Exploring the causal association between serum metabolites and erectile dysfunction: a bidirectional Mendelian randomisation study.

Erectile dysfunction is a common sexual disorder in men. Some studies have found a strong association between some serum metabolites and erectile dysfunction. To investigate this association further, we used bidirectional Mendelian randomisation to investigate causality and possible biological mechanisms.Firstly, this study screened the statistics of genome-wide association studies of serum metabolites and erectile dysfunction to obtain instrumental variables. Inverse variance weighting was used as the primary method for causal effect analysis of instrumental variables in forward or reverse Mendelian randomisation, and the results obtained by MR-Egger regression and the weighted median method were used as references. Subsequently, the metabolites causally associated with erectile dysfunction were subjected to replication analyses and meta-analyses, and the results of the meta-analyses were analysed by pathway analyses to find influential pathways. In this process, Mendelian randomisation results need to be assessed for stability and reliability using sensitivity analysis.It was found that a total of six serum metabolites were causally associated with erectile dysfunction in a forward Mendelian randomisation study. 1,3,7-trimethyluraten (0.85 (0.73-0.99), P = 0.0368), ergothioneine (0.65 (0.45-0.94), P = 0.0226) and gamma-glutamylglutamate (0.63 (0.46-0.88), P = 0.0059) were protective against the development of erectile dysfunction, whereas 2-hydroxyhippurate (1.10 (1.02-1.19), P = 0.0152), N2,N2-dimethylguanosine (1.57 (1.02-2.40), P = 0.0395) and octanoylcarnitine (1.38 (1.06-1.82), P = 0.0183) were able to induce the development of erectile dysfunction. In addition, metabolic pathway analysis showed that 1,3,7-trimethylurate was able to influence the development of erectile dysfunction via the caffeine metabolism pathway (P = 0.0454). On the other hand, reverse Mendelian randomisation analysis showed that erectile dysfunction reduced serum homocitrulline levels (0.99 (0.97-1.00), P = 0.0360). Sensitivity analyses, including heterogeneity tests and pleiotropy tests, confirmed the reliability of the results.In conclusion, this study demonstrated a bidirectional causal relationship between serum metabolites and erectile dysfunction using bidirectional Mendelian randomisation analysis and replication meta-analysis. On this basis, this study provides a new direction of thinking and strong evidence for the therapeutic application and adjunctive diagnosis of serum metabolites in erectile dysfunction, and provides a certain reference value for subsequent related studies.

Causal effects of gut microbiota on the risk of osteomyelitis: a Mendelian randomization study.

Background: Osteomyelitis is characterized by an inflammatory process initiated by microorganisms, leading to infection and subsequent degradation of bone tissue. Several studies have indicated a potential link between gut microbiota and the occurrence of osteomyelitis. Utilizing the benefits of Mendelian randomization, which mitigates issues of confounding and reverse causation, we employed this approach to ascertain the presence of a causal connection between gut microbiota and osteomyelitis. Additionally, we aimed to pinpoint gut microbiota that could potentially exert substantial influence. Methods: We performed a rigorous screening of single nucleotide polymorphisms in GWAS summary statistics for gut microbiota and osteomyelitis. The 2,542 instrumental variables obtained after screening were subjected to MR analyses, including inverse variance weighting, weighted median, weighted mode, MR-Egger, and Mendelian randomization pleiotropy residual sum and outlier test. We then validated the reliability of the results by performing sensitivity analyses on the MR of 196 well-defined gut microbiota. Result: We established a causal relationship between gut microbiota and osteomyelitis through MR analysis. Additionally, we identified a taxon of significant importance and six taxons with nominal significance. Specifically, the family Bacteroidales S24.7 group exhibited an association with a diminished risk of osteomyelitis development. Conversely, the class Bacilli, class Bacteroidia, order Bacteroidales, order Lactobacillales, family Streptococcaceae, and genus Coprococcus3 displayed an increased risk of developing osteomyelitis. The MR outcomes for these seven taxa remained stable throughout a series of sensitivity analyses. Conclusion: This study demonstrated a causal relationship between gut microbiota and osteomyelitis by Mendelian randomization. We hope that this study will provide a new direction for the treatment of osteomyelitis, which has a paucity of therapeutic options.

Atom-Modified gDNA Enhances Cleavage Activity of TtAgo Enabling Ultra-Sensitive Nucleic Acid Testing.

