Gene expression variability in mammalian embryonic stem cells using single cell RNA-seq data

BackgroundGene expression heterogeneity contributes to development as well as disease progression. Due to technological limitations, most studies to date have focused on differences in mean expression across experimental conditions, rather than differences in gene expression variance. The advent of single cell RNA sequencing has now made it feasible to study gene expression heterogeneity and to characterise genes based on their coefficient of variation.MethodsWe collected single cell gene expression profiles for 32 human and 39 mouse embryonic stem cells and studied correlation between diverse characteristics such as network connectivity and coefficient of variation (CV) across single cells. We further systematically characterised properties unique to High CV genes.ResultsHighly expressed genes tended to have a low CV and were enriched for cell cycle genes. In contrast, High CV genes were co-expressed with other High CV genes, were enriched for bivalent (H3K4me3 and H3K27me3) marked promoters and showed enrichment for response to DNA damage and DNA repair.ConclusionsTaken together, this analysis demonstrates the divergent characteristics of genes based on their CV. High CV genes tend to form co-expression clusters and they explain bivalency at least in part.     

Hydroxyl radical formation upon dark oxidation of reduced iron minerals: Effects of iron species and environmental factors

Dark formation of hydroxyl radical upon oxidation of reduced iron minerals plays an important role in the degradation and transformation of organic and inorganic pollutants. Herein, we compared the hydroxyl radical formation from various reduced iron minerals at different redox conditions. OH production was generally observed from the oxidation of reduced iron minerals, following the order: mackinawite (FeS) > reduced nontronite (iron-bearing smectite clay) > pyrite (FeS₂) > siderite (FeCO₃). Structural Fe²⁺ and dissolved O₂ play critical roles in OH production from reduced iron minerals. OH production increases with decreasing pH, and Cl^? has little effect on this process. More importantly, dissolved organic matter significantly enhances OH production, especially under O₂ purging, highlighting the importance of this process in ambient environments. This sunlight-independent pathway in which OH forms during oxidation of reduced iron minerals is helpful for understanding the degradation and transformation of various inorganic and organic pollutants in the redox-fluctuation environments.     

Molecular cloning and expression of an endo-β-1,4-d-glucanase I (avicelase I) gene from Bacillus cellulyticus K-12 and characterization of the recombinant enzyme

Bacillus cellulyticus K-12 Avicelase (Avicelase I; EC 3.2.1.4) gene (ace A) has been cloned in Escherichia coli by using the vector pT7T3U19 and HindIII-HindIII libraries of the chromosomal inserts. The libraries were screened for the expression of avicelase by monitoring the immunoreaction of the antiavicelase (immunoscreening). Positive clones (Ac-3, Ac-5, and Ac-7) contained the identical 3.5-kb HindIII fragment as determined by restriction mapping and Southern hybridization, and expressed avicelase efficiently and constitutively using its own promoter in the heterologous host. From the immunoblotting analysis, a polypeptide that showed a carboxymethylcellulase (CMCase) activity with an M _r , of 64,000 was detected. The recombinant endo 1,4-β- _d -glucanase I was purified to homogeneity from an intracellular fraction of E. coli by DEAE-Toyopearl M650, Phenyl Toyoperal M650, and TSK gel HW50S chromatography. The enzyme had a monomeric structure, its relative molecular mass being 65 kDa by gel filtration and 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI was 5.3 and the optimal pH was 4.6, and the enzyme was stable at pH 4.0–10.5. The enzyme had a temperature optimum of 50°C and was stable at 55°C for 48 h, and retained approx 20% of its activity after 30 min at 70°C. It showed high activity toward carboxymethylcellulose (CMC) as well as p-nitrophenyl-β-d-cellobioside, 4-methylumbelliferyl cellobioside, Avicel, filter paper, and some cellooligosaccharides. K _m values for CMC and Avicel were 7.6 and 85.2 mg/mL, respectively, whereas V _max values were 201 and 9.2 μmol · min⁻¹ · mg⁻¹, respectively. Cellotetraose (G4) was preferentially cleaved into cellobiose (G2) and cellopentaose (G5) was cleaved into G2 + cellotriose (G3), whereas cellohexaose (G6) was cleaved into G4 + G2 and, to a lesser extent, into G3 + G3. G3 was not cleaved at all. G2 was the main product of Avicel hydrolysis. G2 inhibited whereas Mg⁺⁺ stimulated the activity of CMCase and Avicelase. Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars. Based on these results, it appeared that the cellulase should be regarded as endo type, although it hydrolyzed Avicel.     

