Identification of suitable reference genes for gene expression analysis of pork meat quality and analysis of candidate genes associated with the trait drip loss

The aim of this study was to identify a set of stably expressed endogenous control genes for quantitative PCR analysis of mRNA expression in the porcine LTL muscle and to subsequently perform expression analysis of potential candidate genes associated with drip loss. Expression stability of seven commonly used reference genes was examined in n = 60 pigs from three independent populations of different genetic backgrounds. The genes examined were: ACTB, ATP5G1, B2M, GPX1, RPL4, TBP and YWHAZ. GeNorm analysis of expression stability identified B2M, RPL4 and TBP as consistently stable in each breed examined. Analysis of meat samples divergent for water holding capacity identified positive and negative associations between drip loss and gene expression using B2M, RPL4 and TBP as endogenous controls. Specifically, expression of COL1A1 increased significantly with increasing drip loss while expression of CAST decreased significantly with increasing drip loss. This study therefore indicates the use of B2M, RPL4 and TBP as suitable endogenous controls for gene expression analysis of the porcine LTL muscle. Further study is recommended to identify the detailed roles of COL1A1 and CAST with respect to the development of drip loss.     

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.     

Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

Contamination of recreational waters with Escherichia coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the β intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures.     

浅析生物基因原理中所蕴含的产品创新技法

产品创新与很多生物基因原理相似.如果把市场比作大自然,产品比作有生命的物种,那么那些在市场上畅销的产品正是经过激烈的竞争存活下来的优良物种,它们都由优良的产品信息基因所控制的.本文主要浅析从生物基因原理中所折射出来的一些产品创新技法,从而设计出适合市场竞争的优质产品.     

Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan

The purpose of this study was to quantify the occurrence and release of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) into the environment through the effluent and biosolids of different wastewater treatment utilities including an MBR (Membrane Biological Reactor) utility, conventional utilities (Activated Sludge, Oxidative Ditch and Rotatory Biological Contactors-RBCs) and multiple sludge treatment processes (Dewatering, Gravity Thickening, Anaerobic Digestion and Lime Stabilization). Samples of raw wastewater, pre- and post-disinfected effluents, and biosolids were monitored for tetracycline resistant genes (tetW and tetO) and sulfonamide resistant gene (Sul-I) and tetracycline and sulfonamide resistant bacteria. ARGs and ARB concentrations in the final effluent were found to be in the range of ND(non-detectable)-2.33 × 10⁶ copies/100 mL and 5.00 × 10²-6.10 × 10⁵ CFU/100 mL respectively. Concentrations of ARGs (tetW and tetO) and 16s rRNA gene in the MBR effluent were observed to be 1-3 log less, compared to conventional treatment utilities. Significantly higher removals of ARGs and ARB were observed in the MBR facility (range of removal: 2.57-7.06 logs) compared to that in conventional treatment plants (range of removal: 2.37-4.56 logs) (p < 0.05). Disinfection (Chlorination and UV) processes did not contribute in significant reduction of ARGs and ARB (p > 0.05). In biosolids, ARGs and ARB concentrations were found to be in the range of 5.61 × 10⁶-4.32 × 10⁹ copies/g and 3.17 × 10⁴-1.85 × 10⁹ CFU/g, respectively. Significant differences (p < 0.05) were observed in concentrations of ARGs (except tetW) and ARB between the advanced biosolid treatment methods (i.e., anaerobic digestion and lime stabilization) and the conventional dewatering and gravity thickening methods.     

基于神经网络-遗传算法的回流焊参数设定

针对回流焊工艺过程中输入参数难以设定的问题,对多重质量特征值间非线性映射关系进行分析,提出一种基于改进遗传算法的输入参数设定方法。采用实数编码形式,直接表达了各基因表示的意义。应用改进的遗传算子和染色体重启机制,提高了搜索准确程度,避免了早熟现象和陷入局部最优的可能。再结合神经网络预测方案建立完整的参数设置与生产预测模型。以某公司实际生产数据为例,MAPE评估显示预设参数满足企业生产误差精度要求,因此所提出的设定方法可以有效地进行回流焊生产,为企业回流焊生产工艺规划提供指导。     

Short- and long-term dynamics of the toxic potential and genotypic structure in benthic populations of Microcystis

Microcystis colonies are known to overwinter on the surface of the sediment of freshwater ecosystems. However, little is known about the genotypic and toxicological dynamics of Microcystis populations during this benthic life stage. In this study, we report a two-year-long survey of benthic populations of Microcystis, which had spent from a few days to more than six years in the sediment. In order to avoid any interaction with the planktonic proliferations, we chose two deeply buried benthic populations, which could be easily dated. Quantitative PCR on mcyB gene and protein phosphatase inhibition assays were performed to measure their toxic potential, and their genotypic structure was assessed by Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP), based on 16S-23S Intergenic Transcribed Spacer (ITS). The microcystin content of the cells seemed to change sharply during the first few months of benthic survival, whereas this content was low and decreased steadily after several years of benthic life. No genetic selection was observed in either the proportion of potentially toxic clones or the ITS sequences for any of the populations considered. From these results, the benthic life stage of Microcystis appears to preserve the structure and the composition of the population over a far larger time scale than classical overwintering period. Finally, some genotypes were common in both of the benthic populations, even though they originated from planktonic blooms that had developed five years apart, suggesting a major overlap of planktonic proliferations in successive years.     

Identification of Novel Insulin Resistance Related ceRNA Network in T2DM and Its Potential Editing by CRISPR/Cas9

Background: Type 2 diabetes mellitus is one of the leading causes of morbidity and mortality worldwide and is derived from an accumulation of genetic and epigenetic changes. In this study, we aimed to construct Insilco, a competing endogenous RNA (ceRNA) network linked to the pathogenesis of insulin resistance followed by its experimental validation in patients’, matched control and cell line samples, as well as to evaluate the efficacy of CRISPR/Cas9 as a potential therapeutic strategy to modulate the expression of this deregulated network. By applying bioinformatics tools through a two-step process, we identified and verified a ceRNA network panel of mRNAs, miRNAs and lncRNA related to insulin resistance, Then validated the expression in clinical samples (123 patients and 106 controls) and some of matched cell line samples using real time PCR. Next, two guide RNAs were designed to target the sequence flanking LncRNA/miRNAs interaction by CRISPER/Cas9 in cell culture. Gene editing tool efficacy was assessed by measuring the network downstream proteins GLUT4 and mTOR via immunofluorescence. Results: LncRNA-RP11-773H22.4, together with RET, IGF1R and mTOR mRNAs, showed significant upregulation in T2DM compared with matched controls, while miRNA (i.e., miR-3163 and miR-1) and mRNA (i.e., GLUT4 and AKT2) expression displayed marked downregulation in diabetic samples. CRISPR/Cas9 successfully knocked out LncRNA-RP11-773H22.4, as evidenced by the reversal of the gene expression of the identified network at RNA and protein levels to the normal expression pattern after gene editing. Conclusions: The present study provides the significance of this ceRNA based network and its related target genes panel both in the pathogenesis of insulin resistance and as a therapeutic target for gene editing in T2DM.