The DNA-guided (gDNA) Argonaute from Thermus thermophilus (TtAgo) has little potential for nucleic acid detection and gene editing due to its poor dsDNA cleavage activity at relatively low temperature. Herein, the dsDNA cleavage activity of TtAgo is enhanced by using 2'-fluorine (2'F)-modified gDNA and developes a novel nucleic acid testing strategy. This study finds that the gDNA with 2'F-nucleotides at the 3'-end (2'F-gDNA) can promote the assembly of the TtAgo-guide-target ternary complex significantly by increasing its intermolecular force to target DNA and TtAgo, thereby providing ≈40-fold activity enhancement and decreasing minimum reaction temperature from 65 to 60°C. Based on this outstanding advance, a novel nucleic acid testing strategy is proposed, termed FAST, which is performed by using the 2'F-gDNA/TtAgo for target recognition and combining it with Bst DNA polymerase for nucleic acid amplification. By integrating G-quadruplex and Thioflavin T, the FAST assay achieves one-pot real-time fluorescence analysis with ultra-sensitivity, providing a limit of detection up to 5 copies (20 µL reaction mixture) for miR-21 detection. In summary, an atom-modification-based strategy has been developed for enhancing the cleavage activity of TtAgo efficiently, thereby improving its practicability and establishing a TtAgo-based nucleic acid testing technology with ultra-sensitivity and high-specificity.

Causal effects of gut microbiota on the risk of erectile dysfunction: a Mendelian randomization study.

Erectile dysfunction ranks among the prevalent sexual disorders in men. Several studies have indicated a potential link between gut microbiota and erectile dysfunction. To validate this potential association, we were to screen statistical data from genome-wide association studies of gut microbiota and erectile dysfunction. p values of less than 1 × 10-5 were set as the threshold for screening instrumental variables that were strongly associated with gut microbiota. At the same time, in order to obtain more convincing findings, we further excluded instrumental variables with possible chain imbalance, instrumental variables with the presence of palindromes, instrumental variables with F-statistics less than 10, and instrumental variables associated with risk factors for erectile dysfunction. Five methods including inverse-variance weighted method, weighted median method, weighted mode, Mendelian randomization egger method and Mendelian randomization pleiotropy residual sum and outlier test were then used to analyse the 2591 instrumental variables obtained from the screening. We identified correlations between six gut microbiota and the risk of erectile dysfunction. The genus Ruminococcaceae UCG-013 exhibited an inverse association with the risk of developing erectile dysfunction (0.79 (0.65-0.97), P = 0.0214). Conversely, the genus Tyzzerella3 (1.13 (1.02-1.26), P = 0.0225), genus Erysipelotrichaceae UCG-003 (1.18 (1.01-1.38), P = 0.0412), genus LachnospiraceaeNC2004group (1.19 (1.03-1.37), P = 0.0191), genus Oscillibacter (1.23 (1.08-1.41), P = 0.0022), and family Lachnospiraceae (1.26 (1.05-1.52), P = 0.0123) demonstrated positive associations with an increased risk of erectile dysfunction. These sensitivity analyses of the gut microbiota were consistent. This study demonstrated a possible causal relationship between gut microbiota and erectile dysfunction risk through Mendelian randomization analysis, providing new potential possibilities for the prevention and treatment of erectile dysfunction.

Development and application of hepatocellular carcinoma risk prediction model based on clinical characteristics and liver related indexes.

