction (PPI) analysis and lncRNA-miRNA-mRNA regulatory network were performed to explore the correlations between genes and regulatory mechanisms. The relationship between DE-FRGs and differentially expressed mitochondria-related genes (DE-MRGs) was also analyzed. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the hub DE-FRGs in clinical blood samples from acute SCI patients and healthy controls. Consistent with the bioinformatics results, qRT-PCR of the clinical samples indicated similar expression levels of TLR4, STAT3, and HMOX1. This study identified DE-FRGs in blood samples from SCI patients, and the results could improve our understanding of the molecular mechanisms of ferroptosis in SCI. These candidate genes and pathways could be therapeutic targets for SCI. [ABSTRACT FROM AUTHOR]

Aims: CRP is an acute-phase protein that is used as a biomarker to follow severity and progression in infectious and inflammatory diseases. Its pathophysiological mechanisms of action are still poorly defined. CRP in its pentameric form exhibits weak anti-inflammatory activity. The monomeric isoform (mCRP) exerts potent proinflammatory properties in chondrocytes, endothelial cells, and leucocytes. No data exist regarding mCRP effects in human intervertebral disc (IVD) cells. This work aimed to verify the pathophysiological relevance of mCRP in the aetiology and/or progression of IVD degeneration. Methods: We investigated the effects of mCRP and the signalling pathways that are involved in cultured human primary annulus fibrosus (AF) cells and in the human nucleus pulposus (NP) immortalized cell line HNPSV-1. We determined messenger RNA (mRNA) and protein levels of relevant factors involved in inflammatory responses, by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. We also studied the presence of mCRP in human AF and NP tissues by immunohistochemistry. Results: We demonstrated that mCRP increases nitric oxide synthase 2 (NOS2), cyclooxygenase 2 (COX2), matrix metalloproteinase 13 (MMP13), vascular cell adhesion molecule 1 (VCAM1), interleukin (IL)-6, IL-8, and Lipocalin 2 (LCN2) expression in human AF and NP cells. We also showed that nuclear factor-κβ (NF-κβ), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphoinositide 3-kinase (PI3K) are at play in the intracellular signalling of mCRP. Finally, we demonstrated the presence of mCRP in human AF and NP tissues. Conclusion: Our results indicate, for the first time, that mCRP can be localized in IVD tissues, where it triggers a proinflammatory and catabolic state in degenerative and healthy IVD cells, and that NF-κβ signalling may be implicated in the mediation of this mCRP-induced state. Cite this article: Bone Joint Res 2023;12(3):189–198.

ObjectiveTo investigate Mycoplasma pneumoniae （MP） infection and macrolide resistance of hospitalized children in Ningbo Area in 2019‒2021.MethodsA total of 6 782 respiratory throat swab specimens were collected from hospitalized pediatric patients with pneumoniae， admitted in Ningbo Women and Children's Hospital from January， 2019 to December 2021. MP and its mutations in 23S rRNA were detected by real-time polymerase chain reaction.ResultsAmong 6 782 respiratory throat swab specimens from 2019‒2021， 1 290 cases （19.02%） were MP positive， and the positive rate decreased year by year （P0.05）， respectively. The difference of positive rate among the different age was significant （P0.05）.ConclusionThe infection rate of MP in children decreases in Ningbo Area from 2019 to 2021. MP infection may be related with gender， seasonal distribution， age， and the resistance rate of MP to macrolide is high.

Background Hospital-acquired infections (HAIs) are increasing due to the spread of multi-drug-resistant organisms. Gut dysbiosis in an intensive care unit (ICU) patients at admission showed an altered abundance of some bacterial genera associated with the occurrence of HAIs and mortality. In the present study, we investigated the pattern of the gut microbiome in ICU patients at admission to correlate it with the development of HAIs during ICU stay. Methods Twenty patients admitted to an ICU with a cross-matched control group of 30 healthy subjects of matched age and sex. Quantitative SYBR green real-time polymerase chain reaction was done for the identification and quantitation of selected bacteria. Results Out of those twenty patients, 35% developed ventilator-associated pneumonia during their ICU stay. Gut microbiome analysis showed a significant decrease in Firmicutes and Firmicutes to Bacteroidetes ratio in ICU patients in comparison to the control and in patients who developed HAIs in comparison to the control group and patients who did not develop HAIs. There was a statistically significant increase in Bacteroides in comparison to the control group. There was a statistically significant decrease in Bifidobacterium and Faecalibacterium prausnitzii and an increase in Lactobacilli in comparison to the control group with a negative correlation between Acute Physiology and Chronic Health Evaluation (APACHE) II score and Firmicutes to Bacteroidetes and Prevotella to Bacteroides ratios. Conclusions Gut dysbiosis of patients at the time of admission highlights the importance of identification of the microbiome of patients admitted at the ICU as a target for preventing of HAIs.

