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BACKGROUND: Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required. OBJECTIVE: This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings. METHODS: We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively. RESULTS: Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater. CONCLUSIONS: As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

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Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) is a commonly used tool for gene expression analysis. The selection of stably expressed reference genes is required for accurate normalization. The aim of this study was to identify the optimal reference genes for RT-qPCR normalization in various brain regions of rats at different stages of the lithium-pilocarpine model of acquired epilepsy. We tested the expression stability of nine housekeeping genes commonly used as reference genes in brain research: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on four standard algorithms (geNorm, NormFinder, BestKeeper, and comparative delta-Ct), we found that after pilocarpine-induced status epilepticus, the stability of the tested reference genes varied significantly between brain regions and depended on time after epileptogenesis induction (3 and 7 days in the latent phase, and 2 months in the chronic phase of the model). Pgk1 and Ywhaz were the most stable, while Actb, Sdha, and B2m demonstrated the lowest stability in the analyzed brain areas. We revealed time- and region-specific changes in the mRNA expression of the housekeeping genes B2m, Actb, Sdha, Rpl13a, Gapdh, Hprt1, and Sdha. These changes were more pronounced in the hippocampal region during the latent phase of the model and are thought to be related to epileptogenesis. Thus, RT-qPCR analysis of mRNA expression in acquired epilepsy models requires careful selection of reference genes depending on the brain region and time of analysis. For the time course study of epileptogenesis in the rat lithium-pilocarpine model, we recommend the use of the Pgk1 and Ywhaz genes.

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We report a case of pneumonia caused by coinfection with Chlamydia psittaci and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB.1 variant, confirmed using metagenomic next-generation sequencing (mNGS) and quantitative polymerase chain reaction (qPCR). C. psittaci and SARS-CoV-2 were detected in bronchoalveolar lavage fluid using mNGS. Additionally, mNGS detected C. psittaci in blood and nasopharyngeal specimens and was more sensitive than qPCR. The patient recovered after treatment with moxifloxacin. This report highlights the use of coinfections of C. psittaci and SARS-CoV-2, as mNGS has already been recognized to be a diagnostic tool for identifying coinfections.

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Background: The long non-coding RNA (lncRNA) prostate cancer-associated transcript 6 (PCAT6) has been studied in many cancers, yet its relationship with colorectal cancer (CRC) remains poorly defined. Here, we conducted an analysis of The Cancer Genome Atlas (TCGA) database to better clarify the role of PCAT6 in this cancer type. Methods: Wilcoxon rank-sum tests were utilized to assess relative levels of PCAT6 in CRC tumors and normal tissues, while logistic regression analyses were utilized to compare the relationships between PCAT6 levels and clinicopathological findings. Kaplan-Meier curves and Cox regression analyses were used to gauge correlations between PCAT6 and patient survival outcomes, while the biological roles of this lncRNA were investigated via a gene set enrichment analysis (GSEA) approach. The expression level of PCAT6 in CRC cell lines was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: PCAT6 levels were significantly correlated with CRC patient lymph node metastasis (N) stage [odds ratio (OR) =1.8 for N1 & N2 vs. N0], lymphatic invasion [OR =1.9 for yes vs. no), distant metastasis (M stage) (OR =2.1 for M1 vs. M0), carcinoembryonic antigen (CEA) level (OR =1.9 for >5 vs. ≤5), perineural invasion (OR =1.9 for yes vs. no), pathologic stage (OR =1.9 for stage III/IV vs. stage I/II), and neoplasm type (OR =2.1 for rectal adenocarcinoma vs. colon adenocarcinoma) (all P<0.05). CRC patients expressing higher PCAT6 levels exhibited poorer survival outcomes than those expressing low levels of this lncRNA (P=0.017), and in univariate analyses, higher PCAT6 levels were linked to worse overall survival [hazard ratio (HR) =1.540; 95% confidence interval (CI): 1.079-2.199; P=0.017], with this relationship also being preserved in a multivariate analysis (HR =6.892; 95% CI: 1.713-27.727, P=0.007). GSEA revealed high PCAT6 expression to be linked to differential DNA methylation enrichment, with high PCAT6 levels being associated with changes in base excision repair, cellular senescence, G2/M DNA damage checkpoint, chromatin-modifying enzyme, and gene silencing by RNA activity. The high expression of lncRNA PCAT6 in CRC cell lines was demonstrated by PCR experiments. Conclusions: PCAT6 represents a promising prognostic biomarker of poor CRC patient survival outcomes, with DNA methylation and RNA-mediated gene silencing being potentially promising mechanistic pathways whereby this lncRNA may shape patient outcomes.

