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.

Background China is among the largest and fastest aging countries. The elderly population is more vulnerable, with higher proportion of inappropriate sleep duration and risk of mortality, compared with young and middle-aged adults. Single-measured sleep duration has been associated with mortality, but the health effects of long-term sleep duration trajectories remain unknown. This study aimed to explore the prospective associations between sleep duration trajectories and all-cause mortality among Chinese elderly. Methods Participants (n = 3,895; median age: 82 years; females: 53.3%) who reported sleep duration in all three surveys (2005, 2008, and 2011) from the community-based Chinese Longitudinal Healthy Longevity Survey (CLHLS) were followed up until 2019 (about 8 years). We identified sleep duration trajectories by latent class mixed model and explored their association with all-cause mortality using Cox hazard proportional regression and Laplace regression models. Further, stratified analysis by demographic characteristics and lifestyles and sensitivity analysis by lag effect, health-related factors, and inverse probability weighting were used to verify the robustness of the association. In addition, we explored the threshold effect of baseline sleep duration on the risk of all-cause mortality. Results We documented 1,881 all-cause deaths during 16,689 person-years of follow-up. Five sleep duration trajectories were identified: moderately increased trajectory (28.1%), rapidly increased trajectory (7.2%), persistent sleep trajectory of 7 h (33.7%), moderately decreased trajectory (21.3%), and rapidly decreased trajectory (9.7%). Compared with the persistent sleep trajectory of 7 h, the multivariable-adjusted HRs (95%CI) for moderately increased trajectory, rapidly increased trajectory, moderately decreased trajectory, and rapidly decreased trajectory were 1.21 (1.08, 1.36), 1.21 (1.01, 1.44), 0.95 (0.82, 1.10), and 0.93 (0.78, 1.11), respectively; and the corresponding difference in median survival time (95%CI) were -0.53 (-1.01, -0.05), -0.43 (0.16, -1.02), 0.26 (-0.34, 0.86), and 0.25 (-0.51, 1.02), respectively. Stratified and sensitivity analyses showed consistent results. Threshold analysis indicated a sharply increased risk of mortality in participants whose sleep exceeds 9 h (HR = 1.20, 95%CI: 1.11, 1.30). Conclusion Compared with the persistent sleep trajectory of 7 h, moderately and rapidly increased sleep duration trajectories were associated with higher subsequent mortality in Chinese elderly. Those who report sleep exceeding 9 h may be at high risk for all-cause mortality.

Background An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. Methods We report here a new method using a single closed-tube nested quantitative PCR (CN–qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD–PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD–PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. Results Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN–qPCR assay and the standard LD–PCR assay. CN–qPCR successfully made calls for all samples, whereas LD–PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN–qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. Conclusions This new CN–qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.

The pathogenesis of nonalcoholic steatohepatitis (NASH), a severe stage of nonalcoholic fatty liver disease, is complex and implicates multiple cell interactions. However, therapies for NASH that target multiple cell interactions are still lacking. Melatonin (MEL) alleviates NASH with mechanisms not yet fully understood. Thus, we herein investigate the effects of MEL on key cell types involved in NASH, including hepatocytes, macrophages, and stellate cells. In a mouse NASH model with feeding of a methionine and choline-deficient (MCD) diet, MEL administration suppressed lipid accumulation and peroxidation, improved insulin sensitivity, attenuated inflammation and fibrogenesis in the liver. Specifically, MEL reduced proinflammatory cytokines expression and inflammatory signal activation and attenuated CD11C+CD206- M1-like macrophage polarization in the liver of NASH mice. The reduction of proinflammatory response by MEL was also observed in the lipopolysaccharide-stimulated Raw264.7 cells. Additionally, MEL increased liver fatty acid β-oxidation, leading to reduced lipid accumulation, and restored the oleate-loaded primary hepatocytes. Finally, MEL attenuated hepatic stellate cell activation and fibrogenesis in the liver of MCD-fed mice and in LX-2 human hepatic stellate cells. In conclusion, MEL acts on multiple cell types in the liver to mitigate NASH-associated phenotypes, supporting MEL or its analog as potential treatment for NASH.

No abstract