Background: Streptococcus, Bifidobacteria, Lactobacillus and Actinomyces are acidogenic aciduria that may be associated with root caries (RC). The aim of the study was to analyze Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Bifidobacterium spp., Lactobacillus spp. and Actinomyces naeslundii (A. naeslundii) in the saliva of nursing home elderly, to assess the correlation between bacterial composition and RC for five putative catiogenic organisms. Methods: In this study, we collected 43 saliva samples and divided into two groups: the root caries group (RCG, n = 21) and the caries-free group (CFG, n = 22). Bacterial DNA was extracted from the saliva samples. The presence and abundance of the five microorganisms were detected by Quantitative real-time PCR (qPCR). Spearman correlation test was performed to evaluate the relationship between the numbers of root decayed filled surfaces (RDFS) and root caries index (RCI) and salivary levels of the bacteria. Results: The salivary levels of S. mutans, S. sobrinus, Bifidobacterium spp. and Lactobacillus spp. were significantly higher in RCG than in CFG (p < 0.05). RDFS and RCI (RDFS/RCI) were positively associated with salivary levels of S. mutans, S. sobrinus and Bifidobacterium spp. (r = 0.658/0.635, r = 0.465/0.420 and r = 0.407/0.406, respectively). No significant differences in presence and amounts of A. naeslundii was observed between the two groups (p > 0.05). Conclusion: S. mutans, S. sobrinus and Bifidobacterium spp. in saliva appear to be associated with RC in the elderly. Taken together, the findings indicate that specific salivary bacteria may be involved in the progression of RC. [ABSTRACT FROM AUTHOR]

Introduction: Parkinson's disease (PD) is a common adult-onset neurodegenerative disorder caused by a progressive loss of dopaminergic neurons due to the accumulation of α-synuclein in the substantia nigra. Mitochondria are known to play a key role in cell respiratory function and bioenergetics. Indeed, mitochondrial dysfunction causes insufficient energy production required to satisfy the needs of several organs, especially the nervous system. However, the profiling of messenger RNA (mRNA) expression of mitochondrial subunits in PD has not been systematically investigated yet. Material and methods: We explored the mRNA expression of mitochondrial DNA (mtDNA) encoded respiratory chain (RC) subunits in 43 PD patients and 43 normal controls (NC). Next generation sequencing analysis (NGS) was used and quantitative real-time polymerase chain reaction (qRT-PCR) assay was used for confirmation of the NGS results. Results: All tested mitochondrial RC subunits were significantly over-expressed in subjects with PD compared to NC. In qRT-PCR the mean expression of all mitochondrial subunits had an expression level of at least 7 times compared to NC. Conclusions: The over-expression of mitochondrial subunits in PD subjects with respect to NC might be secondary to a degeneration-related alteration of the mitochondrial structure and/or dynamics, or to the occurrence of a compensatory mechanism. The study of specific mRNA by peripheral blood mononuclear cells may provide a further diagnostic frame for early detection PD patients. [ABSTRACT FROM AUTHOR]

Purpose: Haematopoietic stem cell transplant (HSCT) recipients with iatrogenic immunosuppression are highrisk patients for viral infections. The aim of this study was to investigate the incidence of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) infections in HSCT recipients. Materials and Methods: We prospectively monitored 35 patients aged 0-17 years who had allogeneic (n=30) and autologous (n=5) HSCT by quantitative real-time polymerase chain reaction tests for CMV, EBV, and ADV. The monitoring was performed one week before HSCT and weekly for the first 100 days, once a month up to one year after HSCT. In addition, seropositivity for viruses was analysed by Enzyme-Linked Immuno Sorbent Assay a week before transplantation. Results: Before transplantation, all 35 (100%) patients who underwent HSCT were CMV IgG positive, 30 (85.7%-95% CI: 74.1%-97.3%) HSCT recipients were found to be EBV IgG positive. CMV infection was found in 24 (80% - 95% CI: 65.7%-94.3%), ADV infection in 11 (36.7% - 95% CI: 19.4%-53.9%) and EBV infection in 8 (26.7% - 95% CI: 10.8%-42.5%) allogeneic HSCT patients. In this group, CMV DNA viral load in 8 (26.7%) patients, of which one (3.3%) coinfected with EBV DNA and one (3.3%) with ADV DNA, was higher than 1000 copies/mL which was required for pre-emptive treatment. Among 5 autologous HSCT recipients, CMV DNA was detected in 2 patients, EBV DNA in 5 and ADV DNA in 2. Preemptive treatment was given to 11 (%31.4 - 95% CI: 16%-46.8%; 6 CMV, 2 EBV, 1 ADV, 1 CMV-EBV and 1 CMVADV infection) of 35 patients. Thus, the development of viral disease was prevented in 7 (63.6% - 95% CI: 35.2%-92.1%). Of the total 35 patients, only 2 (5.7% - 95% CI: 0.0%-13.4%) died due to viral infection. Conclusion: Early diagnosis of viral infections by prospective monitoring of viral loads in HSCT patients would be effective in preventing morbidity and mortality by ensuring timely initiation of pre-emptive therapy. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan–Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma.

