A spacer design strategy for CRISPR-Cas12f1 with single-nucleotide polymorphism mutation resolution capability and its application in the mutations diagnosis of pathogens.

Infectious diseases remain a major global issue in public health. It is important to develop rapid, sensitive, and accurate diagnostic methods to detect pathogens and their mutations. Cas12f1 is an exceptionally compact RNA-guided nuclease and have the potential to fulfill the clinical needs. Based on the interaction between crRNA-SSDNA binary sequence and Cas12f1, here, we addressed the essential features that determine the recognition ability of CRISPR-Cas12f1 single-nucleotide polymorphism (SNP), such as the length of spacer region and the base pairing region that determines the trans-cleavage of ssDNA. A fine-tuning spacer design strategy is also proposed to enhance the SNP recognition capability of CRISPR-Cas12f1. The optimized spacer confers the Cas12f1 system a strong SNP identification capability for viral or bacterial drug-resistance mutations, with a specificity ratio ranging from 19.63 to 110.20 and an admirable sensitivity up to 100  copy/µL. Together, the spacer screening and CRISPR-Cas12f1 based SNP identification method, is sensitive and versatile, and will have a wide application prospect in pathogen DNA mutation diagnosis and other mutation profiling.

What contributes to the re-positive nucleic acid test results for the omicron variant of SARS-CoV-2 in the shelter cabin hospital in Shanghai, China?

Background: Despite the increasing reports of re-positive SARS-CoV-2 cases after recovery and discharge from hospitals, our knowledge remains very limited regarding the contributing factors of re-positivity and its roles in the transmission and epidemiology of the Omicron variant. Methods: In this retrospective study, re-positivity is defined as the positive nucleic acid result (Ct < 35) following two consecutive negative results during hospitalization. A total of 751 patients from Shanghai Shelter Cabin Hospital were enrolled and divided with a ratio of about 1:2 into the re-positivity group and the non-re-positivity group. Patients required three consecutive negative results daily as the de-isolation criterion. The follow-up time of discharged patients lasted five weeks. Univariate regression analysis was used to compare variables between the re-positivity and non-re-positivity groups, and the single re-positivity and multiple re-positivity groups, with P < 0.05 defined as the statistical significance of differences. Subsequently, variables with P < 0.2 were subjected to multivariate logistic regression analysis to investigate the odds ratio (OR) of re-positivity and the influencing factors of re-positivity of the Omicron variant. Results: The re-positivity group had a higher proportion of males (68.1% vs 58.1%, p = 0.000), a higher education level (31.9% vs 12.7%, p = 0.007), a longer hospitalization duration (13 days vs 8 days, p = 0.000), and a higher Convidecia vaccination rate (6.0% vs 2.4%, p = 0.011). Further multivariable analysis showed male (OR = 2.168, p = 0.000), Convidecia vaccination (OR = 2.634, p = 0.014), hospitalization duration (OR = 2.146, p = 0.000) and education level (OR = 1.595, p = 0.007) were associated with re-positivity. The average rate of re-positivity was 25% during hospitalization and decreased to 0.4% among discharged patients. Re-positivity was more common in the period with a larger number of hospitalized patients and in larger wards with a larger number of patients. Conclusion: A large number of hospitalized patients, large-sized wards, and gender are significant contributing factors to re-positivity. Division of the shelter cabin hospital into small independent wards and requirement of three consecutive results daily as the de-isolation criterion might be more beneficial to the control and prevention of the spread of the Omicron variant.

The Prognostic Value of Serum HBV-RNA during Hepatitis B Virus Infection is Related to Acute-on-Chronic Liver Failure.