BACKGROUND: Hepatocellular carcinoma (HCC) is difficult to diagnose with poor therapeutic effect, high recurrence rate and has a low survival rate. The survival of patients with HCC is closely related to the stage of diagnosis. At present, no specific serological indicator or method to predict HCC, early diagnosis of HCC remains a challenge, especially in China, where the situation is more severe. AIM: To identify risk factors associated with HCC and establish a risk prediction model based on clinical characteristics and liver-related indicators. METHODS: The clinical data of patients in the Affiliated Hospital of North Sichuan Medical College from 2016 to 2020 were collected, using a retrospective study method. The results of needle biopsy or surgical pathology were used as the grouping criteria for the experimental group and the control group in this study. Based on the time of admission, the cases were divided into training cohort (n = 1739) and validation cohort (n = 467). Using HCC as a dependent variable, the research indicators were incorporated into logistic univariate and multivariate analysis. An HCC risk prediction model, which was called NSMC-HCC model, was then established in training cohort and verified in validation cohort. RESULTS: Logistic univariate analysis showed that, gender, age, alpha-fetoprotein, and protein induced by vitamin K absence or antagonist-II, gamma-glutamyl transferase, aspartate aminotransferase and hepatitis B surface antigen were risk factors for HCC, alanine aminotransferase, total bilirubin and total bile acid were protective factors for HCC. When the cut-off value of the NSMC-HCC model joint prediction was 0.22, the area under receiver operating characteristic curve (AUC) of NSMC-HCC model in HCC diagnosis was 0.960, with sensitivity 94.40% and specificity 95.35% in training cohort, and AUC was 0.966, with sensitivity 90.00% and specificity 94.20% in validation cohort. In early-stage HCC diagnosis, the AUC of NSMC-HCC model was 0.946, with sensitivity 85.93% and specificity 93.62% in training cohort, and AUC was 0.947, with sensitivity 89.10% and specificity 98.49% in validation cohort. CONCLUSION: The newly NSMC-HCC model was an effective risk prediction model in HCC and early-stage HCC diagnosis.

Significance of the FGFR3 mutation in Chinese patients with bladder cancer.

Background: Bladder cancer (BC) is the 10th most common malignancy worldwide. The high recurrence rates of BC lead to significant treatment challenges. With the development of molecular biology techniques, research has shown that gene abnormalities are closely related to the occurrence and development of BC. This study analyzed the detection results of gene mutations in the tissue samples of BC patients and explored the relationship between fibroblast growth factor receptor 3 (FGFR3) and the prognosis and recurrence of BC. Methods: This study examined 82 Chinese patients with BC. Of these patients, 34 underwent radical cystectomy (RC), and 48 underwent transurethral resection with intravesical instillation. In addition, multi-gene panel targeted next-generation sequencing (NGS) of the samples was performed. Results: The mutational spectra revealed that C > T was the most common base substitution. Single nucleotide polymorphism (SNP) and deletion (DEL) were the common variant types in our cohort. The top 10 mutant genes were ROS1 (37%), PIK3CA (35%), FGFR3 (34%), BRAF (34%), ERBB2 (32%), ALK (27%), RET (27%), NTRK1 (24%), MET (23%), and EGFR (18%). FGFR3 mutations were detected more frequently in non-muscle-invasive bladder cancer (stages 0a, I) patients than in muscle-invasive bladder cancer (stage II, III, and IV) patients. The top 3 altered types of FGFR3 were p.Ser249Cys, p.Tyr375Cys, and p.Arg248Cys. Conclusions: This study examined the mutated types and frequency of FGFR3 and the prognosis of Chinese BC patients with FGFR mutations. We hope that our findings will enable clinical individualization strategies for BC patients to be optimized.

NDC80 status pinpoints mitotic kinase inhibitors as emerging therapeutic options in clear cell renal cell carcinoma.

∼30% of clear cell renal cell carcinoma (ccRCC) patients present with metastatic disease at the time of diagnosis, causing a dire 5-year survival rate of 13%. Although anti-PD-1 immunotherapy has improved survival, a strong need remains for new therapeutic options. Using integrated network analysis, we identified the mitotic regulator NDC80 as a predictor of ccRCC progression. Overexpression of NDC80 fosters the malignant phenotype by promoting cell cycle progression through S phase as well as boosting glycolysis and mitochondrial respiration. Despite high levels of immune infiltration, particularly derived from tumor resident CD8+T cells with an exhausted phenotype, NDC80 defines a class of ccRCCs that poorly respond to immune checkpoint blockade. Instead, bioinformatics identified NDC80-high ccRCCs as sensitive to inhibitors of mitotic kinases, PLK1 and AURK, therapeutic approaches we validated in cell lines and mouse xenograft studies. Thus, NDC80 status pinpoints mitotic kinase inhibitors as promising therapeutic options in difficult-to-treat ccRCCs.

Establishment and application of a survival rate graph model based on biomarkers and imaging indexes after primary hepatocellular carcinoma resection.