Osteonecrosis of the femoral head (ONFH) is a common disabling disease. Copper has positive effects on cells that regulate bone metabolism. However, the relationship between copper metabolism (CM) and steroid-induced ONFH (SONFH) remains unclear. The GSE123568 dataset was downloaded from the Gene Expression Omnibus. The differentially expressed CM-related SONFH genes (DE-CMR-SONFHGs) were identified via differential analysis and weighted gene coexpression network analysis (WGCNA). Receiver operating characteristic (ROC) analysis was performed for the predictive accuracy of key genes. Targeting drugs and the copper death-related genes (CDRGs) relevant to key genes were investigated. The bioinformatics results were confirmed via quantitative real-time polymerase chain reaction (qRT–PCR) and Western blot (WB) analysis. Two out of 106 DE-CMR-SONFHGs were identified as key genes (PNP and SLC2A1), which had diagnostic value in distinguishing SONFH from control samples and were related to various immune cell infiltrations. Eleven PMP-targeting drugs and five SLC2A1-targeting drugs were identified. The qRT–PCR, as well as WB, results confirmed the downregulation PNP and SLC2A1 and high expression of the CDRGs DLD, PDHB, and MTF1, which are closely related to these two key genes. In conclusion, PNP and SLC2A1 were identified as key genes related to SONFH and may provide insights for SONFH treatment. [ABSTRACT FROM AUTHOR]

Dysbiosis plays an important role in the development of bacterial infections in the gastric mucosa, particularly Helicobacter pylori. The international guidelines for the treatment of H. pylori infections suggest standard triple therapy (STT). Nevertheless, because of the increasing resistance rates to clarithromycin, metronidazole has been widely considered in several countries. Unfortunately, the non-justified administration of antibiotics induces dysbiosis in the target organ. We characterized the gastric microbiota of patients diagnosed with follicular gastropathy and pangastropathy attributed to H. pylori infection, before and after the administration of STT with metronidazole. Dominant relative abundances of Cutibacterium were observed in pre-treatment patients, whereas H. pylori was observed at Cutibacterium acnes and H. pylori with gastric infectious diseases was also evaluated using quantitative real-time polymerase chain reaction. The dominance of C. acnes over H. pylori was observed in gastritis, gastropathies, and non-significant histological alterations. None of the microorganisms were detected in the intestinal metaplasia. Post-treatment alterations revealed an increase in the relative abundances of Staphylococcus, Pseudomonas, and Klebsiella. Non-H. pylori gastrointestinal bacteria can be associated with the initiation and development of gastric diseases, such as pathobiont C. acnes.

Introduction: The SLC25A38 gene produces protein that belongs to the mitochondrial solute carrier family, SLC25. It is implicated in apoptotic pathways, which regulate intrinsic caspase-dependent apoptosis. Aim: To determine level of expression of SLC25A38 gene in patients of Acute Lymphoblastic Leukaemia (ALL). Materials and Methods: A cross-sectional study was done in the Department of Biochemistry, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India, from April 2019 to March 2020. The study included 30 leukaemia patients out of which 25 were adult males and five were adult females and 10 healthy volunteers were included as the control group. Level of expression of SLC25A38 gene normalised to Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH) gene relative to normal healthy volunteers among the ALL patients was measured using quantitative real time polymerase chain reaction. All data collected were analysed using Statistical Package for the Social Sciences (SPSS) version 16.0 software. Results: An average of 5.3 fold expression of SLC25A38 gene normalised to GAPDH relative to normal healthy volunteer among the ALL patients was seen. The fold of expression was determined by 2-∆∆Ct method. There was a positive correlation between blast cell abundance and level of SLC25A38 gene expression (Pearson’s r=0.408, p=0.025). The expression level was found to be associated with the proportion of blast cells in the bone marrow. Conclusion: High expression of SLC25A38 gene is a common feature in ALL and may be a novel biomarker for prognosis and diagnosis, as well as a potential therapeutic target for ALL.