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Although elevated blood levels of trimethylamine N-oxide (TMAO) have been associated with atherosclerosis development in humans, the role of its gut microbiota-derived precursor, TMA, in this process has not been yet deciphered. Taking this into account, and the fact that increased intestinal fatty acid absorption contributes to atherosclerosis onset and progression, this study aimed to evaluate the effect of TMA on fatty acid absorption in a cell line that mimics human enterocytes. Caco-2 cells were treated with TMA 250 µM for 24 h. Fatty acid absorption was assessed by measuring the apical-to-basolateral transport and the intracellular levels of BODIPY-C12, a fluorescently labelled fatty acid analogue. Gene expression of the main intestinal fatty acid transporters was evaluated by real-time quantitative reverse transcription PCR. Compared to control conditions, TMA increased, in a time-dependent manner and by 20-50 %, the apical-to-basolateral transport and intracellular levels of BODIPY-C12 fatty acid in Caco-2 cells. Fatty acid transport protein 4 (FATP4) and fatty acid translocase (FAT)/CD36 gene expression were not stimulated by TMA, suggesting that TMA-induced increase in fatty acid transport may be mediated by an increase in FAT/CD36 and/or FATP4 activity and/or fatty acid passive transport. This study demonstrated that TMA increases the intestinal absorption of fatty acids. Future studies are necessary to confirm if this may constitute a novel mechanism that partially explains the existing positive association between the consumption of a diet rich in TMA sources (e.g. red meat) and the increased risk of atherosclerotic diseases.

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BACKGROUND: The COVID-19 pandemic necessitated rapid real-time surveillance of epidemiological data to advise governments and the public, but the accuracy of these data depends on myriad auxiliary assumptions, not least accurate reporting of cases by the public. Wastewater monitoring has emerged internationally as an accurate and objective means for assessing disease prevalence with reduced latency and less dependence on public vigilance, reliability, and engagement. How public interest aligns with COVID-19 personal testing data and wastewater monitoring is, however, very poorly characterized. OBJECTIVE: This study aims to assess the associations between internet search volume data relevant to COVID-19, public health care statistics, and national-scale wastewater monitoring of SARS-CoV-2 across South Wales, United Kingdom, over time to investigate how interest in the pandemic may reflect the prevalence of SARS-CoV-2, as detected by national testing and wastewater monitoring, and how these data could be used to predict case numbers. METHODS: Relative search volume data from Google Trends for search terms linked to the COVID-19 pandemic were extracted and compared against government-reported COVID-19 statistics and quantitative reverse transcription polymerase chain reaction (RT-qPCR) SARS-CoV-2 data generated from wastewater in South Wales, United Kingdom, using multivariate linear models, correlation analysis, and predictions from linear models. RESULTS: Wastewater monitoring, most infoveillance terms, and nationally reported cases significantly correlated, but these relationships changed over time. Wastewater surveillance data and some infoveillance search terms generated predictions of case numbers that correlated with reported case numbers, but the accuracy of these predictions was inconsistent and many of the relationships changed over time. CONCLUSIONS: Wastewater monitoring presents a valuable means for assessing population-level prevalence of SARS-CoV-2 and could be integrated with other data types such as infoveillance for increasingly accurate inference of virus prevalence. The importance of such monitoring is increasingly clear as a means of objectively assessing the prevalence of SARS-CoV-2 to circumvent the dynamic interest and participation of the public. Increased accessibility of wastewater monitoring data to the public, as is the case for other national data, may enhance public engagement with these forms of monitoring.

