Obesity has emerged as an alarming health crisis due to its association with metabolic risk factors such as diabetes, dyslipidemia, and hypertension. Recent work has demonstrated the multifaceted roles of lncRNAs in regulating mouse adipose development, but their implication in human adipocytes remains largely unknown. Here we present a catalog of 3149 adipose active lncRNAs, of which 909 are specifically detected in brown adipose tissue (BAT) by performing deep RNA-seq on adult subcutaneous, omental white adipose tissue and fetal BATs. A total of 169 conserved human lncRNAs show positive correlation with their nearby mRNAs, and knockdown assay supports a role of lncRNAs in regulating their nearby mRNAs. The knockdown of one of those, lnc-dPrdm16, impairs brown adipocyte differentiation in vitro and a significant reduction of BAT-selective markers in in vivo. Together, our work provides a comprehensive human adipose catalog built from diverse fat depots and establishes a roadmap to facilitate the discovery of functional lncRNAs in adipocyte development.
Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.
ObjectivesColorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays.MethodsWe systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol.ResultsThe yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively.ConclusionsThrough the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.
Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2), has ravaged the pig industry, causing huge economic loss. At present, PCV2b and PCV2d are highly prevalent genotypes worldwide, while in China, in addition to PCV2b and PCV2d, a newly emerged PCV2e genotype detected in the Fujian province has attracted attention, indicating that PCV2 genotypes in China are more abundant. A preliminary study was conducted to better understand the genetic diversity and prevalence of PCV2 genotypes in southern China. We collected 79 random lung samples from pigs with respiratory signs, from 2018 to 2021. We found a PCV2-positivity rate of 29.1%, and frequent co-infections of PCV2 with PCV3, Streptococcus suis (S. suis), and other porcine pathogens. All PCV2-positive samples were sequenced and subjected to whole-genome analysis. Phylogenetic analysis, based on the PCV2 ORF2 gene and complete genomes, found that PCV2 strains identified in this study belonged to genotypes PCV2a (1), PCV2b (6), PCV2d (10), and PCV2e (6). Importantly, PCV2e was identified for the first time in some provinces, including Guangdong and Jiangxi. Additionally, we found two positively selected sites in the ORF2 region, located on the previously reported antigenic epitopes. Moreover, codon 63, one of the positively selected sites, has different types of amino acids in different genotypes. In conclusion, this study shows that PCV2e is an emerging genotype circulating in southern China, which warrants urgent, specific surveillance to aid the development of prevention and control strategies in China.
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