Purpose: The abnormal activation of the sonic hedgehog (SHH) signaling pathway is responsible for the progression of several types of cancers including Gastric Cancer (GC). SHH has been associated with the activation of different signaling pathways. Therefore, in this study, we investigated messenger RNA (mRNA) and protein expression of SHH in gastric malignancies and possible correlation with various clinicopathological parameters. Materials and Methods: A total of 53 surgically resected tumors and adjacent histologically normal tissues from GC patients were investigated in study subjects. A quantitative real-time polymerase chain reaction and immunohistochemistry methods were used for expression analysis of SHH. Results: At mRNA level, SHH was overexpressed in 60% (27/45) of GC cases as compared to their adjacent normal tissues. SHH immunohistochemical analysis revealed abundant cytoplasmic localization and overexpression in 43.39% (23/53) of GC tissues. SHH overexpression was not associated with any of the clinicopathological parameters. Conclusion: Our results showed that SHH is dysregulated in GC and might be considered as a biomarker for GC progression and can be used as a target in cancer therapeutics. [ABSTRACT FROM AUTHOR]

Leaves and aerial parts of Malva neglecta Wallr. have been traditionally used in Anatolia for the treatment of pain, inflammation, hemorrhoids, renal stones, constipation, and infertility. This study investigated the effects of M. neglecta leaves in a rat endometriosis model. The dried plant material was extracted with n-hexane, ethyl acetate, and methanol, successively. Experimental endometriosis was surgically induced in six-week-old female, non-pregnant, Wistar albino rats by autotransplant of endometrial tissue to the abdominal wall. After twenty-eight days, rats were evaluated for a second laparotomy. Endometrial foci areas were assessed, and intraabdominal adhesions were scored. Rats were divided into five groups as control, n-hexane, ethyl acetate, methanol, and aqueous extracts, as well as reference. At the end of the treatment, all rats were sacrificed and endometriotic foci areas and intraabdominal adhesions were re-evaluated and compared with the previous findings. Moreover, peritoneal fluid was collected to detect tumor necrosis factor- α (TNF-α), vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6) levels, and cDNA synthesis, and a quantitative real-time polymerase chain reaction (PCR) test was done. The phytochemical content of the most active extract was determined using High-Performance Liquid Chromatography (HPLC). Both endometrial volume and adhesion score decreased significantly in the group treated with methanol extract. In addition, significant decreases were observed in TNF-α, VEGF, and IL-6 levels in animals administered methanol extract. HPLC results showed that the activity caused by the methanol extract of M. neglecta was due to the polyphenols. Taken together, these novel findings indicate that M. neglecta may be a promising alternative for the treatment of endometriosis. [ABSTRACT FROM AUTHOR]