BACKGROUND: Primary liver cancer (PLC) is a highly malignant disease. This study developed an effective and convenient tool to evaluate survival times of patients after hepatectomy, which can provide a reference point for clinical decisions. METHODS: Clinical and laboratory data of 243 patients with PLC after hepatectomy were collected. Univariate cox regression analysis, Lasso regression analysis and multivariate cox regression analysis were used to determine the best prediction index. Multivariate cox regression analysis was used to construct a survival prediction model. A receiver operating characteristic (ROC) curve, calibration curve and decision curve analysis (DCA) were used to verify the model. The patients in this model were divided into was divided into high-risk and low-risk groups according to the optimal cut-off value of the ROC curve for different prognostic years. Kaplan-Meier survival analysis and log-rank test were used to analyse the survival differences between the two groups. RESULTS: Tumour size, portal vein thrombosis, distant metastasis, alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II levels were independent risk factors for overall survival (OS) after PLC surgery. The area under the concentration-time curve for 2-, 3- and 4-year survival of patients was 0.710, 0.825 and 0.919, respectively, with a good calibration of the Hosmer-Lemeshow test (p > 0.05) and net benefit. The mortality rates in patients with 2, 3 and 4 years of survival were 70.59%, 67.83% and 66.67% for the high-risk group and 21.84%, 22.50% and 22.67% for the low-risk group, respectively. The mortality rate of the high-risk group was significantly higher than that of the low-risk group (p < 0.05). CONCLUSION: The OS model of prognosis after PLC surgery constructed in this study can be used to predict the 2-, 3- and 4-year survival prognoses of patients, and patients with different prognosis years can be re-stratified so that they achieve more accurate and personalised assessment, thereby providing a reference point for clinical decision-making.

Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage.

BACKGROUND: Isothermal amplification is considered to be one of the most promising tools for point-of-care testing molecular diagnosis. However, its clinical application is severely hindered by nonspecific amplification. Thus, it is important to investigate the exact mechanism of nonspecific amplification and develop a high-specific isothermal amplification assay. METHODS: Four sets of primer pairs were incubated with Bst DNA polymerase to produce nonspecific amplification. Gel electrophoresis, DNA sequencing, and sequence function analysis were used to investigate the mechanism of nonspecific product generation, which was discovered to be nonspecific tailing and replication slippage mediated tandem repeats generation (NT&RS). Using this knowledge, a novel isothermal amplification technology, bridging primer assisted slippage isothermal amplification (BASIS), was developed. RESULTS: During NT&RS, the Bst DNA polymerase triggers nonspecific tailing on the 3'-ends of DNAs, thereby producing sticky-end DNAs over time. The hybridization and extension between these sticky DNAs generate repetitive DNAs, which can trigger self-extension via replication slippage, thereby leading to nonspecific tandem repeats (TRs) generation and nonspecific amplification. Based on the NT&RS, we developed the BASIS assay. The BASIS is carried out by using a well-designed bridging primer, which can form hybrids with primer-based amplicons, thereby generating specific repetitive DNA and triggering specific amplification. The BASIS can detect 10 copies of target DNA, resist interfering DNA disruption, and provide genotyping ability, thereby offering 100% accuracy for type 16 human papillomavirus detection. CONCLUSION: We discovered the mechanism for Bst-mediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity.

Pan-Cancer Analysis of Histone Methyltransferase KMT2D with Potential Implications for Prognosis and Immunotherapy in Human Cancer.

BACKGROUND: Pan-cancer analysis is an efficient tool to obtain a panoramic view of cancer- related genes and identify their oncogenic processes, facilitating the development of new therapeutic targets. Lysine methyltransferase 2D (KMT2D), acting as a major enhancer coactivator for mammalian cells, is one of the most frequently mutated genes across various cancer types and is considered an oncogene and a rationale for epigenetic therapeutic targets. OBJECTIVE: This study was designed to explore the potential role of KMT2D in human cancer through a pan-cancer analysis. METHODS: The expression of KMT2D was assessed in normal tissues and cell lines, and pancancers from The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTE) datasets were used to explore its correlation with prognosis, immune cell infiltration, tumor mutation burden, microsatellite instability, and mismatch repair. RESULTS: KMT2D expression was heterogeneous across different cancer types. Increased KMT2D indicated a worse prognosis in adrenocortical carcinoma (ACC), brain lower-grade glioma (LGG), and mesothelioma (MESO), while patients with high KMT2D expression showed better outcomes in renal clear cell carcinoma (KIRC). Moreover, KMT2D expression was positively correlated with immune cell infiltration and negative tumor mutation burden in multiple cancers. In addition, a significant correlation between KMT2D and immune checkpoint-related genes or mismatch repair genes was identified. CONCLUSIONS: These findings support the hypothesis that KMT2D is not only a potential biomarker for prognosis and immunotherapy response prediction but also an essential immune regulator in human cancer.

