Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE

On the validity of the local Fourier analysis

Local Fourier analysis (LFA) is a useful tool in predicting the convergence factors of geometric multigrid methods (GMG). As is well known, on rectangular domains with periodic boundary conditions this analysis gives the exact convergence factors of such methods. In this work, using the Fourier method, we extend these results by proving that such analysis yields the exact convergence factors for a wider class of problems

The transcription factor Ste12-like increases the mycelial abiotic stress tolerance and regulates the fruiting body development of Flammulina filiformis

IntroductionFlammulina filiformis is one of the most commercially important edible fungi worldwide, with its nutritional value and medicinal properties. It becomes a good model species to study the tolerance of abiotic stress during mycelia growth in edible mushroom cultivation. Transcription factor Ste12 has been reported to be involved in the regulation of stress tolerance and sexual reproduction in fungi.MethodsIn this study, identification and phylogenetic analysis of ste12-like was performed by bioinformatics methods. Four ste12-like overexpression transformants of F. filiformis were constructed by Agrobacterium tumefaciens-mediated transformation.Results and DiscussionPhylogenetic analysis showed that Ste12-like contained conserved amino acid sequences. All the overexpression transformants were more tolerant to salt stress, cold stress and oxidative stress than wild-type strains. In the fruiting experiment, the number of fruiting bodies of overexpression transformants increased compared with wild-type strains, but the growth rate of stipes slowed down. It suggested that gene ste12-like was involved in the regulation of abiotic stress tolerance and fruiting body development in F. filiformis

An observational and Mendelian randomisation study on vitamin D and COVID-19 risk in UK Biobank

A growing body of evidence suggests that vitamin D deficiency has been associated with an increased susceptibility to viral and bacterial respiratory infections. In this study, we aimed to examine the association between vitamin D and COVID-19 risk and outcomes. We used logistic regression to identify associations between vitamin D variables and COVID-19 (risk of infection, hospitalisation and death) in 417,342 participants from UK Biobank. We subsequently performed a Mendelian Randomisation (MR) study to look for evidence of a causal effect. In total, 1746 COVID-19 cases (399 deaths) were registered between March and June 2020. We found no significant associations between COVID-19 infection risk and measured 25-OHD levels after adjusted for covariates, but this finding is limited by the fact that the vitamin D levels were measured on average 11 years before the pandemic. Ambient UVB was strongly and inversely associated with COVID-19 hospitalization and death overall and consistently after stratification by BMI and ethnicity. We also observed an interaction that suggested greater protective effect of genetically-predicted vitamin D levels when ambient UVB radiation is stronger. The main MR analysis did not show that genetically-predicted vitamin D levels are causally associated with COVID-19 risk (OR = 0.77, 95% CI 0.55–1.11, P = 0.160), but MR sensitivity analyses indicated a potential causal effect (weighted mode MR: OR = 0.72, 95% CI 0.55–0.95, P = 0.021; weighted median MR: OR = 0.61, 95% CI 0.42–0.92, P = 0.016). Analysis of MR-PRESSO did not find outliers for any instrumental variables and suggested a potential causal effect (OR = 0.80, 95% CI 0.66–0.98, p-val = 0.030). In conclusion, the effect of vitamin D levels on the risk or severity of COVID-19 remains controversial, further studies are needed to validate vitamin D supplementation as a means of protecting against worsened COVID-19

The Optimal Chinese Herbal Injections for Use With Radiotherapy to Treat Esophageal Cancer: A Systematic Review and Bayesian Network Meta-Analysis

