An enzyme-free sensing platform for miRNA detection and in situ imaging in clinical samples based on DNAzyme cleavage-triggered catalytic hairpin assembly.

MicroRNA (miRNA) is demonstrated to be associated with the occurrence and development of various diseases including cancer. Currently, most miRNA detection methods are confined to in vitro detection and cannot obtain information on the temporal and spatial expression of miRNA in relevant tissues and cells. In this work, we established a novel enzyme-free method that can be applied to both in vitro detection and in situ imaging of miRNA by integrating DNAzyme and catalytic hairpin assembly (CHA) circuits. This developed CHA-Amplified DNAzyme miRNA (CHAzymi) detection system can realize the quantitively in vitro detection of miR-146b (the biomarker of papillary thyroid carcinoma, PTC) ranging from 25 fmol to 625 fmol. This strategy has also been successfully applied to in situ imaging of miR-146b both in human PTC cell TPC-1 and clinical samples, showing its capacity as an alternative diagnostic method for PTC. Furthermore, this CHAzymi system can be employed as a versatile sensing platform for various miRNAs by revising the relevant sequences. The results imply that this system may expand the modality of miRNA detection and show promise as a novel diagnostic tool in clinical settings, providing valuable insights for effective treatment and management of the disease.

Design and analysis of self-priming extension DNA hairpin probe for miRNA detection based on a unified dynamic programming framework.

MicroRNAs (miRNAs) are potential biomarkers for cancer diagnosis and prognosis, methods for detecting miRNAs with high sensitivity, selectivity, and stability are urgently needed. Various nucleic acid probes that have traditionally been for this purpose suffer several drawbacks, including inefficient signal-to-noise ratios and intensities, high cost, and time-consuming method establishment. Computing tools used for investigating the thermodynamics of DNA hybridization reactions can accurately predict the secondary structure of DNA and the interactions between DNA molecules. Herein, NUPACK was used to design a series of nucleic acid probes and develop a phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) signal amplification strategy, which enabled the ultrasensitive detection of miR-200a in serum samples. The free and binding energies of the DNA detection probes calculated using NUPACK, as well as the biological experimental results, were considered synthetically to select the best sequence and experimental conditions. A unified dynamic programming framework, NUPACK analysis and the experimental data, were complementary and improved the designed model in all respects. Our study demonstrates the feasibility of using computer technology such as NUPACK to simplify the experimental process and provide intuitive results.

Site-specific detection of circulating tumor DNA methylation in biological samples utilizing phosphorothioated primer-based loop-mediated isothermal amplification.

DNA methylation, a kind of epigenetic alteration, plays a vital role in tumorigenesis and offers a new class of targets for cancer treatment. DNA hypermethylation at the E-Box site (CACGTG, -288 bp) in the SLC22A2 promoter was related to multidrug resistance of renal cell carcinoma (RCC), which can provide the target for both treatment and monitoring. Herein, we developed a novel phosphorothioated primer based loop-mediated isothermal amplification (PS-LAMP) assay to detect circulating tumor DNA (ctDNA) methylation levels in E-Box sites in tumor tissue, urine, and plasma samples from patients with RCC. Bisulfite treatment converted methylated/unmethylated discrepancy to a single base discrepancy (C/U). PS-LAMP amplified the templates to a tremendous amount. One-step strand displacement (OSD) probe provided single base resolution in amplified products and finally realized the specific site methylation detection. Our proposed method provided a linear range from 0% to 100% for methylation levels and was available in samples at low concentrations (102 copies/µL). Visually observable colorimetric detection can be achieved by incorporating the OSD probe with gold nanoparticles (AuNP). Our assay performed better than traditional methods in biological samples with low ctDNA concentration. Further, we found a potential consistency of methylation levels between tumor tissue and plasma sample from the same patient (Spearman's ρ = 0.886, P = 0.019, n = 6). In general, this work provides a PS-LAMP assay combining OSD probes for site-specific methylation detection in various biological samples, offering a method for noninvasive detection.