Aim: The purpose of this study was to compare the antibacterial effects of Allium sativum (garlic extract), calcium hydroxide (Ca [OH]2) and their combination as intracanal medicaments in infected mature anterior teeth using real‐time PCR. Methodology: This prospective double‐blind, controlled, parallel, superiority, randomized clinical trial was carried out on 66 permanent, necrotic incisors associated with asymptomatic apical periodontitis in 66 male patients. Patients were randomly divided into three groups (n = 22) according to the intracanal medications used. After access preparation, four microbiological samples (S) were taken using sterile absorbent paper points as follows: S1: before canal instrumentation and S2: after cleaning and shaping. The third sample (S3) and fourth sample (S4) were taken after the placement of the tested intracanal medications into their corresponding canals for 7 and 14 days, respectively. Total DNA was extracted from microbiological samples and relative quantitative real‐time PCRs were done to quantify the relative gene expression fold change (FC) for Enterococcus faecalis and Streptococcus species. At significance level p ≤.05, the data were statistically analysed in SPSS software using Kruskal–Wallis and Freidman's tests, followed by Dunn–Bonferroni post hoc test for pairwise comparisons. Results: Both bacterial mean FC decreased significantly after mechanical instrumentation (S1 to S2) in all groups. However, no statistically significant differences were found after intracanal medicament placement (from S2 to S3 and from S3 to S4) except in the garlic group. Garlic significantly reduced Enterococcus faecalis FC in S3 and S4 when compared to Ca (OH)2 and Ca (OH)2+ garlic combination. However, garlic and Ca (OH)2 reduced Streptococcus bacteria in S3 similarly. Whilst in S4, garlic showed significantly more reduction than Ca (OH)2. The combination of Ca (OH)2 with garlic extract showed the least significant bacterial reduction. Conclusion: Within the study limitations, garlic intracanal medicament has a comparable anti‐Streptococcus efficiency to Ca (OH)2, whilst it is more effective against Enterococcus faecalis species. When Ca (OH)2 and garlic are combined, their antibacterial effectiveness is reduced. Increasing the time of application for tested intracanal medicaments by more than one week has no additional antibacterial effectiveness. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the significant expression of Peptidyl prolyl cis-trans isomerase (PIN1) as a key regulator of COVID-19 cycle. A quantitative real-time polymerase chain reaction (qRT-PCR) measured the expression levels of PIN1 in the serum of mild and severe patients and evaluated its association with clinical parameters. ROC curve analysis was performed to evaluate the expression of PIN1 for the diagnosis of COVID-19 between mild and severe patients. Expression of the PIN1 gene in severe patients (0.89±0.43) was higher than in mild patients (-2.28±0.34), and this difference was statistically significant between the two groups regardless of other factors (P‑value<0.0001). ROC curve analysis showed that high PIN1 levels in the discrimination of severe from mild patients could be useful. PIN1 expression levels were significantly associated with shortness of breath and cough. PIN1 can be considered an effective factor in the intensification of the symptoms of COVID-19., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Saeideh Gholamzadeh Khoei reports financial support was provided by Qazvin University of Medical Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Objective(s): This study aimed to research the roles of miR-139, miR-221, miR-200c, miR-145, miR-223, miR-424, and miR-377 in endoplasmic reticulum stress (ERS), oxidative stress (OS), fibrosis, and apoptosis processes in chronic pancreatitis (CP) rat model. Materials and Methods: Fourteen rats were randomized into 2 groups (Group 1, sham group (n=7) and Group 2, CP group (n=7)). TGF-beta and malondialdehyde concentrations were measured in rat blood samples. qRT-PCR was used to investigate the expression levels of 7 miRNAs in the pancreas tissues. The correlations of mRNA undergoing significant changes with inflammation (TNF-α, IL-6), ERS (Ire1-α, Perk), apoptosis (Caspase 3, Bcl-2), OS (Cat, Gpx1), and fibrosis (α-Sma) were investigated.Results: The biochemical results and histopathological scores in Group 1 were statistically significantly high compared with Group 2 (p

Aims: Circular RNAs (circRNAs) are a novel type of non-coding RNA that plays major roles in the development of diverse diseases including osteonecrosis of the femoral head (ONFH). Here, we explored the impact of hsa_circ_0066523 derived from forkhead box P1 (FOXP1) (also called circFOXP1) on bone mesenchymal stem cells (BMSCs), which is important for ONFH development. Methods: RNA or protein expression in BMSCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell Counting Kit 8 (CCK8) and 5-ethynyl-2’-deoxyuridine (EdU) were used to analyze cell proliferation. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red S staining were employed to evaluate the osteoblastic differentiation. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pull down, and RNA immunoprecipitation (RIP) assays were combined for exploring molecular associations. Results: Circ_0066523 was upregulated in osteogenic induction process of BMSCs. Silencing circ_0066523 restrained the proliferation and osteogenic differentiation of BMSCs. Mechanistically, circ_0066523 activated phosphatidylinositol-4,5-bisphosphate 3-kinase / AKT serine/threonine kinase 1 (PI3K/AKT) pathway via recruiting lysine demethylase 5B (KDM5B) to epigenetically repress the transcription of phosphatase and tensin homolog (PTEN). Functionally, AKT signalling pathway agonist or PTEN knockdown counteracted the effects of silenced circ_0066523 on BMSC proliferation and differentiation. Conclusion: Circ_0066523 promotes the proliferation and differentiation of BMSCs by epigenetically repressing PTEN and therefore activating AKT pathway. This finding might open new avenues for the identification of therapeutic targets for osteoblast differentiation related diseases such as ONFH.

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients. • We searched for optimal internal reference for sEV miRNAs in sequencing results. • The candidate internal reference was evaluated for expression using qRT-PCR method. • miR-140-3p is the most stably expressed plasma sEV miRNA in lung cancer patients. • miR-140-3p is a better reference gene than U6 to evaluate miRNA expression profile. [ABSTRACT FROM AUTHOR]

