meHOLMES: A CRISPR-cas12a-based method for rapid detection of DNA methylation in a sequence-independent manner.

Aberrant DNA methylation is closely associated with various diseases, particularly cancer, and its precise detection plays an essential role in disease diagnosis and monitoring. In this study, we present a novel DNA methylation detection method (namely meHOLMES), which integrates both the TET2/APOBEC-mediated cytosine deamination step and the CRISPR-Cas12a-based signal readout step. TET2/APOBEC efficiently converts unmethylated cytosine to uracil, which is subsequently changed to thymine after PCR amplification. Utilizing a rationally designed crRNA, Cas12a specifically identifies unconverted methylated cytosines and generates detectable signals using either fluorescent reporters or lateral flow test strips. meHOLMES quantitatively detects methylated CpG sites with or without Protospacer Adjacent Motif (PAM) sequences in both artificial and real biological samples. In addition, meHOLMES can complete the whole detection process within 6 h, which is much faster than traditional bisulfite-based sample pre-treatment method. Above all, meHOLMES provides a simpler, faster, more accurate, and cost-effective approach for quantitation of DNA methylation levels in a sequence-independent manner.

Construction and validation of a predictive model for hepatocellular carcinoma based on serum markers.

BACKGROUND: Early hepatocellular carcinoma (HCC) detection with non-invasive biomarkers remains an unmet clinical need. We aimed to construct a predictive model based on the pre-diagnostic levels of serum markers to predict the early-stage onset of HCC. METHODS: A total of 339 HCC patients (including 157 patients from Changzhou cohort and 182 patients from Wuxi cohort) were enrolled in our retrospective study. Levels of 25 baseline serum markers were collected. Propensity score matching (PSM) analysis was conducted to balance the distributions of patients' gender, age, and the surveillance time between HCC group and control group. Then, Receiver operating characteristic (ROC) and Logistic regression analysis were performed to screen the independent predictive variables and construct a non-invasive predictive model. Subsequently, ROC curve and Kaplan-Meier (K-M) curve were used to evaluate the predictive values of the model. Clinical net benefit of the model was demonstrated by decision curve analysis (DCA) and clinical impact curve. RESULTS: Five independent predictive variables for HCC onset and two general characteristics of patients (age and gender) were incorporated into the score model. ROC and DCA curves showed that the score model had better predictive performance in discrimination and clinical net benefit compared with single variable or other score systems, with the area under the curve (AUC) of 0.890 (95% CI 0.856-0.925) in Changzhou cohort and 0.799 (95% CI 0.751-0.849) in Wuxi cohort. Meanwhile, stratification analysis indicated that the score model had good predictive values for patients with early tumor stage (AJCC stage I) or small tumors (< 2 cm). Moreover, the score of HCC patient began to increase at 30 months before clinical diagnosis and reach a peak at 6 months. CONCLUSION: Based on this model, we could optimize the current risk stratification at an early stage and consider further intensive surveillance programs for high-risk patients. It could also help clinicians to evaluate the progression and predict the prognosis of HCC patients.

Evaluation of enterovirus nucleic acids detection method based on ultra-fast real-time fluorescence RT-PCR technology-A pilot study.

The outbreak of COVID-19 epidemic has enabled the establishment and application of various rapid detection methods. It is particularly important to establish a fast and accurate detection method for enterovirus, which will be beneficial for clinical diagnosis, epidemic prevention and control, and timely traceability. Through establishing an ultra-fast reverse transcription-polymerase chain reaction (RT-PCR) equipment, this study aimed to evaluate the sensitivity and specificity of the testing method of enterovirus nucleic acids based on ultra-fast real-time fluorescence RT-PCR technology. A total of 61 cases were sampled, which were then transported and preserved. After the nucleic acid extraction, the nucleic acids of the same sample were tested with the enterovirus nucleic acid detection kit produced by Guangzhou Da An Gene Company and the ultra-fast RT-PCR equipment system established in this study. ABI7500Fast and Ahram biosystems S1 fast equipment were used for amplification detection. If the sample had an S-shaped amplification curve in the FAM channel and the Ct value ≤40.00, the result was positive. The sensitivity, precision, and accuracy of the detection method were then verified. This study established a novel testing method to achieve enterovirus nucleic acid detection within 24 min. The sensitivity detection limit of the method was 1.0 × 102 copies/ml. The coefficients of variation for repeated detection of the high, medium, and low concentration samples were 2.644%, 1.674%, and 4.281%, respectively, with good detection repeatability. In addition, a total of 29 cases were positive by the ultra-fast RT-PCR detection method in 61 suspected samples, which was consistent with the conventional fluorescent RT-PCR method. The established rapid detection method can greatly shorten the time for providing a detection report, which may greatly improve the efficiency of diagnosis and treatment.