Plasma total cholesterol concentration and risk of higher-grade prostate cancer: A nested case-control study and a dose-response meta-analysis.

Our previous publication found an increased risk of higher-grade (Gleason sum ≥7) prostate cancer for men with high total cholesterol concentration (≥200 mg/dl) in the Health Professionals Follow-up Study (HPFS). With additional 568 prostate cancer cases, we are now able to investigate this association in more detail. For the nested case-control study, we included 1260 men newly diagnosed with prostate cancer between 1993 and 2004, and 1328 controls. For the meta-analyses, 23 articles studied the relationship between total cholesterol level and prostate cancer incidence were included. Logistic regression models and dose-response meta-analysis were performed. An increased risk of higher-grade (Gleason sum ≥4 + 3) prostate cancer for high vs low quartile of total cholesterol level was observed in the HPFS (ORmultivariable = 1.56; 95% CI = 1.01-2.40). This finding was compatible with the association noted in the meta-analysis of highest vs lowest group of total cholesterol level, which suggested a moderately increased risk of higher-grade prostate cancer (Pooled RR =1.21; 95%CI: 1.11-1.32). Moreover, the dose-response meta-analysis indicated that an increased risk of higher-grade prostate cancer occurred primarily at total cholesterol levels ≥200 mg/dl, where the RR was 1.04 (95%CI: 1.01-1.08) per 20 mg/dl increase in total cholesterol level. However, total cholesterol concentration was not associated with the risk of prostate cancer overall either in the HPFS or in the meta-analysis. Our primary finding, as well as the result of the meta-analysis suggested a modest increased risk of higher-grade prostate cancer, at total cholesterol concentrations exceeding 200 mg/dl.

Phosphorothioated and phosphate-terminal dumbbell (PP-TD) probe-based rapid detection of polynucleotide kinase activity.

Polynucleotide kinase (PNK), a bifunctional enzyme with 5'-kinase and 3'-phosphatase activities, plays an important role in DNA repair and is associated with various diseases. Here, we developed a primer-free, sensitive, and isothermal quantitative assay to detect PNK activity. In the presence of PNK, the 3'-phosphate group of the substrate was digested with 3'-OH, initiating the amplification reaction. Elongated dsDNA binds to the dsDNA-specific fluorescent dye EvaGreen, leading to a significant enhancement in fluorescence intensity. The limit of detection (LOD) of this method was 7.7 × 10-7 U µL-1, which is comparable or even superior to that of previously reported methods. This approach also showed good quantitative ability in complex cell lysates, indicating potential for biological sample analysis. Additionally, this facile and sensitive assay can be used to screen for PNK inhibitors. The proposed method provides a promising platform for sensitive PNK activity monitoring and inhibition screening for drug discovery and clinical treatment.

A putative causality of vitamin D in common diseases: A mendelian randomization study.

Backgrounds: Vitamin D is considered as a nutrient protecting individuals against an array of diseases based on observational studies. Such a protective effect, however, has not been demonstrated by randomized controlled trials. This study aims to explore a putative causal role of vitamin D in common diseases through a two-sample Mendelian randomization (MR) framework. Methods: Circulating vitamin D was predicted by 41 genetic variants discovered in European populations. Common diseases were verified through two ways, using information from Japanese patients of Biobank Japan and using information from European patients of FinnGen project. We additionally validated the results by replacing vitamin D-associated instrumental variables (IVs) of European population with that of an independent Japanese population and of an independent Indian population. Inverse-variance weighted method was used as the primary analytical approach while a series of MR methods including MR-Egger regression, weighted median, maximum likelihood, MR-PRESSO and multivariate MR were adopted to guarantee MR model assumptions and to detect horizontal pleiotropy. Results: Genetically predicted vitamin D was significantly associated with an increased risk of Graves' disease (OR = 1.71, 95%CI: 1.25-2.33, P = 0.001) and cataract (OR = 1.14, 95%CI: 1.03-1.28, P = 0.016); while with a decreased risk of esophageal cancer (OR = 0.66, 95%CI: 0.46-0.93, P = 0.019). This significant causal link between vitamin D and cataract was validated replacing IVs identified in the European population with those from Japanese population. No notable associations of vitamin D with other diseases were observed. Conclusions: Our findings indicate a potential causal role of vitamin D in common diseases, which needs further validation.