Nucleic acid amplification with specific signal filtration and magnification for ultrasensitive colorimetric detection.

Recently, sensitive, fast and low cost nucleic acid isothermal amplification technologies (such as loop-mediated isothermal amplification, LAMP) have attracted great attention in the urgent needs of point-of-care testing (POCT) and regular epidemic prevention and control. However, unlike PCR which usually employs TaqMan probe to report specific signals, specific-signal-output strategies in isothermal amplification are immature and visual detection even rare, which limits their popularity in POCT. We hypothesize to address this issue by designing a visual-signal-report system to both filtrate and magnify the target information in isothermal amplification. In this work, we developed a specific signal filtration and magnification colorimetric isothermal sensing platform (SFMC for short) for ultrasensitive detection of DNA and RNA. SFMC consists of two processes: an isothermal amplification with specific signal filtration and a self-replication catalyzed hairpin assembly (SRCHA) for rapid target-specific signal magnification and outputting. With these unique properties, this biosensing platform could detect target DNA as low as 5 copies per reaction and target RNA as low as 10 copies per reaction by naked eyes. Benefited from the excellent colorimetric detection performance, this biosensing platform has been successfully used for African swine fever virus (ASFV) and SARS-CoV-2 detection.

DLEU7-AS1 promotes renal cell cancer by silencing the miR-26a-5p/coronin-3 axis.

Long non-coding RNAs (lncRNAs) have been implicated in the progression and development of many types of cancer by interacting with RNA, DNA and proteins, including DLEU7-AS1. However, the function of DLEU7-AS1 in renal cell cancer (RCC) remains unclear. In this study, two in silico prediction algorithms were used to discover the potential target of miR-26a-5p, which was determined to be a tumor suppressor gene, possibly DLEU7-AS1, through the downregulation of coronin-3 in RCC. Thus, we hypothesized that DLEU7-AS1 promotes RCC by silencing the miR-26a-5p/coronin-3 axis. To test our hypothesis, we confirmed that DLEU7-AS1 directly targets miR-26a-5p using the pmirGLO dual-luciferase reporter assay. Next, we observed that DLEU7-AS1 expression was markedly upregulated in RCC samples and inversely correlated with clinical prognosis and miR-26a-5p levels. Knockdown of DLEU7-AS1 significantly suppressed the growth and metastasis of RCC cells in vitro and attenuated tumor growth in vivo. Interestingly, exogenous expression of coronin-3 or miR-26a-5p inhibitor treatment almost completely rescued the DLEU7-AS1 knockdown-induced inhibitory effects on cell proliferation, migration and invasion. In conclusion, our data demonstrate that DLEU7-AS1 is an oncogene in RCC capable of regulating the growth and metastasis of RCC by silencing the miR-26a-5p/coronin-3 axis, suggesting that DLEU7-AS1 can be employed as a potential therapeutic target and prognostic biomarker for RCC.

Is radiotherapy helpful for the prognosis of upper urinary tract urothelial carcinoma? A meta-analysis.

BACKGROUND: At present, the guidelines of the European Society of Urology regarding the use of adjuvant radiotherapy for patients with upper tract urothelial carcinoma (UTUC) are not clear, and there are many contradictions in previous research. In addition, several studies on the effect of radiotherapy alone on UTUC patients have been published in recent years, but their results are still inconsistent. To address these problems, we conducted the current meta-analysis. METHOD: Articles were searched in 3 electronic databases: PubMed, Embase and the Cochrane Library. Studies were filtered according to a selection strategy, and data were extracted. Finally, Stata software was used for sensitivity analysis, and Egger's test was used to calculate the stability of the results and determine whether there was publication bias. RESULT: In total, 17 studies involving and 25906 patients were included, adjuvant radiotherapy had no significant effect on patients with UTUC (OS: HR = 1.38 [0.94, 2.03], = P 0.10; CSS: HR = 1.43 [0.92, 2.25], P = 0.11; LRFS: HR = 1.40 [0.99, 1.98], P = 0.06; RFS: HR = 1.19 [0.52, 2.73], P = 0.68; MFS: HR = 1.37 [0.35, 5.31], P = 0.65). Radiotherapy alone had adverse effects on patients with UTUC (OS: HR = 1.25 [1.09, 1.43], P = 0.001, CSS: HR = 1.47 [1.37, 1.59], P < 0.00001). CONCLUSION: Our results show that adjuvant radiotherapy has no significant effect on the prognosis of patients with UTUC, while radiotherapy alone will have an adverse effect on patients with UTUC. In the future, the determination of the tumor stage of patients receiving adjuvant radiotherapy may be an important research direction.