Background: One of the amazing and economical spices is Chilli (Capsicum annum L.) and also called "wonder spice," and it gives great significance to traditional household use and export as well as such a remarkable cash crop in India. However, the important constraints in chilli production are chilli leaf curl disease which has been caused by whitefly (Bemisia tabaci) transmitted by begomoviruses and leads to a major decline in yield. Aim: To explore the stability of the insect resistance gene (tryptophan decarboxylase [TDC]) in resistant genotypes after being challenged with a known number of whiteflies. Since, biotic stress defense allied mechanism regulated during tryptophan biosynthesis. Materials and Methods: The presence of endogenous genes was verified using the polymerase chain reaction (PCR), cloning (T/A vector), and gene expression level (quantitative real-time PCR). Results: Amplification of mitochondrial coxI gene fragment using the primer (C1-J-2195 and L2-N-3014) produced B. tabaci specific ~ 800 bp band. Besides, the whitefly sequence was aligned with the NCBI blast where 92% of identity was observed. TDC gene expression was greater in P2 accession leaves at 48 h post-infestation with 20 number of B. tabaci after feeding and downregulation in less TDC gene expression level at moderately resistant accession ACC 20. Further, all the amplified gene sequences were aligned using NCBI BLAST. The expression of TDC genes in chillies could demoralize the B. tabaci fitness causes mortality. Conclusion: The satisfactory control of viral disease may be achieved with the application of molecular biology, DNA, and RNA-based technologies include the cloning of insect resistance genes. [ABSTRACT FROM AUTHOR]

Introduction Parkinson’s disease (PD) is a common adult-onset neurodegenerative disorder caused by a progressive loss of dopaminergic neurons due to the accumulation of α-synuclein in the substantia nigra. Mitochondria are known to play a key role in cell respiratory function and bioenergetics. Indeed, mitochondrial dysfunction causes insufficient energy production required to satisfy the needs of several organs, especially the nervous system. However, the profiling of messenger RNA (mRNA) expression of mitochondrial subunits in PD has not been systematically investigated yet. Material and methods We explored the mRNA expression of mitochondrial DNA (mtDNA) encoded respiratory chain (RC) subunits in 43 PD patients and 43 normal controls (NC). Next generation sequencing analysis (NGS) was used and quantitative real-time polymerase chain reaction (qRT-PCR) assay was used for confirmation of the NGS results. Results All tested mitochondrial RC subunits were significantly over-expressed in subjects with PD compared to NC. In qRT-PCR the mean expression of all mitochondrial subunits had an expression level of at least 7 times compared to NC. Conclusions The over-expression of mitochondrial subunits in PD subjects with respect to NC might be secondary to a degeneration-related alteration of the mitochondrial structure and/or dynamics, or to the occurrence of a compensatory mechanism. The study of specific mRNA by peripheral blood mononuclear cells may provide a further diagnostic frame for early detection PD patients.

Objective The purpose of the study was to compare results and evaluate the agreement between the endpoint fluorescence (EPF) method and quantitative real-time polymerase chain reaction (QPCR) during molecular monitoring of patients with chronic myeloid leukemia (CML) receiving treatment. Materials and Methods The study was conducted at Molecular Lab of Riphah International University, Islamabad, Pakistan, from January 2017 to December 2018. A total of 150 blood specimens from 30 patients with CML were analyzed at regular intervals during therapy. The detection/quantification of transcript mRNA was done simultaneously using QPCR and the EPF method. Results Out of a total of 150 RNA specimens analyzed, 117 (78%) specimens were positive, whereas 33 (22%) were negative for the transcript using both methods at various stages of treatment. Strong linear negative correlations between the cycle threshold and relative fluorescence unit values were observed with P  <.0001 at 0, 3, 6, 9, and 12 months of treatment. No significant difference (P  >.05) between the means of the BCR-ABL percentage was observed in either method at all stages of treatment. The bias between the 2 methods was calculated as 0.069 ± 3.50, and 95% limits of agreement were 6.92% to –6.79%. Conclusion We found that EPF is s simple method to detect/quantify BCR-ABL mRNA expression during treatment with comparable results to QPCR. [ABSTRACT FROM AUTHOR]

Background: Circulating microRNAs (miRNAs) are a group of noncoding RNAs with promising potential as minimal invasive biomarkers for noncommunicable diseases. However, challenges exist in the preparation of these miRNAs from peripheral blood samples for quantification purposes. The low quality of miRNA extracts presents an obstacle. Acknowledging the superior performance of quantitative real-time polymerase chain reaction (qPCR) as gold standard for gene expression analysis, we conducted this study to observe the capabilities of qPCR using the Taqman® protocol in amplifying circulating miRNAs from miRNA extracts with low purity and yield. Methods: miRNAs were extracted from thirty-six plasma samples that were obtained from public subjects. Four selected miRNAs were quantified using the Taqman® protocol in an integrated fluidic circuit chip that was optimized from a previous study. The amplification graph and Cq values were obtained to observe any abnormal amplification signs and expression levels, respectively. Results: The qualitative observation of the amplification of the four miRNAs showed no sign of abnormality, thereby indicating the successful amplification of the miRNAs without enzymatic inhibition. Furthermore, the miRNAs were quantified in high expression levels. Conclusion: The circulating miRNA extracts with low purity and yield were practical for the study of circulating miRNA expression based on the Taqman® protocol as the method of detection.