A Novel Helper qPCR Assay for the Detection of miRNA Using Target/Helper Template for Primer Formation.

A novel, simple, and sensitive quantitative polymerase chain reaction (qPCR) technology, which is termed as helper qPCR, was established to detect miRNA. In this assay, the target miRNA sequence was introduced as helper template for a reaction switch preforming two-step real-time qPCR strategy. Firstly, the reverse primer was reverse transcribed to form "mediator primer" after binding to the target miRNA. Then, the mediator primer was further extended to form "active template" with annealing to the mediator template. In the end, the active template was amplified and detected by the qPCR reaction system with the help of reverse and forward primers. The SYBR Green dye was used for fluorescence quantification, which is quicker and cheaper than the fluorescent probes, as the detection limit of this assay was 1 pM. This helper qPCR system can be used for different miRNAs detection by redesigning reverse primer for target, indicating this strategy could afford good performance in detecting multiple miRNAs and has a promising application prospect.

Protective Effects of Lactobacillus plantarum CCFM8610 against Acute Toxicity Caused by Different Food-Derived Forms of Cadmium in Mice.

Cadmium (Cd) is an environmental pollutant that is toxic to almost every human organ. Oral supplementation with lactic acid bacteria (LAB) has been reported to alleviate cadmium toxicity. However, research on the mitigation of cadmium toxicity by LAB is still limited to inorganic cadmium, which is not representative of the varied forms of cadmium ingested daily. In this study, different foodborne forms of cadmium were adopted to establish an in vivo toxicity model, including cadmium-glutathione, cadmium-citrate, and cadmium-metallothionein. The ability of Lactobacillus plantarum CCFM8610 to reduce the toxic effects of these forms of cadmium was further investigated. The 16S rRNA gene sequencing and metabolomics technologies based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) were adopted for the exploration of relevant protective mechanisms. The results demonstrated that the consumption of CCFM8610 can reduce the content of cadmium in mice and relieve the oxidative stress caused by different food-derived forms of cadmium, indicating that CCFM8610 has a promising effect on the remediation of the toxic effects of cadmium food poisoning. Meanwhile, protective effects on gut microflora and serum metabolites might be an important mechanism for probiotics to alleviate cadmium toxicity. This study provides a theoretical basis for the application of L. plantarum CCFM8610 to alleviate human cadmium poisoning.

Comparative Genomic Analysis Determines the Functional Genes Related to Bile Salt Resistance in Lactobacillus salivarius.

Lactobacillus salivarius has drawn attention because of its promising probiotic functions. Tolerance to the gastrointestinal tract condition is crucial for orally administrated probiotics to exert their functions. However, previous studies of L. salivarius have only focused on the bile salt resistance of particular strains, without uncovering the common molecular mechanisms of this species. Therefore, in this study, we expanded our research to 90 L. salivarius strains to explore their common functional genes for bile salt resistance. First, the survival rates of the 90 L. salivarius strains in 0.3% bile salt solutions were determined. Comparative genomics analysis was then performed to screen for the potential functional genes related to bile salt tolerance. Next, real-time polymerase chain reaction and gene knockout experiments were conducted to further verify the tolerance-related functional genes. The results indicated that the strain-dependent bile salt tolerance of L. salivarius was mainly associated with four peptidoglycan synthesis-related genes, seven phosphotransferase system-related genes, and one chaperone-encoding gene involved in the stress response. Among them, the GATase1-encoding gene showed the most significant association with bile salt tolerance. In addition, four genes related to DNA damage repair and substance transport were redundant in the strains with high bile salt tolerance. Besides, cluster analysis showed that bile salt hydrolases did not contribute to the bile salt tolerance of L. salivarius. In this study, we determined the global regulatory genes, including LSL_1568, LSL_1716 and LSL_1709, for bile salt tolerance in L. salivarius and provided a potential method for the rapid screening of bile salt-tolerant L. salivarius strains, based on PCR amplification of functional genes.