A meta-analysis of the platelet-lymphocyte ratio: A notable prognostic factor in renal cell carcinoma.

The platelet-lymphocyte ratio (PLR) has been assessed in some studies on renal cell carcinoma (RCC), but the results have been inconsistent. This meta-analysis aims to review and report the latest data regarding the prognostic role of the PLR in RCC patients. Articles were searched in the PubMed, EMBASE, and Cochrane Library electronic databases. Studies were filtered according to a selection strategy, and data corresponding to the index of interest were extracted. A fixed-effects model or random-effects model was selected based on heterogeneity. The sensitivity analysis was carried out by eliminating the studies one by one. Finally, funnel plots and Egger's test were used to assess publication bias, and the trim and fill method was used to assess the impact of bias on the results. In total, 15,193 patients with RCC from 44 studies were included in this meta-analysis. The pooled analysis indicated that the higher the PLR was, the poorer the prognosis for RCC patients in terms of overall survival (hazard ratio (HR) = 1.01 (95% confidence interval (CI) 1.00, 1.02), P = 0.010), cancer-special survival (CSS) (HR = 1.21 (95% CI 1.00, 1.46), P = 0.05), progression-free survival (HR = 1.44 (95% CI 1.28, 1.62), P < 0.00001), recurrence-free survival (HR = 1.73 (95% CI 1.11, 2.71), P = 0.02), disease-free survival (HR = 1.63 (95% CI 0.91, 2.94), P = 0.01) and metastasis-free survival (HR = 1.223 (95% CI 0.712, 2.099), P = 0.466). In the subgroup analysis of high PLR, targeted treatment, TKI use, nivolumab use, surgical treatment, clear cell RCC, metastasis, Asian race, and high PLR were related to poor prognosis. This study showed that a high PLR was associated with the poor prognosis of RCC patients, but more studies are needed to confirm the value of the PLR.

Metabolic targeting of cancer by a ubiquinone uncompetitive inhibitor of mitochondrial complex I.

SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI. SMIP004-7 and an improved chemical analog selectively engage NDUFS2 in vivo to inhibit the growth of triple-negative breast cancer transplants, a response mediated at least in part by boosting CD4+ and CD8+ T cell-mediated immune surveillance. Thus, SMIP004-7 defines an emerging class of ubiquinone uncompetitive CI inhibitors for cell autonomous and microenvironmental metabolic targeting of mitochondrial respiration in cancer.

A novel microfluidic RNA chip for direct, single-nucleotide specific, rapid and partially-degraded RNA detection.

Direct RNA detection is critical for providing the RNA insights into gene expression profiling, noncoding RNAs, RNA-associated diseases and pathogens, without reverse transcription. However, classical RNA analysis usually requires RT-PCR, which can cause bias amplification and quantitation errors. To address this challenge, herein we report a microfluidic RNA chip (the microchip prototype) for direct RNA detection, which is primarily based on RNA extension and labeling with DNA polymerase. This detection strategy is of high specificity (discriminating against single-nucleotide differences), rapidity, accuracy, nuclease resistance, and reusability. Further, we have successfully detected disease-associated RNAs in clinical samples, demonstrating its great potentials in biomedical research and clinical diagnosis.

LGALS1 was related to the prognosis of clear cell renal cell carcinoma identified by weighted correlation gene network analysis combined with differential gene expression analysis.

