CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output.

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

Metagenomics as New Tool for Diagnosis of Scrub Typhus: Two Case Reports.

Scrub typhus is a vector-borne infectious disease caused by Orientia tsutsugamushi. Accurate and timely diagnosis at the early infection stage could save the patients' lives. Traditional technologies were limited to rapidly and successfully detecting Orientia tsutsugamushi due to poor specificity, especially in the condition of atypical symptoms. The technology of Metagenomic next-generation sequencing (mNGS) is amenable to finding the real pathogen because it holds potential as a diagnostic platform for unbiased pathogen identification and precision medicine. Herein, we reported two clinical case reports relative to the Orientia tsutsugamushi infection diagnosed by mNGS. We hope these two cases will improve clinical diagnosis.

m6A-Related lncRNAs Predict Overall Survival of Patients and Regulate the Tumor Immune Microenvironment in Osteosarcoma.

Background: m6A-related lncRNAs have demonstrated great potential tumor diagnostic and therapeutic targets. The goal of this work was to find m6A-regulated lncRNAs in osteosarcoma patients. Method: The Cancer Genome Atlas (TCGA) database was used to retrieve RNA sequencing and medical information from osteosarcoma sufferers. The Pearson's correlation test was used to identify the m6A-related lncRNAs. A risk model was built using univariate and multivariable Cox regression analysis. Kaplan-Meier survival analysis and receiver functional requirements were used to assess the risk model's performance (ROC). By using the CIBERSORT method, the associations between the relative risks and different immune cell infiltration were investigated. Lastly, the bioactivities of high-risk and low-risk subgroups were investigated using Gene Set Enrichment Analysis (GSEA). Result: A total of 531 m6A-related lncRNAs were obtained from TCGA. Seven lncRNAs have demonstrated prognostic values. A total of 88 OS patients were separated into cluster 1, cluster 2, and cluster 3. The overall survival rate of OS patients in cluster 3 was more favorable than that of those in cluster 1 and cluster 2. The average Stromal score was much higher in cluster 1 than in cluster 2 and cluster 3 (P < 0.05). The expression levels of lncRNAs used in the construction of the risk prediction model in the high-risk group were generally lower than those in the low-risk group. Analysis of patient survival indicated that the survival of the low-risk group was higher than that of the high-risk group (P < 0.0001) and the area under the curve (AUC) of the ROC curve was 0.719. Using the CIBERSORT algorithm, the results revealed that Macrophages M0, Macrophages M2, and T cells CD4 memory resting accounted for a large proportion of immune cell infiltration. By GSEA analysis, our results implied that the high-risk group was mainly involved in unfolded protein response, DNA repair signaling, and epithelial-mesenchymal transition signaling pathway and glycolysis pathway; meanwhile, the low-risk group was mainly involved in estrogen response early and KRAS signaling pathway. Conclusion: Our investigation showed that m6A-related lncRNAs remained tightly connected to the immunological microenvironment of osteosarcoma tumors, potentially influencing carcinogenesis and development. The immune microenvironment and immune-related biochemical pathways can be changed by regulating the transcription of M6A modulators or lncRNAs. In addition, we looked for risk-related signaling of m6A-related lncRNAs in osteosarcomas and built and validated the risk prediction system. The findings of our current analysis will facilitate the assessment of outcomes and the development of immunotherapies for sufferers of osteosarcomas.

Internal fixation using fully threaded cannulated compression screws for fresh femoral neck fractures in adults.

OBJECTIVES: Internal fixation with multiple cannulated compression screws is an optional treatment for femoral neck fracture. Recently, fully threaded cannulated compression screws (FTCCS) have been introduced to fix fresh femoral neck fractures (FNF). The purpose of this study was to investigate the effectiveness of FTCCS. PATIENTS AND METHODS: Patients with FNF fixed by multiple FTCCS from February 1st, 2014 to August 31st, 2017 were included in this study. They were followed for at least 12 months postoperatively. Nonunion, osteonecrosis of the femoral head (ONFH), fixation failure, reoperation, and femoral neck shortening (FNS) were used to evaluate the outcomes. Risk factors including age, sex, fracture side, fracture displacement, fracture stability, fixation configuration, and screw numbers were analyzed. RESULTS: A total of 113 patients including 67 males and 46 females with an average age of 48.4 ± 13.4 years were included. The mean duration of follow-up was 27.1 months (range: 12-51 months). The incidence of nonunion, ONFH, fixation failure, and reoperation was 15.9%, 22.1%, 8.8%, and 24.8%, respectively. The rates of nonunion and reoperation were significantly higher in displaced fractures and unstable fractures. And patients with an unstable fracture had a higher risk of internal fixation failure. The median length of FNS was 2.9 mm (interquartile range: 0.9-6.5 mm, range: 0-17.5 mm). Age was a significant risk factor for FNS. CONCLUSIONS: The screw fixation method with FTCCS provided encouraging clinical results which may be a rational choice for the treatment of fresh FNF. Displaced fractures and unstable fractures were attributed to the higher incidence of complications. TRIAL REGISTRATION: ChiCTR, ChiCTR1800017200. Registered 17 July 2018-Retrospectively registered, http: www.chictr.org.cn/showprojen.aspx?proj=29182 .