Abstract The European eel, Anguilla anguilla, is classified as critically endangered by the IUCN. To protect what remains of the European eel population, accurate monitoring methods for this species are important. Environmental DNA (eDNA) techniques are gaining popularity for ecological monitoring of aquatic organisms because they are sensitive and noninvasive. This study directly compared catch data from a standardized fyke‐net fishing survey with a single species A. anguilla eDNA survey in five freshwater lakes in Ireland. The eDNA was recovered by the filtration of water samples and amplified by quantitative real‐time polymerase chain reaction (qPCR). European eel eDNA was reliably determined in 83% (70/84) of surface water samples collected from lakes classified as having high, medium, and low eel populations. In addition, there was a positive association between the eDNA concentrations recovered and the eel population classification with lower eDNA concentrations in lakes classified as low eel population lakes. Similar amounts of A. anguilla eDNA were detected in water samples collected from open water and shore‐side, suggesting shore sampling is an adequate method for eel detection. Together, the results demonstrate that eDNA sampling is more sensitive for detecting eel presence in low eel population environments than standard survey methods and may be a useful noninvasive tool for monitoring A. anguilla species distribution.

Objective: Currently, application of oncolytic-virus in cancer treatment of clinical trials are growing. Oncolytic-reovirus is an attractive anti-cancer therapeutic agent for clinical testing. Many studies used mesenchymal stem cells (MSCs) as a carrier cell to enhance the delivery and quality of treatment with oncolytic-virotherapy. But, biosynthetic capacity and behavior of cells in response to viral infections are different. The infecting process of reoviruses takes from two-hours to one-week, depends on host cell and the duration of different stages of virus replication cycle. The latter includes the binding of virus particle, entry, uncoating, assembly and release of progeny-viruses. We evaluated the timing and infection cycle of reovirus type-3 strain Dearing (T3D), using one-step replication experiment by molecular and conventional methods in MSCs and L929 cell as control. Materials and Methods: In this experimental study, L929 and adipose-derived MSCs were infected with different multiplicities of infection (MOI) of reovirus T3D. At different time points, the quantity of progeny viruses has been measured using virus titration assay and quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the ability of these cells to support the reovirus replication. One-step growth cycle were examined by 50% cell culture infectious dose (CCID50) and qRT-PCR. Results: The growth curve of reovirus in cells shows that MOI: 1 might be optimal for virus production compared to higher and lower MOIs. The maximum quantity of virus production using MOI: 1 was achieved at 48-hours post-infection. The infectious virus titer became stationary at 72-hours post-infection and then gradually decreased. The virus cytopathic effect was obvious in MSCs and this cells were susceptible to reovirus infection and support the virus replication. Conclusion: Our data highlights the timing schedule for reovirus replication, kinetics models and burst size. Further investigation is recommended to better understanding of the challenges and opportunities, for using MSCs loaded with reovirus in cancer-therapy.

Objective: MicroRNA (miRNA), a short noncoding RNA, is claimed to be a potential blood-based biomarker. We aimed to identify and evaluate miRNAs as diagnostic biomarkers for non-small cell lung cancer (NSCLC). Methods: Profiles of 745 miRNAs were screened in the serum of 8 patients with NSCLC and 8 age- and sex-matched controls using TaqMan low-density arrays (TLDAs) and validated in 25 patients with NSCLC and 30 with other lung diseases (OLs) as well as in 19 healthy persons (HPs). The diagnostic performance of the candidate miRNAs was assessed in 117 cases of NSCLC and 113 OLs using quantitative real-time polymerase chain reaction (qRT-PCR). Differences in miRNA expression between patients with NSCLC and controls were assessed using the Mann–Whitney U test. The area under receiver operating characteristic (ROC) curve (AUC) was obtained based on the logistic regression model. Results: Ten miRNAs were found to be differentially expressed between patients with NSCLC and controls, including miR-769, miR-339-3p, miR-339-5p, miR-519a, miR-1238, miR-99a#, miR-134, miR-604, miR-539, and miR-342. The expression of miR-339-3p was significantly higher in patients with NSCLC than in those with OLs (P < 0.001) and HPs (P = 0.020). ROC analysis revealed an miR-339-3p expression AUC of 0.616 [95% confidence interval (CI): 0.561–0.702]. The diagnostic prediction was increased (AUC = 0.706, 95% CI: 0.649–0.779) in the model combining miR-339-3p expression and other known risk factors (i.e., age, smoking status, and drinking status). Conclusions: MiR-339-3p was significantly upregulated in patients with NSCLC compared with participants without cancer, suggesting a diagnostic prediction value for high-risk individuals. Therefore, miR-339-3p expression could be a potential blood-based biomarker for NSCLC.