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The genetic diversity of coronaviruses (CoVs) is high, and their infection in animals has not yet been fully revealed. By RT-PCR detection of the partial RNA-dependent RNA polymerase (RdRp) gene of CoVs, we screened a total of 502 small mammals in the Dali and Nujiang prefectures of Western Yunnan Province, China. The number of overall CoV positives was 20, including ß-CoV (n = 13) and α-CoV (n = 7), with a 3.98% prevalence in rectal tissue samples. The identity of the partial RdRp genes obtained for 13 strains of ß-CoV was 83.42-99.23% at the nucleotide level, and it is worth noting that the two strains from Kachin red-backed voles showed high identity to BOV-36/IND/2015 from Indian bovines and DcCoV-HKU23 from dromedary camels (Camelus dromedarius) in Morocco; the nucleotide identity was between 97.86 and 98.33%. Similarly, the identity of the seven strains of α-CoV among the partial RdRp sequences was 94.00-99.18% at nucleotide levels. The viral load in different tissues was measured by quantitative RT-PCR (qRT-PCR). The average CoV viral load in small mammalian rectal tissue was 1.35 × 106 copies/g; differently, the mean CoV viral load in liver, heart, lung, spleen, and kidney tissue was from 0.97 × 103 to 3.95 × 103 copies/g, which revealed that CoV has extensive tropism in rectal tissue in small mammals (p < 0.0001). These results revealed the genetic diversity, epidemiology, and infective tropism of α-CoV and ß-CoV in small mammals from Dali and Nujiang, which deepens the comprehension of the retention and infection of coronavirus in natural hosts.

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Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.

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Background: Osteosarcoma (OS) is the most common primary malignancy in bone tissues, and effective therapeutics remain absent in clinical practice. Traditional Chinese medicines (TCM) have been used for thousands of years, which provide great insights into OS management. Gallic acid (GA) is a natural phenolic acid enriched in various foods and herbs. Several pharmacological activities of GA such as anti-oxidation and anti-inflammation have been well-established. However, its biological function in OS remains not fully understood. Methods: The potential anti-cancer properties of GA were evaluated in 143 â€‹B, U2OS and MG63 â€‹cells. Its effects on cell growth, cell cycle, apoptosis and migration were examined in these OS cells. The lncRNA H19 and Wnt/ß-catenin signaling were detected by qPCR, luciferase activity and Western blotting assays. The in vivo effect of GA on tumor growth was investigated using an orthotopic mouse model. Results: In the present study, GA was found to suppress the tumor growth in vitro via inducing cell cycle arrest and apoptosis in OS cells, and inhibit the invasion and metastasis as well. Using the orthotopic animal model, GA was also found to suppress tumorigenesis in vivo. Long noncoding RNA (lncRNA) H19 was demonstrated to be down-regulated by GA, and thus disrupted the canonical Wnt/ß-catenin signaling in OS cells. Furthermore, the ectopic expression of H19 rescued the GA-induced suppressive effects on tumor growth and metastasis, and partially reversed the inactivation of Wnt/ß-catenin signaling. Conclusions: Taken together, our results indicated that GA inhibited tumor growth through an H19-mediated Wnt/ß-catenin signaling regulatory axis in OS cells. The translational potential of this article: The information gained from this study provides a novel underlying mechanism of GA mediated anti-OS activity, suggesting that GA may be a promising drug candidate for OS patients.

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OBJECTIVE: Endometriosis (EMS) is a chronic gynecological disorder with an urgent need of a reliable non-invasive diagnostic strategy. Recently, there has been increasing interest in using the contents of exosomes, especially exosomal microRNAs (miRNAs), as potential biomarkers for various types of diseases. In this study, we assessed the differentially expressed miRNAs in exosomes derived from primary normal and ectopic endometrial cells. METHODS: We used miRNA microarray analysis to identify differentially expressed exosomal miRNAs. Among the selected exosomal miRNAs, we focused on hsa-miR-202-3p and hsa-miR-202-5p and validated their expression levels using quantitative reverse transcription polymerase chain reaction analysis. We then further examined their expression in exosomes derived from vaginal discharge (leukorrhea) from patients with EMS and the negative control group. RESULTS: The data show that hsa-miR-202-3p and hsa-miR-202-5p were expressed significantly higher in leukorrhea-derived exosomes from EMS patients compared with the negative controls. CONCLUSION: Taken together, our results suggest that leukorrhea-derived exosomal hsa-miR-202 could serve as a potential useful biomarker for diagnosing EMS.