聚离子亚基化合物结构影响外源基因转染效率

合成了带正电荷密度不同的聚离子亚基化合物,探讨了聚合物结构对外源基因转染真核细胞的影响规律。从实验结果看,聚离子亚基化合物结构影响外源基因转染效率,但其影响程度与靶细胞的种类相关,对ＨｅＬａ细胞,ＮＩＨ３Ｔ３细胞,聚离子亚基化合物的正电荷密度加大,其促基因转移功能加强;而对ＰＡ３１７包装细胞,结果相反。     

Dissolved oxygen concentration affects the accumulation of HIV-1 recombinant proteins in Escherichia coli

A central problem in aerobic growth of any culture is the maintenance of dissolved oxygen concentration (DOC) above growth-limiting levels especially in high-cell density fermentations that are usually of the fed-batch type. Fermentor studies have been conducted to determine the influence of DOC on the production of heterologous proteins in Escherichia coli. The results demonstrated that there is a significant degree of product-to-product variation in the response of heterologous protein accumulation to DOC. For translational fusions of the human immunodeficiency virus-1 (HIV-1) proteins p24Gag and Env41, the imposition of a dissolved oxygen (DO) limitation resulted in 100 and 15% increases in the respective product yields. On the other hand, the imposition of a DO limitation had no effect on the production of a similar translational fusion of the HIV-1 protein p55Gag, and a large negative effect on the production of an influenza protein (C13). The stimulatory effects of DOC on p24Gag production were investigated further. The results of my studies suggested that the stimulatory effect observed at reduced agitation rates on p24Gag accumulation was owing to an oxygen effect and not a shear effect. Furthermore, the results of my investigations indicated that the effect a DOC had on the production of p24Gag was strongly influenced by the cell density at which the culture was induced.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Analysis on First-stage Ignition of n-Heptane at Low Temperatures with a Lumped Skeletal Mechanism

Two-stage ignition exists in the low-temperature combustion process of n-heptane and the first-stage ignition also shows a negative temperature coefficient(NTC) phenomenon. To study key reactions and understand chemical principles affecting the first-stage ignition of n-heptane, a lumped skeletal mechanism with 62 species is obtained based on the detailed NUIGMech1.0 mechanism using the directed relation graph method assisted by sensitivity analysis and isomer lumping. The lumped mechanism shows good performance on ignition delay time under wide conditions. The study revealed that the temperature after the first-stage ignition is higher and a larger amount of fuel is consumed at lower initial temperatures. The temperature at the first-stage ignition is relatively insensitive to the initial temperature. Further sensitivity analysis and reaction path analysis carried out based on the lumped mechanism show that the decomposition of RO₂ to produce alkene and HO₂is the most important reaction to inhibit the first-stage ignitions. The chain branching explosion closely related to the first-stage ignition will be terminated when the rate constant for the RO₂ decomposition is larger than that of the isomerization of RO₂ to produce QOOH. The NTC behavior as well as other characteristics of the first-stage ignition can be rationalized from the competition between these two reactions.     

Graphene Nanocomposites Based Electrochemical Sensing Platform for Simultaneous Detection of Multi-drugs

Three reduced graphene oxide nanocomposites were employed to achieve the simultaneous electrochemical determination of multi-drugs including acetaminophen (ACTM), carbendazim (CB) and ciprofloxacin (CFX). All nanocomposite modified electrodes showed improved current responses for three drugs. Notably cauliflower-like platinum nanoparticles decorated reduced graphene oxide modified electrode (or Pt−RGO/GCE) exhibited the best performance in terms of electrochemical stability. Using Pt−RGO/GCE, the linear detect ranges of 30–120 μM, 25–115 μM and 10–25 μM, and detection limit values of 3.49, 2.96, and 1.53 μM were achieved for ACTM, CB and CFX respectively. The electrode was further used for the successful determination of above drugs in tap and river water using differential pulse voltammetry. From the obtained results, we believe that Pt-RGO/GCE is highly promising for the fabrication of robust electrochemical sensors for simultaneously determining ACTM, CB and CFX or similar types of drugs in the future.     

电子能量对沿面介质阻挡放电等离子体化学产物的影响

基于光电倍增管研制了一套无触发信号的双通道光学探测系统, 并组装了测试样机, 实现了对沿面介质阻挡放电(SDBD)等离子体约化电场的测量, 进而通过BOLSIG+软件获得了电子能量. 采用原位紫外吸收光谱和傅里叶变换红外光谱, 研究了SDBD等离子体在不同电压和频率下的化学产物的浓度变化, 并结合空气等离子体化学反应揭示了产物相互作用的微观机理. 结果表明, 电子能量能够改变电子碰撞反应的速率系数, 调控化学反应的源头活性粒子的浓度, 进而影响到化学产物的生成和猝灭.