ObjectiveCurrent non-invasive early detection of colorectal cancer (CRC) requires improvement. We aimed to identified a fecal Clostridium symbiosum-based biomarker for early and advanced colorectal cancer detection.DesignIn the test stage, the relative abundance of Clostridium symbiosum (C. symbiosum) was measured by qPCR in 781 cases including 242 controls, 212 colorectal adenoma (CRA) patients, 109 early CRC (tumor restricted to the submucosa) patients, 218 advanced CRC patients. The prediction accuracy was compared to Fusobacterium nucleatum (F. nucleatum), fecal immunochemical test (FIT) and CEA (carcinoembryonic antigen) and validated in an independent cohort of 256 subjects. Current status of the trial:ongoing/still enrolling. Primary endpoint:June, 2017 (Clinicaltrials.gov Identifier NCT02845973).ResultsSignificant stepwise increase of C. symbiosum abundance was found in CRA, early CRC and advanced CRC (P < 0.01). C. symbiosum outperformed all the other markers in early CRC prediction performance. The combination of C. symbiosum and FIT achieved better performance (0.803 for test cohort and 0.707 for validation cohort). For overall discrimination of CRCs, the combination of all above markers achieved the performance of 0.876.ConclusionsFecal C. symbiosum is a promising biomarker for early and noninvasive detection of colorectal cancer, being more effective than F. nucleatum, FIT and CEA. Combining C. symbiosum and FIT or CEA may improve the diagnosis power.

High-throughput screening is a key technique in discovery and engineering of enzymes. In vitro compartmentalization based fluorescence-activated cell sorting (IVC-FACS) has recently emerged as a powerful tool for ultrahigh-throughput screening of biocatalysts. However, the accuracy of current IVC-FACS assays is severely limited by the wide polydispersity of micro-reactors generated by homogenizing. Here, an improved protocol based on membrane-extrusion technique was reported to generate the micro-reactors in a more uniform manner. This crucial improvement enables ultrahigh-throughput screening of enzymatic activity at a speed of >108 clones/day with an accuracy that could discriminate as low as two-fold differences in enzymatic activity inside the micro-reactors, which is higher than similar IVC-FACS systems ever have reported. The enzymatic reaction in the micro-reactors has very similar kinetic behavior compared to the bulk reaction system and shows wide dynamic range. By using the modified IVC-FACS, E. coli cells with esterase activity could be enriched 330-fold from large excesses of background cells through a single round of sorting. The utility of this new IVC-FACS system was further illustrated by the directed evolution of thermophilic esterase AFEST. The catalytic activity of the very efficient esterase was further improved by ∼2-fold, resulting in several improved mutants with k cat/K M values approaching the diffusion-limited efficiency of ∼108 M−1s−1.

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