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Objective: Knee osteoarthritis (KOA) is a highly prevalent musculoskeletal disorder characterized by degeneration of cartilage and abnormal remodeling of subchondral bone (SCB). Teriparatide (PTH (1-34)) is an effective anabolic drug for osteoporosis (OP) and regulates osteoprotegerin (OPG)/receptor activator of nuclear factor ligand (RANKL)/RANK signaling, which also has a therapeutic effect on KOA by ameliorating cartilage degradation and inhibiting aberrant remodeling of SCB. However, the mechanisms of PTH (1-34) in treating KOA are still uncertain and remain to be explored. Therefore, we compared the effect of PTH (1-34) on the post-traumatic KOA mouse model to explore the potential therapeutic effect and mechanisms. Methods: In vivo study, eight-week-old male mice including wild-type (WT) (n â€‹= â€‹54) and OPG-/- (n â€‹= â€‹54) were investigated and compared. Post-traumatic KOA model was created by destabilization of medial meniscus (DMM). WT mice were randomly assigned into three groups: the sham group (WT-sham; n â€‹= â€‹18), the DMM group (WT-DMM; n â€‹= â€‹18), and the PTH (1-34)-treated group (WT-DMM â€‹+ â€‹PTH (1-34); n â€‹= â€‹18). Similarly, the OPG-/- mice were randomly allocated into three groups as well. The designed mice were executed at the 4th, 8th, and 12th weeks to evaluate KOA progression. To further explore the chondro-protective of PTH (1-34), the ATDC5 chondrocytes were stimulated with different concentrations of PTH (1-34) in vitro. Results: Compared with the WT-sham mice, significant wear of cartilage in terms of reduced cartilage thickness and glycosaminoglycan (GAG) loss was detected in the WT-DMM mice. PTH (1-34) exhibited cartilage-protective by alleviating wear, retaining the thickness and GAG contents. Moreover, the deterioration of the SCB was alleviated and the expression of PTH1R/OPG/RANKL/RANK were found to increase after PTH (1-34) treatment. Among the OPG-/- mice, the cartilage of the DMM mice displayed typical KOA change with higher OARSI score and thinner cartilage. The damage of the cartilage was alleviated but the abnormal remodeling of SCB didn't show any response to the PTH (1-34) treatment. Compared with the WT-DMM mice, the OPG-/--DMM mice caught more aggressive KOA with thinner cartilage, sever cartilage damage, and more abnormal remodeling of SCB. Moreover, both the damaged cartilage from the WT-DMM mice and the OPG-/--DMM mice were alleviated but only the deterioration of SCB in WT-DMM mice was alleviated after the administration of PTH (1-34). In vitro study, PTH (1-34) could promote the viability of chondrocytes, enhance the synthesis of extracellular matrix (ECM) (AGC, COLII, and SOX9) at the mRNA and protein level, but inhibit the secretion of inflammatory cytokines (TNF-α and IL-6). Conclusion: Both wear of the cartilage was alleviated and aberrant remodeling of the SCB was inhibited in the WT mice, but only the cartilage-protective effect was observed in the OPG-/- mice. PTH (1-34) exhibited chondro-protective effect by decelerating cartilage degeneration in vivo as well as by promoting the proliferation and enhancing ECM synthesis of chondrocytes in vitro. The current investigation implied that the rescue of the disturbed SCB is dependent on the regulation of OPG while the chondro-protective effect is independent of modulation of OPG, which provides proof for the treatment of KOA. The translational potential of this article: Systemic administration of PTH (1-34) could exert a therapeutic effect on both cartilage and SCB in different mechanisms to alleviate KOA progression, which might be a novel therapy for KOA.

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As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin-biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.

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OBJECTIVE: The primary goals of this research were to analyze the relationship between ABO blood types and the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and investigate the effect of vaccination in Iraq. METHODS: Data and outcomes were gathered from the medical records of 200 patients. Patients were categorized by blood group and vaccination status in the analysis. RESULTS: In total, 200 hospitalized patients (125 men and 75 women) with confirmed SARS-CoV-2 infection and blood group (ABO) and clinical data were enrolled. Of the 200 patients, 155 (77.5%) were vaccinated against SARS-CoV-2. The results illustrated that 25 patients died, which might have been attributable to a lack of vaccination or older age. Our analysis revealed that blood group O individuals were much less likely to be infected by SARS-CoV-2 than non-O subjects, whereas blood group A individuals carried a higher risk of infection. CONCLUSIONS: Our findings illustrated that immunization significantly reduces COVID-19 risk across all age groups, but there has been an increase in the number of cases because of decreased vaccine efficacy in older patients and persons with comorbidities. However, 45% vaccination coverage lowered the outbreak's peak.