Glutamine deficiency links clindamycin-induced dysbiosis and intestinal barrier dysfunction in mice.

Antibiotics rank as the most powerful weapons against bacterial infection, but their use is often limited by antibiotic-associated diarrhoea (AAD). Here, we reported that glutamine deficiency might act as a new link between clindamycin-induced dysbiosis and intestinal barrier dysfunction during AAD progression. Using a mouse model, we demonstrated that glutamine became a conditionally essential amino acid upon persistent therapeutic-dose clindamycin exposure, evidenced by a dramatic decrease in intestinal glutamine level and glutaminase expression. Mechanistically, clindamycin substantially confounded the abundance of butyrate-producing strains, leading to the deficiency of faecal butyrate which is normally a fundamental fuel for enterocytes, and in turn increased the compensatory use of glutamine. In addition to its pivotal roles in colonic epithelial cell turnover, glutamine was required for nitric oxide production in classic macrophage-driven host defence facilitating pathogen removal. Importantly, oral administration of glutamine effectively attenuated clindamycin-induced dysbiosis and restored intestinal barrier dysfunction in mice. Collectively, the present study highlighted the importance of gut microbiota in host energy homoeostasis and provided a rationale for introducing glutamine supplementation to patients receiving long-term antibiotic treatment.

Screening of Lactobacillus salivarius strains from the feces of Chinese populations and the evaluation of their effects against intestinal inflammation in mice.

Lactobacillus salivarius is a species of lactic acid bacteria with probiotic potency. Compared to such well-known probiotics as L. rhamnosus and L. casei, the genomic characteristics and health-beneficial effects of L. salivarius are inadequately researched. For this study, a medium with enhanced selectivity for the isolation of L. salivarius was developed by optimizing the carbon source and antibiotics in the medium. Seventy-three L. salivarius strains were isolated from 472 fecal samples from Chinese populations, and their pan-genomic and phylogenetic characterizations were analyzed. Three strains (L. salivarius HN26-4, NT4-8, and FXJCJ7-2) that were clearly categorized in different sub-phylotypes of the phylogenetic tree were randomly selected for further studies. Compared to the other two tested strains, L. salivarius FXJCJ7-2 showed higher tolerance to simulated gastrointestinal tract conditions and more significant anti-inflammatory effects in lipopolysaccharides (LPS)-treated RAW264.7 murine macrophages. This strain was also more effective in reversing LPS-induced alterations in gut barrier function, colonic histopathology, Treg/Th-17 balance, immunomodulatory indicators, nuclear factor kappa B pathway activation, and the intestinal microenvironment of the mice than the other two tested strains. Comparative genomic analysis indicated that these protective effects may be related to the specific genes of L. salivarius FXJCJ7-2 that were involved in the tolerance to the gastrointestinal environment, short-chain fatty acid production, and host-bacterium interaction.

A new method for evaluating the bioaccessibility of different foodborne forms of cadmium.

The bioabsorption and biotoxicity of cadmium are closely related to its binding form. Currently, total concentration is used as the indicator for evaluating cadmium toxicity in food, but it might not accurately reflect cadmium's toxic effects. This study attempted to evaluate the toxicity of the different forms of cadmium including cadmium-malate, cadmium-glutathione, and cadmium-metallothionein that are commonly found in food. The in vitro physiologically based extraction test (PBET) combined with Visual MINTEQ modeling was used to predict the toxicity of different forms of cadmium, and acute toxicity testing was performed in mice for validating their results. The in vivo experimental results showed that different forms of cadmium had diverse biotoxicities of which PBET was a good predictor. In particular, the simulation of cadmium ions in PBET using the MINTEQ software revealed that the free cadmium ion content in the simulated intestinal fluid had a superior linear relationship than the total cadmium concentration with the toxicology indexes. Verification using the other two forms of cadmium confirmed the accuracy of the prediction of their biotoxicity. These findings hopefully provide an important reference for a more accurate and rapid safety assessment of cadmium in food.