Background: Serum HBV-RNA levels can predict antiviral response in chronic hepatitis B (CHB) patients; however, its role in HBV-related ACLF (HBV-ACLF) remains unclear. Here, we determined its implications for HBV-ACLF. Methods: Baseline serum HBV-RNA levels were retrospectively detected in HBV-ACLF and CHB patients. The association of serum HBV-RNA level with clinical outcomes was evaluated by performing multiple logistic regression. A nomogram was developed to formulate an algorithm incorporating serum HBV-RNA for predicting the survival of HBV-ACLF patients. After being discharged from the hospital, the HBV-ACLF patients were followed up for 36 weeks. Results: In this study, 82 HBV-ACLF patients and 33 CHB patients were included. Serum HBV-RNA levels were significantly higher in CHB patients than in HBV-ACLF patients (4.15 ± 2.63 log10 copies/mL VS 5.37 ± 2.02 log10 copies/mL) (P < 0.05). Among the HBV-ACLF cases, patients with poor outcomes had lower serum HBV-RNA levels, but the difference was not significant. The area under the receiver operating characteristic curve of the serum HBV-RNA inclusive model was 0.745, superior to 0.66 from MELD scores (P < 0.05). During the follow-up for four weeks, the serum HBV-RNA levels, especially in the survival group, were found to be lower than the baseline levels. Conclusions: Serum HBV-RNA levels were associated with disease severity and might predict the long-term clinical outcome of HBV-ACLF patients.

A Highly Sensitive and Specific Detection Method for Mycobacterium tuberculosis Fluoroquinolone Resistance Mutations Utilizing the CRISPR-Cas13a System.

Objectives: CRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear. Methods: A combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed. Results: A set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated Mycobacterium tuberculosis strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated M. tuberculosis, the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays. Conclusion: These results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.

Application of TGF-ß1, TIMP-1 and TIMP-2 small interfering RNAs can alleviate CCl4-induced hepatic fibrosis in rats by rebalancing Th1/Th2 cytokines.

The present study aimed to investigate the effects of TGF-ß1, tissue inhibitor of metalloproteinase (TIMP)-1 small interfering (si)RNA and TIMP-2 siRNA on hepatic fibrosis in rats and explore the T helper (Th)1/Th2 balance. Moreover, IFN-γ, IL-4 and IL-13 are the main cytokines associated with Th1/Th2 responses and have significant influence on the progression of hepatic fibrosis. The expression levels of IFN-γ, IL-4 and IL-13 in rats with hepatic fibrosis that were treated with siRNAs against the aforementioned molecules were measured using various techniques including immunohistochemical staining, western blotting and reverse transcription-quantitative PCR. The principal outcomes revealed the downregulation of IFN-γ and the upregulation of IL-4 and IL-13 in the model group compared with the normal group. Moreover, the expression of IFN-γ was significantly increased, while IL-4 and IL-13 demonstrated no significant difference in the TGF-ß1 siRNA treatment group compared with the model group. The TIMP-1 and TIMP-2 siRNA treatment groups exhibited significantly increased expression levels of IFN-γ, but lower expression levels of IL-4 and IL-13 compared with the model group. These results indicated that TIMP-1 and TIMP-2 were improved antifibrotic targets compared with TGF-ß1.

Functions and Mechanisms of Lysine Glutarylation in Eukaryotes.

Lysine glutarylation (Kglu) is a newly discovered post-translational modification (PTM), which is considered to be reversible, dynamic, and conserved in prokaryotes and eukaryotes. Recent developments in the identification of Kglu by mass spectrometry have shown that Kglu is mainly involved in the regulation of metabolism, oxidative damage, chromatin dynamics and is associated with various diseases. In this review, we firstly summarize the development history of glutarylation, the biochemical processes of glutarylation and deglutarylation. Then we focus on the pathophysiological functions such as glutaric acidemia 1, asthenospermia, etc. Finally, the current computational tools for predicting glutarylation sites are discussed. These emerging findings point to new functions for lysine glutarylation and related enzymes, and also highlight the mechanisms by which glutarylation regulates diverse cellular processes.

Diagnostic and prognostic value of ABC transporter family member ABCG1 gene in clear cell renal cell carcinoma.