Prediction of early recurrence and response to adjuvant Sorafenib for hepatocellular carcinoma after resection.

BACKGROUND: Early recurrence of hepatocellular carcinoma (HCC) is a major obstacle to improving the prognosis, and no widely accepted adjuvant therapy guideline for patients post-liver resection is available. Currently, all available methods and biomarkers are insufficient to accurately predict post-operation HCC patients' risk of early recurrence and their response to adjuvant therapy. METHODS: In this study, we downloaded four gene expression datasets (GSE14520, GSE54236, GSE87630, and GSE109211) from the Gene Expression Omnibus database and identified 34 common differentially expressed genes associated with HCC dysregulation and response to adjuvant sorafenib. Then, we constructed a novel 11-messenger RNA predictive model by using ROC curves analysis, univariate Cox regression analysis, and LASSO Cox regression analysis. Furthermore, we validated the predictive values of the risk model in GSE14520 and TCGA-LIHC cohorts by using Kaplan-Meier survival analysis, multivariable Cox regression analysis, and decision curve analysis, respectively. RESULTS: The risk score model could identify patients with a high risk of HCC recurrence at the early stage and could predict the response of patients to adjuvant sorafenib. Patients with a high risk score had a worse recurrence rate in training cohorts (2-year: p < 0.0001, hazard ratio (HR): 4.658, confidence interval 95% CI [2.895-7.495]; 5-year: p < 0.0001, HR: 3.251, 95% CI [2.155-4.904]) and external validation cohorts (2-year: p < 0.001, HR: 3.65, 95% CI [2.001-6.658]; 5-year: p < 0.001, HR: 3.156, 95% CI [1.78-5.596]). The AUC values of the risk score model for predicting tumor early recurrence were 0.746 and 0.618, and that of the risk score model for predicting the response to adjuvant sorafenib were 0.722 and 0.708 in the different cohort, respectively. Multivariable Cox regression analysis and decision curve analysis also showed that the risk score model was superior to and independent of other clinicopathologic characteristics. Moreover, the risk score model had excellent abilities to predict the overall survival and HCC recurrence of patients with the same tumor stage category. CONCLUSIONS: Our risk model is a reliable and superior predictive tool. With this model, we could optimize the risk stratification based on early tumor recurrence and could evaluate the response of patients to adjuvant sorafenib after liver resection.

miR-34a in extracellular vesicles from bone marrow mesenchymal stem cells reduces rheumatoid arthritis inflammation via the cyclin I/ATM/ATR/p53 axis.

Extracellular vesicles (Evs) participate in the development of rheumatoid arthritis (RA), but the mechanisms remain unclear. This study aimed to determine the mechanism by which microRNA-34a (miR-34a) contained in bone marrow mesenchymal stem cell (BM-MSC)-derived Evs functions in RA fibroblast-like synoviocytes (RA-FLSs). BM-MSC-derived Evs and an Evs inhibitor were extracted. A rat model of RA was established. miR-34a gain- and loss-of-function experiments were performed, and the inflammation in rat synovial fluid and tissues was detected. The role of miR-34a in RA-FLSs was also measured in vitro. The target gene of miR-34a was predicted using the online software TargetScan and identified using a dual-luciferase reporter gene assay, and the activation of the ATM/ATR/p53 signalling pathway was assessed. BM-MSC-derived Evs mainly elevated miR-34a expression, which reduced RA inflammation in vivo and inhibited RA-FLS proliferation and resistance to apoptosis in vitro, while inhibited miR-34a expression enhanced RA development. In addition, miR-34a could target cyclin I to activate the ATM/ATR/p53 signalling pathway, thus inhibiting abnormal RA-FLS growth and RA inflammation. Our study showed that miR-34a contained in BM-MSC-derived Evs could reduce RA inflammation by inhibiting the cyclin I/ATM/ATR/p53 signalling pathway.

Downregulated exosomal microRNA-148b-3p in cancer associated fibroblasts enhance chemosensitivity of bladder cancer cells by downregulating the Wnt/ß-catenin pathway and upregulating PTEN.