Ethnopharmacological relevance: Esophageal cancer is one of the most common human cancers, and its incidence is highly endemic in China. The combination of Chinese herbal injections (CHIs) and radiotherapy should be informed by the best available evidence.Aim of the study: To update and expand on previous work in order to compare and rank the efficacy and safety of CHIs in combination with radiotherapy to treat esophageal cancer.Materials and Methods: We searched several electronic databases to identify randomized controlled trials (RCTs) regarding CHIs to treat esophageal cancer from their inception to March 15, 2017. In a network meta-analysis (NMA), the bias of the included trials was assessed by two individuals independently in accordance with the Cochrane Handbook for Systematic Reviews of Interventions. Outcomes such as the clinical effectiveness rate, performance status, adverse reactions (ADRs), and survival rate were evaluated. We performed a random-effects NMA to obtain estimates of efficacy and safety outcomes, and we present these estimates as odds ratios (ORs) and corresponding 95% confidence intervals (CIs) calculated via Stata 13.1 and WinBUGS 1.4 software. Furthermore, the surface under cumulative the ranking curve (SUCRA) was used to rank the efficacy and safety of different CHIs in relation to each outcome.Results: Of 685 identified trials, 55 were eligible for inclusion in the study. These 55 trials included 12 CHIs and 4,114 participants. The cluster analysis results suggested that Compound kushen injection therapy is the optimal CHI treatment for patients with esophageal cancer in terms of improving the clinical effectiveness rate and performance status. Huachansu and Kangai injection are superior in improving 1-year and 2-year survival rates. Lentinan injection may be considered a favorable choice for reliving ADRs, and Compound kushen injection may provide treatment benefits by reducing both gastrointestinal reaction and radiation esophagitis.Conclusions: The current clinical evidence indicated that Compound kushen injection combined with radiotherapy is the most preferable and beneficial option for patients with esophageal cancer in terms of efficacy and safety. However, the results of our study should be interpreted with caution given the limitations of the sample size and the methodological quality of the included trials

Immunomodulatory Effects of Cinobufagin on Murine Lymphocytes and Macrophages

Cinobufagin (CBG), a major bioactive component of the traditional Chinese medicine ChanSu, has been reported to have potent pharmacological activity. In this study, we aimed to elucidate the effects of CBG on the activity of immune cells in mice. Peritoneal macrophages and splenocytes from mice were prepared and cultured in RPMI1640 supplemented with 10% fetal bovine serum. Concanavalin (ConA), lipopolysaccharide (LPS), and CBG (0.0125, 0.05, 0.15, or 0.25 μg/mL) were added to the culture medium, and the phagocytic activity of macrophages was detected by MTT assays. Additionally, lymphocyte secretion of interleukin- (IL-)2 and IL-10 was detected by enzyme-linked immunosorbent assay, and the cell cycle distribution and cell surface markers were detected by flow cytometry. Our results demonstrated that CBG promoted lymphocyte proliferation; this effect was suppressed by combined treatment with ConA or LPS. Moreover, CBG also significantly improved the CD4+/CD8+ ratio in spleen lymphocytes and increased the percentage of spleen lymphocytes in the S phase. Finally, we found that CBG enhanced the secretion of IL-2 and IL-10 and increased the phagocytosis ability of macrophages. In summary, CBG could enhance activity of immune cells

Identification of Potential Key Genes Associated With the Pathogenesis and Prognosis of Gastric Cancer Based on Integrated Bioinformatics Analysis

Background and Objective: Despite striking advances in multimodality management, gastric cancer (GC) remains the third cause of cancer mortality globally and identifying novel diagnostic and prognostic biomarkers is urgently demanded. The study aimed to identify potential key genes associated with the pathogenesis and prognosis of GC.Methods: Differentially expressed genes between GC and normal gastric tissue samples were screened by an integrated analysis of multiple gene expression profile datasets. Key genes related to the pathogenesis and prognosis of GC were identified by employing protein–protein interaction network and Cox proportional hazards model analyses.Results: We identified nine hub genes (TOP2A, COL1A1, COL1A2, NDC80, COL3A1, CDKN3, CEP55, TPX2, and TIMP1) which might be tightly correlated with the pathogenesis of GC. A prognostic gene signature consisted of CST2, AADAC, SERPINE1, COL8A1, SMPD3, ASPN, ITGBL1, MAP7D2, and PLEKHS1 was constructed with a good performance in predicting overall survivals.Conclusion: The findings of this study would provide some directive significance for further investigating the diagnostic and prognostic biomarkers to facilitate the molecular targeting therapy of GC