Objectives: Diagnosis of active cytomegalovirus (CMV) infection in hematopoietic stem cell (HSC)-transplanted patients is essential because of the serious complications it causes, especially CMV disease. Such diagnosis allows the initiation of a preemptive antiviral therapy, which avoids the development of such disease. In this retrospective study made as a part of routine work for transplanted patients, we compared two diagnosis testing for CMV-active infection such as pp65 CMV antigenemia and a quantitative real-time polymerase chain reaction (QPCR). Our purpose was to assess the contribution of these tools to the diagnosis and monitoring of active CMV infections. Materials and Methods: Five hundred and fifty-eight and 156 samples belonging to 24 HSC transplanted patients were, respectively, tested by antigenemia and QPCR. Results: For the 156 samples analyzed by both testing, a number of positive samples by a QPCR were higher than that by antigenemia (44.8% vs. 13%), with a statistically significant difference (P < 0.0001). A statistically significant correlation between viral loads of these two testing was also found (rs = 0.765). In median, QPCR becomes positive 14 days before antigenemia (P < 0.0001) and its positivity lasts for 91 days, whereas antigenemia positivity lasts only for 56 days (P < 0.001). No statistically significant difference was found between the results of these two testing in CMV disease cases. Conclusions: QPCR is a rapid, standardized assay, which permits a precocious detection of CMV DNA. Kinetic of DNA evolution is a reliable diagnostic tool and more effective than antigenemia-based assays in monitoring CMV infection.

Osteonecrosis of the femoral head (ONFH) is a common disabling disease. Copper has positive effects on cells that regulate bone metabolism. However, the relationship between copper metabolism (CM) and steroid-induced ONFH (SONFH) remains unclear. The GSE123568 dataset was downloaded from the Gene Expression Omnibus. The differentially expressed CM-related SONFH genes (DE-CMR-SONFHGs) were identified via differential analysis and weighted gene coexpression network analysis (WGCNA). Receiver operating characteristic (ROC) analysis was performed for the predictive accuracy of key genes. Targeting drugs and the copper death-related genes (CDRGs) relevant to key genes were investigated. The bioinformatics results were confirmed via quantitative real-time polymerase chain reaction (qRT–PCR) and Western blot (WB) analysis. Two out of 106 DE-CMR-SONFHGs were identified as key genes (PNP and SLC2A1), which had diagnostic value in distinguishing SONFH from control samples and were related to various immune cell infiltrations. Eleven PMP-targeting drugs and five SLC2A1-targeting drugs were identified. The qRT–PCR, as well as WB, results confirmed the downregulation PNP and SLC2A1 and high expression of the CDRGs DLD, PDHB, and MTF1, which are closely related to these two key genes. In conclusion, PNP and SLC2A1 were identified as key genes related to SONFH and may provide insights for SONFH treatment.

Summary: Background: Colorectal cancer (CRC) is among the most widespread malignancies in the world. MicroRNA (miRNA) has been identified as an important modulator of the biological processes of the cells. This group of noncoding RNAs also has a pivotal function in the growth and development of human cancers, including CRC. Among these miRNAs, miR-196, miR-132, miR-146a, and miR-134 have fundamental impacts on the regulation of cancers. The current study aimed to investigate the involvement of these miRNAs in CRC patients. Methods: In this study, 50 pairs of tumor and tumor margin samples of CRC patients were investigated to assess the expression levels of miR-196, miR-132, miR-146a, and miR-134 in this cancer. For this purpose, firstly, quantitative real-time PCR (qRT-PCR) was applied. Also, KRAS mutation and clinicopathological characteristics of the CRC patients were analyzed in the study groups. Results: The findings demonstrated the overexpression of miR-196 (P-value = 0.0045) and miR-146a (P-value = 0.0033) in tumor tissues compared to controls. Conversely, the expression levels of miR-132 (P-value = 0.00032) and miR-134 (P-value < 0.0001) were downregulated in tumor tissues. Also, miR-146a was the only miRNA with significant expression change in the case of the KRAS gene mutation. Interestingly, the expression ratio of these miRNAs was significantly associated with some of the clinicopathological features of the patients, such as lymph node and distant metastases. Conclusion: Our data demonstrated that these miRNAs appear to be promising novel biomarkers for early diagnosis of CRC and may pave the way for the future establishment of novel therapeutic options for CRC. [ABSTRACT FROM AUTHOR]

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