A MnO2 nanosheet-mediated CRISPR/Cas12a system for the detection of organophosphorus pesticides in environmental water.

Nowadays, easy, convenient, and sensitive sensing strategies are still critical for organophosphorus pesticides in environmental water samples. Herein, a novel organophosphorus pesticide (OP) assay based on acetylcholinesterase (AChE) and a MnO2 nanosheet-mediated CRISPR/Cas12a reaction is reported. The single-strand DNA (ssDNA) activator of CRISPR/Cas12a was simply adsorbed on the MnO2 nanosheets as the nanoswitches of the assay. In the absence of target OPs, AChE hydrolyzed acetylcholine (ATCh) to thiocholine (TCh), which reduced the MnO2 nanosheets to Mn2+, resulting in the release of the activator followed by activation of the CRISPR/Cas12a system. The activated Cas12a thereafter nonspecifically cleaved the FAM/BHQ1-labeled ssDNA (FQ-reporter), producing a fluorescence signal. Upon the addition of target OPs, the hydrolysis of ATCh by AChE was inhibited owing to OPs combining with AChE, and thus effective quantification of OPs could be achieved by measuring the fluorescence changes of the system. As a proof of concept, dichlorvos (DDVP) was chosen as a model OP analyte to address the feasibility of the proposed method. Attributed to the excellent trans-cleavage activity of Cas12a, the fluorescent biosensor exhibits a satisfactory limit of detection (LOD) for DDVP at 0.135 ng mL-1. In addition, the excellent recoveries for the detection of DDVP in environmental water samples demonstrate the applicability of the proposed assay in real sample research.

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA.

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/µL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/µL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.

Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine.

A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/µL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.

Effect of Lactobacillus plantarum LP-Onlly on gut flora and colitis in interleukin-10 knockout mice.

BACKGROUND AND AIM: Probiotics are used in the therapy of inflammatory bowel disease. This study aimed to determine the effects of probiotic Lactobacillus plantarum LP-Onlly (LP) on gut flora and colitis in interleukin-10 knockout (IL-10(-/-) ) mice, a model of spontaneous colitis. METHODS: IL-10(-/-) and wild-type mice were used at 8 weeks of age and LP by gavage was administered at a dose of 10(9) cells/day per mice for 4 weeks. Mice were maintained for another one week without LP treatment. The colonic tissues were collected for histological and ultrastructural analysis at death after 4 weeks treatment of LP, and the feces were collected at 1-week intervals throughout the experiment for the analysis of gut flora and LP using selective culture-based techniques. RESULTS: Compared with control mice, IL-10(-/-) mice developed a severe intestinal inflammation and tissue damage, and had an abnormal composition of gut microflora. LP administration attenuated colitis with the decreased inflammatory scoring and histological injury in the colon of IL-10(-/-) mice. In addition, LP administration increased the numbers of beneficial total bifidobacteria and lactobacilli, and decreased the numbers of potential pathogenic enterococci and Clostridium perfringens, although the decrease of coliforms was not significant after LP treatment in IL-10(-/-) mice. CONCLUSIONS: Oral administration of LP was effective in the treatment of colitis, with the direct modification of gut microflora in IL-10(-/-) mice. This probiotic strain could be used as a potential adjuvant in the therapy of inflammatory bowel disease, although further studies are required in human.

Lactobacillus plantarum ameliorates colonic epithelial barrier dysfunction by modulating the apical junctional complex and PepT1 in IL-10 knockout mice.

