Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge

The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. Sewage treatment plants (STPs) are considered as important reservoirs for antibiotic resistance genes (ARGs) and activated sludge characterized with high microbial density and diversity facilitates ARG horizontal gene transfer (HGT) via mobile genetic elements (MGEs). However, little is known regarding the pool of ARGs and MGEs in sludge microbiome. In this study, the transposon aided capture (TRACA) system was employed to isolate novel plasmids from activated sludge of one STP in Hong Kong, China. We also used Illumina Hiseq 2000 high-throughput sequencing and metagenomics analysis to investigate the plasmid metagenome. Two novel plasmids were acquired from the sludge microbiome by using TRACA system and one novel plasmid was identified through metagenomics analysis. Our results revealed high levels of various ARGs as well as MGEs for HGT, including integrons, transposons and plasmids. The application of the TRACA system to isolate novel plasmids from the environmental metagenome, coupled with subsequent high-throughput sequencing and metagenomic analysis, highlighted the prevalence of ARGs and MGEs in microbial community of STPs.     

Manganese binds to Clostridium difficile Fbp68 and is essential for fibronectin binding

Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficile-associated disease is recognized. In this study, we identified Fbp68, one of the colonization factors that bind to fibronectin (Fn), as a manganese-binding protein (K(D) = 52.70 ± 1.97 nM). Furthermore, the conformation of Fbp68 changed dramatically upon manganese binding. Manganese binding can also stabilize the structure of Fbp68 as evidenced by the increased T(m) measured by thermodenatured circular dichroism and differential scanning calorimetry (CD, T(m) = 58-65 °C; differential scanning calorimetry, T(m) = 59-66 °C). In addition, enhanced tolerance to protease K also suggests greatly improved stability of Fbp68 through manganese binding. Fn binding activity was found to be dependent on manganese due to the lack of binding by manganese-free Fbp68 to Fn. The C-terminal 194 amino acid residues of Fbp68 (Fbp68C) were discovered to bind to the N-terminal domain of Fn (Fbp68C-NTD, K(D) = 233 ± 10 nM, obtained from isothermal titration calorimetry). Moreover, adhesion of C. difficile to Caco-2 cells can be partially blocked if cells are pretreated with Fbp68C, and the binding of Fbp68C on Fn siRNA-transfected cells was significantly reduced. These results raise the possibility that Fbp68 plays a key role in C. difficile adherence on host cells to initiate infection.     

混杂试验的多元分析

本文首次提出混杂试验的多元统计分析方法,并以香茅肥料试验为实例,论述平方和与乘积和矩阵的分解及总自由度的裂分方法,各种WilksA统计量的构造及其显著性测验法．最后给出了笔者编制的SAS统计分析程序．     

Tumor necrosis factor-alpha enhances inhibitory effects of interleukin-1 beta on Leydig cell steroidogenesis

Human recombinant tumor necrosis factor-alpha (rTNF alpha) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNF alpha with human recombinant interleukin-1 beta (rIL-1 beta) enhanced the inhibitory effects of rIL-1 beta. The rIL-1 beta dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNF alpha had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNF alpha in a dose dependent manner. In the presence of both rTNF alpha and rIL-1 beta, hCG-induced cyclic AMP formation and binding of [125I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNF alpha and IL-1 may be produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.     

血管成形术后原癌基因c－fos、c－myc表达的研究

ｃ－ｆｏｓ、ｃ－ｍｙｃ是两种参与细胞增殖的原癌基因。动脉血管成形术（ＰＴＡ）后的血管再狭窄,是由于血管壁平滑肌细胞增生所致。利用大白兔建立的试验动物模型,提取ＰＴＡ后不同时间间隔的动脉壁总ＲＮＡ,并用斑点杂交法分析两种原癌基因的表达情况,发现了ｃ－ｆｏｓ和ｃ－ｍｙｃ的基因分别在术后１５分钟内转录活性提高,并分别在３０分钟和第２小时达到高峰。     

Chromosomal distribution and organization of three cervid satellite DNAs in Chinese water deer (Hydropotes inermis)

The species-specific profile and centromeric heterochromatin localization of satellite DNA in mammalian genomes imply that satellite DNA may play an important role in mammalian karyotype evolution and speciation. A satellite III DNA family, CCsatIII was thought to be specific to roe deer (Capreolus capreolus). In this study, however, this satellite DNA family was found also to exist in Chinese water deer (Hydropotes inermis) by PCR-Southern screening. A satellite III DNA element of this species was then generated from PCR-cloning by amplifying this satellite element using primer sequences from the roe deer satellite III clone (CCsatIII). The newly generated satellite III DNA along with previously obtained satellite I and II DNA clones were used as probes for FISH studies to investigate the genomic distribution and organization of these three satellite DNA families in centromeric heterochromatin regions of Chinese water deer chromosomes. Satellite I and II DNA were observed in the pericentric/centric regions of all chromosomes, whereas satellite III was distributed on 38 out of 70 chromosomes. The distribution and orientation of satellite DNAs I, II and III in the centromeric heterochromatin regions of the genome were further classified into four different types. The existence of a Capreolus-like satellite III in Chinese water deer implies that satellite III is not specific to the genus Capreolus (Buntjer et al., 1998) and supports the molecular phylogeny classification of Randi et al. (1998) which suggests that Chinese water deer and roe deer are closely related.     

Protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism

Pyruvate, an endogenous metabolite of glycolysis, is an anti-toxicity agent. Recent studies have suggested possible roles for pyruvate in protecting CNS neurons from excitotoxic and metabolic insults. Utilizing cultures derived from embryonic rat cortex, the studies presented in this paper indicate that an astroglia-mediated mechanism is involved in the neuroprotective effects of pyruvate against glutamate toxicity. Glutamate-induced toxicity could be reversed by pyruvate in a mixed culture of cortex cells. Importantly, in pure neuronal cultures from the same tissue, pyruvate failed to protect against glutamate toxicity. Addition of astroglia to the pure neuronal cultures restores the ability of pyruvate to protect neurons from glutamate-induced toxicity. Our results further suggest that pyruvate can induce glia to up-regulate the synthesis of glutathione (GSH), an antioxidant that protects cells from toxins such as free radicals. Taken together, our data suggest that astroglia in mixed cultures are essential for mediating the effects of pyruvate, revealing a novel mechanism by which pyruvate, an important intermediate of tricarboxylic acid cycle in the body, may act to protect neurons from damage during insults such as brain ischemia.