As the most common histologic subtype of renal cancer, clear cell renal cell carcinoma (ccRCC) poses a serious threat to public health. However, there are no specific molecular-targeted drugs for ccRCC at present. Human ATP-binding cassette (ABC) transporter family plays an important role in homeostasis maintenance. This study aimed to evaluate the potential diagnostic value of ABC genes in ccRCC. A total of 952 samples of ccRCC patients (707) and controls (245) from three different datasets were included for analysis. Receiver operating characteristic analysis and t-test were used to analyze the differential expression of ABC genes in ccRCC patients and control samples at mRNA level during screening and validations. The Cancer Genome Atlas (TCGA-ccRCC) dataset was utilized to investigate the correlation between ABC genes expression and prognostic value in ccRCC. We then investigated the interactions between ABCG1 and proteins in the Comparative Toxicogenomics Database (CTD). Finally, we found that ATP-binding cassette transporter G member 1 (ABCG1) was over-expressed in ccRCC patients compared with healthy samples at mRNA level. Cox regression analysis and Kaplan-Meier analysis showed that ccRCC patients with high ABCG1 expression had better overall survival (OS) than those patients with low expression (hazard ratio (HR) = 0.662, p = 0.007). This study demonstrated that ABCG1 is a potential diagnostic and prognostic biomarker in ccRCC and discussed the molecular mechanisms underlying the relationship between ccRCC and ABCG1, which might provide guidance for better management and treatment of ccRCC in the future.

CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia.

Emerging infectious diseases, the persistent potential for destabilising pandemics, remain a global threat leading to excessive morbidity and mortality. The current outbreak of pneumonia caused by 2019 novel coronavirus (COVID-19) illustrated difficulties in lack of effective drugs for treatment. Accurate and rapid diagnostic tools are essential for early recognition and treatment of infectious diseases, allowing timely implementation of infection control, improved clinical care and other public health measures to stop the spread of the disease. CRISPR-Cas technology speed up the development of infectious disease diagnostics with high rapid and accurate. In this review, we summarise current advance regarding diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13, in the development of fast, accurate and portable diagnostic tests and highlight the potential of CRISPR-Cas13 in COVID-19 Pneumonia and other emerging infectious diseases diagnosis.

Multiple-Scales Integrative Analysis of MicroRNAs Unveils Biomarkers and Key Regulatory Connections for Hepatocellular Carcinoma.

Hepatocellular carcinoma is the sixth most common liver cancer worldwide and the third leading cause of cancer mortality. For these reasons, early diagnostic, prognostic, and therapeutic biomarkers are extremely urgent. MicroRNAs are small noncoding single-stranded RNA molecules that are reported to be involved in a variety of physiological and pathological processes during carcinogenesis. In this study, the expression characteristics, functions, and validated targets of microRNAs in HCC were summarized and potential microRNA biomarkers were confirmed from clinical specimens. Multiple-scales integrative analysis was used to predict the diagnostic, prognostic, and therapeutic potential of microRNAs in blood and tissue of HCC patients, providing novel insight into HCC diagnosis and treatment using microRNA biomarkers.

Quasispecies characters of hepatitis B virus in immunoprophylaxis failure infants.

Hepatitis B vaccination prevents 80-95% of transmission and reduces the incidence of HBV in children. The variations in the a determinant of HBV surface antigen (HBsAg) have been reported to be the most prevalent cause for vaccine or antibody escape. There is a conflicting evidence on as to whether escape mutants arise de novo in infected infants or whether the mutants, that have preexisted maternally, subsequently undergo selective replication in the infant under immune pressure. Here, we report that nearly 65% (55 of 85) vaccination failure in child patients has no amino acid substitution in a determinant as seen by Sanger sequencing. We further employed an Illumina sequencing platform-based method to detect HBV quasispecies in four immunoprophylaxis failure infants and their mothers. In our data, the substitution rate of amino acid located at a determinant is relatively low (< 10%), I/T126A, C124S, F134Y, K141Q, Q129H, D144A, G145V, and N146K, which showed no statistical difference to their mothers, proving that these vaccine escape mutants preexist maternally as minor variants. Besides that, bioinformatical analysis showed that the binding affinity of high variation epitopes (amino acid divergence in mother and their infants > 20%) to related HLA molecules was generally decreased, these traces of immune escape suggesting that immune pressure was present and was effective in all samples.

[Effect of TGF-b1 siRNA-mediated silencing on Smad proteins in hepatic fibrosis rats].