OBJECTIVE: Exosomes derived from cancer-associated fibroblasts (CAFs) are known as important drivers of tumor progression. Previously, microRNA (miR)-148b-3p has been found to be upregulated in bladder cancers as well as in body fluids (blood, urine) of bladder cancer patients. Here, we aimed to explore the role of CAF-derived exosome miR-148b-3p in bladder cancer progression and chemosensitivity. METHODS: Transwell, MTT, flow cytometry and colony formation assays were applied to assess the effects of CAF-derived exosomes on bladder cancer cell metastasis, epithelial-mesenchymal transition (EMT) and chemosensitivity. A dual luciferase reporter assay was employed to evaluate the targeting relationship between miR-148b-3p and PTEN. Gain- and loss- of function assays were conducted to explore the roles of miR-148b-3p and PTEN in the behavior of bladder cancer cells. The role of PTEN in the metastasis, EMT and chemosensitivity of bladder cancer cells was assessed both in vivo and in vitro. RESULTS: We found that CAF-derived exosomes promoted the metastasis, EMT and drug resistance of bladder cancer cells. We also found that CAF-derived exosomes could directly transport miR-148b-3p into bladder cancer cells. In a xenograft mouse model we found that CAF-derived exosomes increased miR-148b-3p expression levels and promoted tumor proliferation, metastasis and drug resistance. PTEN was validated as a target of miR-148b-3p. Concordantly, we found that PTEN overexpression inhibited EMT, metastasis and chemoresistance in bladder cancer cells, reversing the tumor promoting effects of miR-148b-3p via the Wnt/ß-catenin pathway. CONCLUSIONS: Our results suggest that miR-148b-3p downregulation in CAF-derived exosomes, thereby inhibiting the Wnt/ß-catenin pathway and promoting PTEN expression, may offer potential opportunities for bladder cancer treatment.

Functional polymorphisms in the gene encoding macrophage migration inhibitory factor (MIF) are associated with active pulmonary tuberculosis.

OBJECTIVE: The role of the cytokine, macrophage migration inhibition factor (MIF) was assessed in tuberculosis. This case-control study investigated whether commonly occurring functional MIF polymorphisms are associated with active tuberculosis as well as with serum levels of MIF, IFN-γ and TNF-α. METHODS: Two MIF promoter polymorphisms, a functional -794 CATT5-8 microsatellite repeat (rs5844572) and a -173G/C single-nucleotide polymorphism (rs755622), were analysed by PCR and PCR-RFLP, respectively, in 47 patients and 50 healthy subjects. The mRNA level of MIF was performed by real-time PCR (RT-PCR), and MIF, IFN-γ and TNF-α serum levels were determined by ELISA. RESULTS: A significant increase of MIF mRNA expression and MIF protein level were found in patients compared to healthy controls. Meanwhile, the increase of IFN-γ and TNF-α serum levels were confirmed. According to the profile of genetic model, a significant association was found of genotypes carrying the -794 CATT 7 or 8 and -173 C risk alleles with susceptibility to active tuberculosis and with a significant increase of MIF, IFN-γ and TNF-α. CONCLUSIONS: These data suggested a distinct genetic and immunopathogenic basis for tuberculosis at the MIF locus. Serum MIF, IFN-γ and TNF-α profiles distinguish tuberculosis from the more inflammatory phenotype and may play a role in pathogenesis and as biomarkers of active tuberculosis.

Quantitation of plasma circulating DNA using quantitative PCR for the detection of hepatocellular carcinoma.