Probiotics are efficacious in the treatment of inflammatory bowel disease. However, the precise mechanisms remain unknown. To determine whether probiotic Lactobacillus plantarum (LP) ameliorates colonic epithelial barrier dysfunction present in interleukin-10 knockout (IL-10⁻(/)⁻) mice, IL-10⁻(/)⁻ and wild-type mice received LP or the vehicle for 4 wk. Colitis was assessed by histological scores and clinical manifestation, and gut paracellular permeability was measured by Ussing chamber. Oligopeptide transporter 1 (PepT1)-mediated transepithelial transport was evaluated by measuring the plasma cephalexin concentration. The expression and distribution of apical junctional complex (AJC) proteins and PepT1 were determined by Western blotting and immunofluorescence and their mRNA by reverse transcriptase-PCR. Spontaneous colitis was observed in all IL-10⁻(/)⁻ mice in which paracellular permeability was increased, in conjunction with decreased expression and redistribution of zonula occludens-1, occludin, claudin-1, and ß-catenin. PepT1 expression was increased, accompanied with an enhanced cephalexin transport. Colonic epithelial barrier dysfunction was further confirmed by increased bacterial translocation and proinflammatory cytokine production. Treatment with LP decreased colonic paracellular permeability with restoration of expression and distribution of AJC proteins and partially prevented PepT1 expression and cephalexin transport in IL-10⁻(/)⁻ mice. Moreover, treatment with LP also prevented bacterial translocation and proinflammatory cytokine production in IL-10⁻(/)⁻ mice. Results from this study indicated that treatment with LP may ameliorate colonic epithelial barrier dysfunction in IL-10⁻(/)⁻ mice, by modulating the AJC- and PepT1-mediated transepithelial transport.

Lactobacillus plantarum consumption increases PepT1-mediated amino acid absorption by enhancing protein kinase C activity in spontaneously colitic mice.

Although probiotic consumption has generally been shown to have many beneficial effects for the prevention and treatment of inflammatory bowel disease, the effects of Lactobacillus plantarum (LP) on intestinal nutrient absorption, particularly oligopeptide transporter 1 (PepT1)-mediated absorption of dietary protein under inflammatory conditions, has not yet been characterized. In this study, we first investigated the effects of LP consumption on plasma amino acid concentrations and PepT1-mediated absorption of cephalexin in the small intestine of wild-type (WT) mice and interleukin-10 knockout (IL-10(-/-)) mice, a model of spontaneous colitis. We then analyzed expression and distribution of PepT1 and protein kinase C (PKC) activity in the jejunum of these mice. LP consumption (10(9) colony-forming units/0.5 mL) delivered by gavage once per day for 4 wk increased the total plasma amino acid concentration and the concentration of plasma cephalexin through enhancement of PepT1-mediated uptake in LP treated IL-10(-/-) mice compared with IL-10(-/-) mice. However, Western blotting and quantitative PCR analysis revealed no significant differences in PepT1 protein and mRNA expression between LP-treated and untreated mice. Additionally, immunofluorescence analysis showed that PepT1 did not appear to be mislocalized in IL-10(-/-) mice. Interestingly, IL-10(-/-) mice had significantly lower PKC activity and expression of phosphorylated PKC compared with WT mice, and these decreases could be prevented by LP treatment. These data suggest that consumption of LP enhances PepT1-mediated amino acid absorption, likely through alterations in PKC activity, as opposed to changes in expression or distribution of PepT1 in the small intestine of IL-10(-/-) mice.

[Analysis of the probiotic Bifidobacterium and Lactobacillus community in child intestinal flora].

To investigate the distribution of child intestinal flora and the composition of its key probiotics community, study on intestinal flora of 21 Chinese children (age 2 - 5) was conducted, which included bacteria isolation and counting, 16S rDNA sequencing and homology analysis. For identification of the key probiotics such as Bifidobacterium and Lactobacillus in children feces at the species level, the specific primers Im26/Im3 and L159/L677 for PCR amplification of partial 16S rDNA were used. The results show that the composition of child intestinal flora is was relatively stable and almost same to the intestinal flora of the youth (age 20 - 25). Culture-based approaches show that the key probiotic community in feces at the species level was highly different in composition and numbers from individual to individual. B. longum and B. pseudocatenulatum, which are detected at levels of 10(7) CFU/g (wet) in samples and the detection rates are 90.48% and 85.71% respectively, are believed to be major bifidobacterial species in child intestinal microbiota. In addition, B. adolescentis, B. bifidum, B. infantis and B. thermacidophium have also been found. L. mucosae, L. fermentum, L. salivarius, L. ruminis, L. gasseri and L. plantarum are isolated from the stools. L. mucosae (3.68 log10 CFU/g (wet), detection rate 71.43%) and L. fermentum (3.97 log10 CFU/g (wet), detection rate 52.38%) are two dominant species of Lactobacillus. Study on Chinese child intestinal flora, especially on the compositions and numbers of key probiotics in the feces will be very helpful to the development of effective probiotics in future.