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Increasing globalization, agricultural intensification, urbanization, and climatic changes have resulted in a significant recent increase in emerging infectious zoonotic diseases. Zoonotic diseases are becoming more common, so innovative, effective, and integrative research is required to better understand their transmission, ecological implications, and dynamics at wildlife-human interfaces. High-throughput sequencing (HTS) methodologies have enormous potential for unraveling these contingencies and improving our understanding, but they are only now beginning to be realized in livestock research. This study investigates the current state of use of sequencing technologies in the detection of livestock pathogens such as bovine, dogs (Canis lupus familiaris), sheep (Ovis aries), pigs (Sus scrofa), horses (Equus caballus), chicken (Gallus gallus domesticus), and ducks (Anatidae) as well as how it can improve the monitoring and detection of zoonotic infections. We also described several high-throughput sequencing approaches for improved detection of known, unknown, and emerging infectious agents, resulting in better infectious disease diagnosis, as well as surveillance of zoonotic infectious diseases. In the coming years, the continued advancement of sequencing technologies will improve livestock research and hasten the development of various new genomic and technological studies on farm animals.

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ADP-ribosylation factor (Arf)-GTPase-activating protein (GAP) with coiled-coil, ankyrin repeat and PH domains 1 (ACAP1) has been reported to serve as an adaptor for clathrin coat complex playing a role in endocytic recycling and cellular migration. The potential role of ACAP1 in lung adenocarcinoma (LUAD) has not been yet completely defined. We performed the comprehensive analyses, including gene expression, survival analysis, genetic alteration, function enrichment, and immune characteristics. ACAP1 was remarkably downregulated in tumor tissues, and linked with the clinicopathologic features in LUAD patients. Prognostic analysis demonstrated that low ACAP1 expression was correlated with unsatisfactory overall survival (OS) and disease specific survival (DSS) in LUAD patients. Moreover, ACAP1 could be determined as a prognostic biomarker according to Cox proportional hazard model and nomogram model. We also confirmed that ACAP1 was downregulated in two LUAD cell lines, comparing to normal lung cell. Overexpression of ACAP1 caused a profound attenuation in cell proliferation, migration, invasion, and promoted cell apoptosis. Additionally, functional enrichment analyses confirmed that ACAP1 was highly correlated with T cell activation and immune response. Then, we further conducted immune landscape analyses, including single cell RNA sequencing, immune cells infiltration, and immune checkpoints. ACAP1 expression was positively associated with the infiltrating level of immune cells in TME and the expression of immune checkpoint molecules. This study first comprehensively analyzed molecular expression, clinical implication, and immune landscape features of ACAP1 in LUAD, suggesting that ACAP1 was predictive of prognosis and could serve as a potential biomarker predicting immunotherapy response for LUAD patients.

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MicroRNAs (miRNAs) are potential clinical biomarkers for the detection of various diseases. However, their quantification has not been implemented in clinical practice given the inconsistencies in their variable recovery rate and accuracy of the results. Thus, we utilized a technique based on bioluminescent enzyme immunoassay (BLEIA) to perform fully automated miRNA quantification using magnetic particles conjugated with antibodies targeting DNA-RNA hybrids, biotinylated DNA probes specific to miRNAs, and firefly luciferase-labeled streptavidin. This method enabled direct use of diluted serum and automation of all processes within 1 hr. The results revealed a wide linear range between 10 fmol/L and 1 nmol/L, high sensitivity with a detection limit of 6.3 fmol/L or below, high specificity with a false positive rate under 2.4%, and high reproducibility in intra- and inter-experimental observations (under CV 10% and r = 0.9965, respectively). Furthermore, a significant correlation was revealed between quantitative reverse transcription-polymerase chain reaction and BLEIA assay for the quantification of synthetic miRNA (r = 0.9993) and endogenous miRNA in healthy serums (r = 0.8203), respectively. Overall, we developed a fully automated miRNA quantification method based on BLEIA, which can be adopted in a clinical setting. However, further studies are needed to assess its clinical performance.