Circulating DNA is a potential biomarker for tumor diagnosis and prognosis. This study was aimed to quantify the circulating DNA in plasma from patients with hepatocellular carcinoma (HCC) using quantitative PCR and evaluate its potential clinical value. Blood samples were collected from 72 patients with HCC, 37 with liver cirrhosis or chronic hepatitis and 41 healthy volunteers. Plasma DNA was extracted and quantified by a real-time quantitative PCR method. The diagnostic and prognostic value of plasma DNA analysis for HCC was evaluated. DNA levels in the HCC plasma (median: 173 ng/mL) were significantly higher than those in the healthy controls (9 ng/mL) or control benign patients (46 ng/mL) (P < 0.001). The area under the receiver-operation characteristic (ROC) curve (AUC) assessing plasma DNA was 0.949 for healthy controls and 0.874 for control patients. Plasma DNA detection could discriminate HCC from normal controls with 90.2% sensitivity and 90.3% specificity at the cut-off value of 18.2 ng/mL. Combined ROC analyses using plasma DNA and serum AFP revealed an elevated AUC of 0.974 with 95.1% sensitivity and 94.4% specificity in discriminating HCC from normal controls. The plasma DNA levels were positively associated with tumor size (P = 0.012), and were significantly elevated in HCC patients with intrahepatic spreading or vascular invasion (P = 0.035). The overall survival time of patients with high plasma DNA levels showed a shortened tread when compared with that of patients with low plasma DNA concentrations (P = 0.071). Plasma DNA may be a valuable noninvasive tool for the detecting and predicting the metastasis potential of HCC; and the prognostic value of plasma DNA needed further investigation.

TNRC9/LOC643714 polymorphisms are not associated with breast cancer risk in Chinese women.

Variants in several genes are known to confer increased susceptibility to breast cancer, but the frequencies of these mutations in the population are very rare and different between race or ethnic groups. Genetic variation in trinucleotide repeat containing 9 (TNRC9) and the hypothetical gene LOC643714 (TNRC9/LOC643714) is a newly described risk factor for breast cancer. Here, we investigated the association among the three single nucleotide polymorphisms (SNPs) in TNRC9/LOC643714 and breast cancer risk and clinico-pathological parameters in a hospital-based Chinese population. Genomic DNA was extracted from peripheral blood lymphocytes. Polymerase chain reaction-ligation detection reaction was used to genotype 321 breast cancer cases and 330 controls. In addition, the representative polymerase chain reaction products were subjected to direct DNA sequencing to confirm the accuracy of this method. The odds ratios and 95% confidence intervals were calculated by an unconditional logistic regression model. There was no significant association between the risk of breast cancer and three SNPs of TNRC9/LOC643714 gene polymorphisms and their haplotypes. No significant correlation was found between the TNRC9/LOC643714 polymorphisms and age at diagnosis, lymph node metastases, estrogen receptor status, or progesterone receptor status, respectively. None of these three SNPs of TNRC9/LOC643714 gene is associated with individual susceptibility to breast cancer in Chinese women.

[Construction and immunogenicity evaluation of chimerical DNA vaccine of human papillomavirus type 11].

OBJECTIVE: To construct chimerical DNA vaccine plasmid of human papillomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. METHODS: Molecular cloning techniques were used to construct recombinant plasmid pcDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection.IL-2 and gamma-INF secreted by immunized spleens lymphocyte and HPV 11 L1 or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. RESULTS: The chimerical DNA plasmid of pcDNA3 L1-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and gamma-INF were detected in vaccinated mice. CONCLUSION: Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.

ERCC1 polymorphism, expression and clinical outcome of oxaliplatin-based adjuvant chemotherapy in gastric cancer.

AIM: To determine the influence of excision repair cross complementing group 1 (ERCC1) codon 118 polymorphism and mRNA level on the clinical outcome of gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy. METHODS: Eighty-nine gastric cancer patients treated with oxalipatin-based adjuvant chemotherapy were included in this study. ERCC1 codon 118 C/T polymorphism was tested by polymerase chain reaction-ligation detection reaction (PCR-LDR) method in peripheral blood lymphocytes of those patients; and the intratumoral ERCC1 mRNA expression was measured using reverse transcription PCR in 62 patients whose tumor tissue specimens were available. RESULTS: No significant relationship was found between ERCC1 codon 118 polymorphism and ERCC1 mRNA level. The median relapse-free and overall survival period was 20.1 mo and 28.4 mo, respectively. The relapse-free and overall survivals in patients with low levels of ERCC1 mRNA were significantly longer than those in patients with high levels (P<0.05), while there was no significant association found between ERCC1 118 genotypes and the disease prognosis. Multivariate analysis also showed that ERCC1 mRNA level was a potential predictor for relapse and survival in gastric cancer patients treated with oxaliplatin-based adjuvant chemotherapy (P<0.05). CONCLUSION: ERCC1 codon 118 polymorphism has no significant impact on ERCC1 mRNA expression, and the intratumoral ERCC1 mRNA level but not codon 118 polymorphism may be a useful predictive parameter for the relapse and survival of gastric cancer patients receiving oxaliplatin-based adjuvant chemotherapy.