[Influences of enteral nutrition combined with probiotics on the gut microecology and barrier function of the rats with abdominal infection].

OBJECTIVE: To investigate the influences of enteral nutrition (EN), parenteral nutrition (PN) and probiotics supplement on the intestinal microecology, and barrier function of the rats with abdominal infection. METHODS: Twenty-one Sprague-Dawley (SD) rats with abdominal infection were randomly divided into three groups, and received PN (PN group, n=7), PN+ EN (PN+ EN group, n=7) or PN+ EN+ probiotics (probiotics group, n=7) respectively with isonitrogen and isocaloric nutrition. The rats were sacrificed after six days. The feces in cecum were cultured for anaerobic bacterial growth and DNA fingerprint spectrum was analyzed by randomly amplified polymorphic DNA technique. The transmembrane binding protein (occludin) and IgA levels in colon and terminal ileum were detected by immunohistochemistry method. The bacterial translocation rate and endotoxin level were also measured. RESULTS: The germ numbers of different species were both higher in PN+ EN and probiotic group than those in PN group. The bands of DNA fingerprint spectrum were significantly decreased in PN group, but the bands in both PN+ EN group and probiotic group were similar to that in the normal rats. The expression levels of occludin and IgA in the intestine and colorectum were higher in both PN+ EN group and probiotic group compared with those of PN group (P< 0.05, P< 0.01, respectively), the expression level of occludin was higher in probiotic group than that in PN+ EN group (P< 0.05). The overall bacterial translocation rates and endotoxin levels were significantly reduced in both probiotic and PN+ EN group (P< 0.05), but there was no difference between probiotic group and EN group. CONCLUSION: EN combined with probiotics can increase occluding and IgA expressions, improve the intestinal microecology,maintain the gut barrier function, and decrease the incidence of gut bacterial translocation.

Effect of lactobacillus on the gut microflora and barrier function of the rats with abdominal infection.

AIM: To investigate the effect of probiotics supplemented by gut on the tight junctions of epithelial cells, barrier function and the microflora of rats with abdominal infection. METHODS: After the model of cecal ligation and perforation established, SD rats were divided into two groups: parenteral nutrition (PN) group and PN+probiotics (probiotics) group, PN solution was supplemented by neck vein and probiotics was delivered via the jejunostomy tube for five days. Vena cava blood and the homogenated tissue of liver, lung and mesenteric lymph nodes were cultured to determine the bacterial translocation rate (BTR). The ultra-structure of epithelial tight junctions and microvilli of the gut were observed by electron microscopy; occluding expression was measured by indirect-immune fluorescence method; anaerobic bacterial growth by anaerobic culture and DNA fingerprint of bacterial colonies of the feces by PCR. RESULTS: The quantity of lactobacteria and bifydobacteria in probiotics group was higher than that of PN group. The profiles of DNA fingerprint expression in probiotics group were similar to that in the normal group, a new 16S rDNA sequence appeared in the profile in PN group. The occludin expression, the integrality of the gut epithelial tight junction and microvilli in probiotics group were improved as compared with PN group. The BTR and endotoxin in blood were reduced more significantly in probiotics group as compared with PN group. CONCLUSION: The probiotics could improve the gut microflora disturbance, increase occludin expression, maintain the gut epithelial tight junction and decrease the bacterial translocations rate.

[An in vitro study of cholesterol-lowering properties of probiotics isolated from the human feces].

21 strains of Lactobacillus and Bifidobacterium, isolated from feces of healthy youth and children feces and identified by molecular biological methods, together with 6 strains of probiotics preserved in Onlly lab were studied in the experiments, including removal cholesterol from media, bile-tolerance and acid-tolerance. The results demonstrated that all strains could remove cholesterol from media and removal rates of 5 strains were more than 40%. Meanwhile these 5 strains had high removal effectiveness. The bile-tolerance and acid-tolerance were varied from strain to strain. Among 27 strains, Bm26 demonstrated higher ability of removal cholesterol, bile-tolerance and bile-tolerance than other strains.