High-Fidelity CRISPR/Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.

Prevalence of qnrVC Genes in Pseudomonas aeruginosa Clinical Isolates from Guangdong, China.

Pseudomonas aeruginosa is a serious nosocomial pathogen with high morbidity and mortality due to the increasing resistance to antibiotics in recent years. qnrVC genes have been proven as a source of antibiotic resistance, but relationship with Pseudomonas aeruginosa remains not clear. We aimed to investigate the prevalence and molecular characteristics of qnrVC genes in P. aeruginosa clinical isolates. A total of 874 nonduplicate clinical isolates were collected in Guangdong, China, between January 2011 and June 2015. The presence of qnrVC genes and their genotypes were determined using PCR amplification and DNA sequencing. Antibiotic susceptibilities were tested, and the genetic relatedness of qnrVC-positive isolates were analyzed by multi-locus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). Consequently, we found 2.3% of P. aeruginosa isolates were present with qnrVC genes, displaying more resistant to various antibiotics. Phylogenetic analysis of qnrVC-positive strains revealed that antibacterial resistance among qnrVC-positive P. aeruginosa isolates in Guangdong probably emerged from multiple sources and was not spread by clonal strains.

Orthogonal Screening of Anticancer Drugs Using an Open-Access Microfluidic Tissue Array System.

Screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems, due to the requirement of flexibility in liquid handling. To overcome this limitation, we present here an open-access microfluidic tissue array system specifically designed for drug combination screening. The microfluidic chip features a key structure in which a nanoporous membrane is sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach takes advantage of the characteristics of the nanoporous membrane: on one side, this membrane permits the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution; on the other side, it allows diffusion-based media exchange and thus mimics the endothelial layer. In synergy with a liquid-transferring platform, the open-access microfluidic system enables complex multistep operations involving long-term cell culture, medium exchange, multistep drug treatment, and cell-viability testing. By using the microfluidic protocol, a 10 × 10 tissue array was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays indicated that the resultant tumor tissue was faithful to that in vivo. Drug-testing assays showed that the incorporation of the nanoporous membrane further decreased killing efficacy of the anticancer agents, indicating its function as an endothelial layer. Robustness of the microfluidic system was demonstrated by implementing a three-factor, three-level orthogonal screening of anticancer drug combinations, with which 67% of the testing (9 vs. 27) was saved. Experimental results demonstrated that the microfluidic tissue system presented herein is flexible and easy-to-use, thus providing an ideal tool for performing complex multistep cell assays with high efficiencies.

Simultaneous Identification and Antimicrobial Susceptibility Testing of Multiple Uropathogens on a Microfluidic Chip with Paper-Supported Cell Culture Arrays.

A microfluidic chip was developed for one-step identification and antimicrobial susceptibility testing (AST) of multiple uropathogens. The polydimethylsiloxane (PDMS) microchip used had features of cell culture chamber arrays connected through a sample introduction channel. At the bottom of each chamber, a paper substrate preloaded with chromogenic media and antimicrobial agents was embedded. By integrating a hydrophobic membrane valve on the microchip, the urine sample can be equally distributed into and confined in individual chambers. The identification and AST assays on multiple uropathogens were performed by combining the spatial resolution of the cell culture arrays and the color resolution from the chromogenic reaction. The composite microbial testing assay was based on dynamic changes in color in a serial of chambers. The bacterial antimicrobial susceptibility was determined by the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Using three common uropathogenic bacteria as test models, the developed microfluidic approach was demonstrated to be able to complete the multiple colorimetric assays in 15 h. The accuracy of the microchip method, in comparison with that of the conventional approach, showed a coincidence of 94.1%. Our data suggest this microfluidic approach will be a promising tool for simple and fast uropathogen testing in resource-limited settings.

Paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells.

Tumor tropism metastasis is a multi-step process that involves interactions between tumor cells and the microenvironment. Due to the limitations of experimental techniques, current studies are not able to gain insight into the dynamic process of such tropism migration. To overcome this issue, we developed a paper-supported co-culture system for dynamic investigations of the lung-tropic migration of breast cancer cells. This co-culture system contains a tumor layer, a recruitment layer, and several invasion layers between these two parts. The tumor and recruitment layers are impregnated with breast cancer cells and lung cells, respectively. Stacking these layers forms a co-culture device that comprises interactions between breast cancer and lung, destacking such a device represents cancer cells at different stages of the migration process. Thus, the paper-supported co-culture system offers the possibility of investigating migration from temporal and spatial aspects. Invasion assays using the co-culture system showed that breast cancer cells induced lung fibroblasts to convert to cancer-associated fibroblasts (CAFs), and the CAFs, in turn, recruited breast cancer cells. During migration, the local invasion of the cancer cells is a collective behavior, while the long-distance migration comes from individual cell behaviors. Breast cancer cells experienced repetitive processes of migration and propagation, accompanied by epithelial-mesenchymal and mesenchymal-epithelial transitions, and changes in stemness and drug resistance. Based on these results, the lung-tropic migration of breast cancer is interpreted as a process of bilateral interaction with the local and host-organ microenvironment. The developed paper-supported co-culture system offers the possibility of dynamically investigating tropism migration under the pre-metastatic niche, thus providing an advantageous tool for studying tumor metastasis.

Ultrasensitive detection of serum hepatitis B virus by coupling ultrafiltration DNA extraction with real-time PCR.

BACKGROUND: A simple and reliable DNA extraction of hepatitis B virus (HBV) is critical in developing an ultrasensitive detection method for HBV infection. Current commercially available serum Hepatitis B Virus (HBV) DNA extraction methods are time-consuming, expensive and/or require specialized equipment, which hinders wide adoption of clinical laboratories. This study offers a report on an ultrasensitive HBV DNA detection method by coupling serum HBV DNA extraction by ultrafiltration (UF) with real-time PCR (qPCR) detection. METHODS: Serum proteins were precipitated by phenol to release HBV DNA in the supernatant which was then transferred to the UF devices. The resultant DNA concentrate was eluted and released into qPCR pre-mixture. The UF-qPCR assay performance, including recovery rate, linearity, detection sensitivity, precision and diagnostic accuracy that compared to the CAP-CTM V2.0 assay by analyzing batched low viral load clinical samples was evaluated. RESULTS: The recovery rate of the UF-based HBV DNA extraction method was above 80%. The assay linearity was demonstrated with a slope of 0.95 and R2 values of 0.99. Limit-of-detection (LOD) of the UF-qPCR assay was determined to be 12.1IU/ml. The coefficient of variation (CV) of HBV quantitation for high, low and limit titer samples was 2.28%, 5.77% and 25.59%, respectively. Accuracy of the UF-qPCR assay was confirmed with the reference panel, and there was a strong correlation between these two methods (R2 = 0.55, p < 0.01). CONCLUSIONS: The UF-qPCR assay is reliable, highly sensitive, affordable and time-saving, and the method can be used for ultrasensitive detection of serum HBV.