OBJECTIVE: To investigate the changes in Smad 2, 3, 4 and 7 of the transforming growth factor-beta 1 (TGF-b1)/Smad signaling pathways in carbon tetrachloride (CCL4)-induced hepatic fibrosis rats treated with TGF-b1 small interfering (si)RNA. METHODS: Rats were randomly divided among five groups: non-fibrotic (normal); fibrosis-induced (model); fibrotic treated with 0.125 mg/kg TGF-b1 siRNA; fibrotic treated with 0.250 mg/kg TGF-b1 siRNA; and fibrotic treated with negative control TGF-b1 siRNA. The expression of Smad 2, 3, 4 and 7 was detected by real-time polymerase chain reaction (for mRNA), immunohistochemistry and Western blotting (for protein). RESULTS: The mRNA and protein levels of Smad 2, 3 and 4 were significantly lower in the the fibrotic rats treated with either 0.250 mg/kg or 0.125 mg/kg TGF-b1 siRNA than in the fibrotic model or the negative control TGF-b1 siRNA rats (P less than 0.01). Moreover, the mRNA and protein expression levels of Smad 2, 3 and 4 were significantly lower in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05). Comparing the 0.250 mg/kg and 0.125 mg/kg TGF-b1 siRNA groups to the model group and the TGF-b1 siRNA negative control group showed significantly increased levels of mRNA and protein expression of Smad 7 (P less than 0.01). In addition, the expression levels of Smad 7 were significantly higher in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05). CONCLUSION: siRNA-mediated silencing of TGF-b1 in rats led to significantly reduced expression of Smad 2, 3 and 4, but significantly increased expression of Smad 7. TGF-b1 regulation of Smad signaling molecules may contribute to hepatic fibrosis in rats and represent a target of future therapeutic intervention.

The antifibrotic effects of TGF-ß1 siRNA on hepatic fibrosis in rats.

BACKGROUND/AIMS: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-ß1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-ß1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). METHODS: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-ß1 siRNA 0.125mg/kg treatment group, TGF-ß1 siRNA 0.25mg/kg treatment group and TGF-ß1 siRNA negative control group (0.25mg/kg). CCL4 and a high-fat diet were used for 8weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-ß1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-ß1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-ß1 by quantitative real-time polymerase chain reaction. RESULTS: Comparing the TGF-ß1 siRNA 0.25mg/kg treatment group to the model group, the TGF-ß1 siRNA negative control group and the TGF-ß1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the mRNA expression of TGF-ß1, type I collagen and type III collagen (P<0.01). CONCLUSIONS: Using siRNA to target TGF-ß1 can inhibit the expression of TGF-ß1 and attenuate rat hepatic fibrosis induced by a high-fat diet and CCL4. A possible mechanism is through the down-regulation of TGF-ß1 expression, which could inhibit HSC activation, as well as the proliferation and collagen production of collagen reducing, so that collagen deposition in the liver is reduced.

[Construction and identification of siRNA eukaryotic expression vectors targeting on TGFß1, TIMP-1 and TIMP-2 genes in vitro].

OBJECTIVE: To construct the siRNA eukaryotic expression vectors targeting on TGFß1, TIMP-1 and TIMP-2 and to investigate the inhibitory efficiency of target genes expression on rat hepatic stellate cell in vitro. METHODS: The siRNA cDNA sequences of TGFß1, TIMP-1 and TIMP-2 were designed, synthesized and inserted into plasmid pGenesil-1 respectively to generate eukaryotic expression plasmids. The plasmids were transfected into HSC T6 cells in vitro and the inhibitory efficiency of target genes expression was observed with real-time PCR and Western blot. RESULTS: The eukaryotic expression vectors were constructed successfully. The expressions of TGFß1 mRNA, TIMP-1 mRNA and TIMP-2mRNA in siRNA-transfected groups were decreased by 63.4% ± 8.0%, 64.5% ± 9.0% and 55.0% ± 17.0% respectively and the expressions of TGFß1 protein, TIMP-1 protein and TIMP-2 protein were decreased by 57.8% ± 3.0%, 55.1% ± 5.0%, 49.3% ± 1.0% respectively as compared to the control groups. CONCLUSIONS: The siRNA eukaryotic expression vectors constructed targeting on TGFß1, TIMP-1 and TIMP-2 could reduce the expressions of target genes and they might be able to used for the exploration of new anti-fibrosis drugs genetically.