Understanding the molecular mechanism of clear cell renal cell carcinoma (ccRCC) is essential for predicting the prognosis and developing new targeted therapies. Our study is to identify hub genes related to ccRCC and to further analyze its prognostic significance. The ccRCC gene expression profiles of GSE46699 from the Gene Expression Omnibus (GEO) database and datasets from the Cancer Genome Atlas Database The Cancer Genome Atlas were used for the Weighted Gene Co-expression Network Analysis (WGCNA) and differential gene expression analysis. We screened out 397 overlapping genes from the four sets of results, and then performed Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathways. In addition, the protein-protein interaction (PPI) network of 397 overlapping genes was mapped using the STRING database. We identified ten hub genes (KNG1, TIMP1, ALB, C3, GPC3, VCAN, P4HB, CHGB, LGALS1, EGF) using the CytoHubba plugin of Cytoscape based on the Maximal Clique Centrality (MCC) score. According to Kaplan-Meier survival analysis, higher expression of LGALS1 and TIMP1 was related to poorer overall survival (OS) in patients with ccRCC. Univariate and multivariate Cox proportional hazard analysis showed that the expression of LGALS1 was an independent risk factor for poor prognosis. Moreover, the higher the clinical grade and stage of ccRCC, the higher the expression of LGALS1. LGALS1 may play an important role in developing ccRCC and may be potential a biomarker for prognosis and treatment targets.

Hsa_circ_0030586 promotes epithelial-mesenchymal transition in prostate cancer via PI3K-AKT signaling.

Circular RNAs (CircRNAs) gain importance as regulatory molecules in prostate cancer (PCa), but molecular mechanism of most circRNAs in pathogenesis of PCa remains to be studied. This study aimed to explore the role of hsa_circ_0030586 in PCa. Gene Expression Omnibus database (GSE77661) was used to screen out candidate circRNAs. Quantitative real-time PCR was used to verify the relative expressions of circRNAs, miRNAs, and genes in PCa cells. A CCK-8 assay was used to evaluate the cells' proliferation. Transwell and wound healing assay were used to determine the cells' migration and invasion. Western blotting and immunohistochemistry were used to detect the protein expression of PI3K/AKT signaling proteins and epithelial-mesenchymal transition (EMT) markers. Furthermore, a nude mice tumorigenesis experiment in vivo was conducted to determine the function of hsa_circ_0030586 on PCa. Our results showed that hsa_circ_0030586 is significantly upregulated in PCa cells (p < 0.05). Its circular structure was confirmed via agarose gel electrophoresis and Sanger sequencing. Interfering with hsa_circ_0030586 in PC3 cells inhibited cell proliferation, migration, and invasion and led to the significant upregulation of E-cadherin and the significant downregulation of p-AKT/AKT, IKKα, PIK3CB, and Twist (all p < 0.05). Conversely, the hsa_circ_003058 interference fragment combined with the transfection of a miR-145-3p inhibitor could reverse the above effects. In vivo tumorigenesis of the xenograft model confirmed that interfering with hsa_circ_0030586 suppressed tumor cell proliferation and inhibited PI3K-AKT signaling and EMT in PC3 cells. Hsa_circ_0030586 is significantly upregulated in PCa cells and may promote EMT via PI3K-AKT signaling.

Signal Extraction, Transformation, and Magnification for Ultrasensitive and Specific Detection of Nucleic Acids.

Nucleic acid noises caused by the background and nonspecificity amplifications can jeopardize accurate polymerization and detection of nucleic acids, especially when they are analyzed in low copies. We hypothesize to reduce the noises by designing a system for specific signal extraction, transformation, and magnification to improve the specificity and sensitivity. Herein, by developing an extractor-trigger complex (ET-Combo) for the system, we have established isothermal and hybridizing combined amplifications: a one-pot detection system with two-step amplification coupled by ET-Combo. To our surprise, the signal extraction is only successful when ET-Combo is included in the first amplification. Our signal extracting, filtering, and relaying system with ET-Combo is rapid and specific, removing the noises generated during the isothermal amplification under elevated temperatures. To match the first amplification, we have designed and established a hybridizing chain reaction at high temperature. This one-pot system can resist disruption of background noises and allow detection of DNA up to five copies (single digit). With the high sensitivity, specificity, and noise resistance, our system has been successfully used to diagnose clinical samples of human papillomavirus (HPV) with the genotyping specificity.

Selenium atom on phosphate enhances specificity and sensitivity of DNA polymerization and detection.

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.