Shortcut barcoding and early pooling for scalable multiplex single-cell reduced-representation CpG methylation sequencing at single nucleotide resolution.

DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.

Characteristics of Renal Function in Patients Diagnosed With COVID-19: An Observational Study.

Objective: The aim of the study was to analyze the characteristics of renal function in patients diagnosed with COVID-19. Methods: In this retrospective, single-center study, we included all confirmed cases of COVID-19 in a tertiary hospital in Guangdong, China from January 20, 2020 to March 20, 2020. Blood and urine laboratory findings related to renal function were summarized, and the estimated glomerular filtration rate (eGFR) and endogenous creatinine clearance (Ccr) were also calculated to assess the renal function. Results: A total of 12 admitted hospital patients were diagnosed with COVID-19, included 3 severe cases, and 9 common cases. Serum creatinine (Scr) was not abnormally elevated in all of the patients, and blood urea nitrogen (BUN) was abnormally elevated in only 25.0% of the patients. However, compared with the recovery period, the patient's Scr and BUN increased significantly in peak of disease (p-scr = 0.002 & p-bun < 0.001). By observing the fluctuations in Scr and BUN from admission to recovery, it was found that the peak of Scr and BUN appeared within the first 14 day of the course of the disease. Urinary microprotein detection indicated that the abnormally elevated rates of urine microalbumin (UMA), α1-microglobulin (A1M), urine immunoglobulin-G (IGU), and urine transferring (TRU) standardized by urinary creatinine in peak of disease were 41.7, 41.7, 50.0, and 16.7%, respectively. The abnormal rates of the calculated eGFR and Ccr were 66.7 and 41.7%. Conclusion: Scr and BUN were generally increased during the course of COVID-19. Detection of urinary microproteins and application of multiple indicators assessment could be helpful for discovering abnormal renal function in patients with COVID-19. However, the evidence is limited due to the small sample size and observational nature. Additional studies, especially large prospective cohort studies, are required to confirm these findings.

Metabolic Response in Rats following Electroacupuncture or Moxibustion Stimulation.

Electroacupuncture and moxibustion are traditional Chinese medicine practices that exert therapeutic effects through stimulation of specific meridian acupoints. However, the biological basis of the therapies has been difficult to establish; thus the current practices still rely on ancient TCM references. Here, we used a rat model to study perturbations in cortex, liver, and stomach metabolome and plasma hormones following electroacupuncture or moxibustion treatment on either stomach meridian or gallbladder meridian acupoints. All treatment groups, regardless of meridian and mode of treatment, showed perturbation in cortex metabolome and increased phenylalanine, tyrosine, and branched-chain amino acids in liver. In addition, electroacupuncture was found to increase ATP in cortex, creatine, and dimethylglycine in stomach and GABA in liver. On the other hand, moxibustion increased plasma enkephalin concentration, as well as betaine and fumarate concentrations in stomach. Furthermore, we had observed meridian-specific changes including increased N-acetyl-aspartate in liver and 3-hydroxybutyrate in stomach for gallbladder meridian stimulation and increased noradrenaline concentration in blood plasma following stimulation on stomach meridian. In summary, the current findings may provide insight into the metabolic basis of electroacupuncture and moxibustion, which may contribute towards new application of acupoint stimulation.

Metabolomics analysis of herb-partitioned moxibustion treatment on rats with diarrhea-predominant irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, which is commonly treated with antidiarrhoeal, antispasmodics, serotonergic agents or laxative agents. These treatments provide relief for IBS symptoms but may also lead to undesired side effects. Previously, herb-partitioned moxibustion (HPM) treatment has been demonstrated to be effective in ameliorating symptoms of IBS. However, the underlying mechanism of this beneficial treatment is yet to be established. The aim of the current study was to systematically assess the metabolic alterations in response to diarrhea-predominant IBS (IBS-D) and therapeutic effect of HPM. METHODS: Proton nuclear magnetic resonance spectroscopy (1H NMR)-based metabolomics approach was used to investigate fecal and serum metabolome of rat model of IBS-D with and without HPM treatment. RESULTS: The current results showed that IBS-induced metabolic alterations in fecal and serum sample include higher level of threonine and UDP-glucose together with lower levels of aspartate, ornithine, leucine, isoleucine, proline, 2-hydroxy butyrate, valine, lactate, ethanol, arginine, 2-oxoisovalerate and bile acids. These altered metabolites potentially involve in impaired gut secretory immune system and intestinal inflammation, malabsorption of nutrients, and disordered metabolism of bile acids. Notably, the HPM treatment was found able to normalize the Bristol stool forms scale scores, fecal water content, plasma endotoxin level, and a number of IBS-induced metabolic changes. CONCLUSIONS: These findings may provide useful insight into the molecular basis of IBS and mechanism of the HPM intervention.