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OBJECTIVE: Chromodomain helicase DNA-binding 5 (CHD5) acts as a tumor suppressor gene in some cancers. CHD5 expression levels may affect an individual's susceptibility to hepatocellular carcinoma (HCC). This study aimed to evaluate the methylation pattern of the CHD5 promoter region and the gene's corresponding mRNA expression in HCC patients compared with healthy individuals. METHODS: In this case-control study, CHD5 mRNA gene expression levels and DNA methylation patterns were analyzed in 81 HCC patients and 90 healthy individuals by quantitative reverse transcription polymerase chain reaction and methylation-specific polymerase chain reaction, respectively. RESULTS: The CHD5 gene was hypermethylated in 61.8% of the HCC patients and 54.4% of the controls, and this difference was statistically significant. The CHD5 mRNA expression levels were significantly lower in the HCC patient group. CONCLUSIONS: Hypermethylation of the CHD5 promoter region may significantly lower the expression of this gene, affecting the incidence and severity of HCC. The methylation status of CHD5 can also be further studied as a prognostic factor in HCC.

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OBJECTIVE: Even with our best practices, we are frequently unable to prevent slow and stalled wound healing-particularly in people with impaired circulation and conditions such as diabetes. As a result, greater insight into the nature of wound healing and alternative treatment approaches is needed. An avenue that may be of particular promise is increasing understanding of the role of secretory leukocyte protease inhibitor (SLPI) as there is evidence that it enhances wound healing, its expression increases in response to inflammation and infection, and it exhibits anti-protease, anti-inflammatory, antiviral antibacterial and antifungal activities. METHOD: The response of SLPI levels to wounding and skin injury was assessed by taking punch skin biopsies from healthy volunteers and assessing the levels of SLPI at the site of injury at the time of wounding (baseline) as well as one, two, three, four, seven, nine and 12 weeks later. RESULTS: A total of 35 volunteers took part in the study. Significant elevations were found: levels of SLPI were greatly increased, 12 times that at baseline, and remained elevated at three weeks despite re-epithelialisation having occurred. CONCLUSION: These findings not only suggest that levels of SLPI rise rapidly following wounding, but that these elevations are sustained, and continue to increase even when re-epithelialisation has occurred. These results suggest that the role and potential benefits of this protease inhibitor deserve further exploration.

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Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log-fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

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Background: Primary Sjögren's syndrome (pSS) is a relatively common diffuse connective tissue disease that often invades exocrine glands, such as the lacrimal and salivary glands, and manifests as dry eyes and dry mouth. At present, the molecular mechanism of pSS is not clear. This study was designed to explore the internal mechanism of pSS from the gene level and screen out the immune-related diagnostic markers of pSS. Methods: The gene expression profiles GSE84844, GSE7451, and GSE40611 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified with R software. Then, the DEGs were intersected with the immune genes obtained from the ImmPort database to acquire differentially expressed immune-related genes (DEIRGs), and functional enrichment analyses were performed. The DEIRGs were screened through the least absolute shrinkage and selection operator (LASSO) logistic regression algorithm to obtain the optimal immune-related genes (IRGs). Expression levels of the optimal IRGs were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to obtain the key genes. Next, gene chips GSE7451 and GSE40611, from other tissues, were selected as the training sets to verify the sensitivity and specificity of the diagnosis of the key genes by receiver operating characteristic (ROC) analysis. Results: A total of 54 DEIRGs were obtained. The functional enrichment analysis results showed that they play an important role in immune and inflammatory responses. Nine optimal IRGs were screened from the DEIRGs by the LASSO logistic regression algorithm. After qRT-PCR verification, eight out of nine optimal IRGs (IL-18, JAK2, TBK1, EED, TNFSF10, TNFSF13B, CYSLTR1, and ICOS) were significantly highly expressed in pSS patients and were defined as key genes. ROC analysis identified that TNFSF13B and CYSLTR1 had high sensitivity and specificity. Finally, the lack of previous research on EED and CYSLTR1 in pSS suggests that these IRGs may be regarded as new gateways to explore the diagnosis and pathogenesis of pSS. Conclusions: The key DEIRGs play a decisive role during the occurrence and development of pSS.