The solution structure of the invasive tip complex from Afa/Dr fibrils

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.     

Gene selection using support vector machines with non-convex penalty

MOTIVATION: With the development of DNA microarray technology, scientists can now measure the expression levels of thousands of genes simultaneously in one single experiment. One current difficulty in interpreting microarray data comes from their innate nature of 'high-dimensional low sample size'. Therefore, robust and accurate gene selection methods are required to identify differentially expressed group of genes across different samples, e.g. between cancerous and normal cells. Successful gene selection will help to classify different cancer types, lead to a better understanding of genetic signatures in cancers and improve treatment strategies. Although gene selection and cancer classification are two closely related problems, most existing approaches handle them separately by selecting genes prior to classification. We provide a unified procedure for simultaneous gene selection and cancer classification, achieving high accuracy in both aspects. RESULTS: In this paper we develop a novel type of regularization in support vector machines (SVMs) to identify important genes for cancer classification. A special nonconvex penalty, called the smoothly clipped absolute deviation penalty, is imposed on the hinge loss function in the SVM. By systematically thresholding small estimates to zeros, the new procedure eliminates redundant genes automatically and yields a compact and accurate classifier. A successive quadratic algorithm is proposed to convert the non-differentiable and non-convex optimization problem into easily solved linear equation systems. The method is applied to two real datasets and has produced very promising results. AVAILABILITY: MATLAB codes are available upon request from the authors.     

Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance

Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.     

Improve islet yields and quality when clinical grade pancreata are preserved by the two-layer method

Background: Research grade pancreata preserved by the two-layer method (TLM) yield significantly greater numbers of islets than organs stored with University of Wisconsin solution (UW). The goal of this study was to determine whether this would hold true for pancreata that meet selection criteria for clinical grade organs. Methods: Pancreata were chosen based upon a pre-defined set of criteria used for selecting clinical grade pancreata. Thirteen of these organs were preserved in UW and five pancreata were preserved by the TLM. Islets were isolated and evaluated according to the Edmonton protocol. Results: The average preservation time was significantly longer for organ preserved with TLM (9.5 + 2.0 h) as compared to UW (5.8 + 0.6 h, p = 0.015). The pancreata of TLM group resulted in a significant increase in islet yields (3588 ± 500 vs. 2536 ± 312 IE/g pancreas, p<0.05). Visual scoring of islets indicated that islets were better from TLM group (8.3 ± 0.3 vs. 7.3 ± 0.2), and islet survival rates after culture were higher from organs stored with the TLM (87 ± 17 vs. 55 ± 7.4, p<0.05). Other parameters such as viability, insulin content, and stimulation index were similar between the two groups. All the preparations from the TLM group, but only 54% of preparations from the UW group, qualified for islet transplantation. The two recipients receiving islets from TLM group, daily insulin requirements were reduced and C-peptide levels were increased. Conclusion: Compared to storage with UW, exposure of pancreata to the TLM resulted in greater islet yields and improved quality of islets despite longer preservation period. Consequently, pancreata that meet clinical grade status should be preserved by the TLM prior to islet isolation.     

Apelin protects myocardial injury induced by isoproterenol in rats

We aimed to explore the change in level of apelin and its receptor APJ during myocardial injury and the therapeutic effects of apelin in myocardial injury. Rat myocardial injury was induced by subcutaneous injection of a high dose of isoproterenol (ISO); apelin and APJ mRNA levels were determined by RT-PCR; APJ protein was determined by Western blot; EIA and RIA were used to measure the apelin content and receptor binding, respectively. Plasma lactate dehydrogenase (LDH) activity and myocardial and plasma malondialdehyde (MDA) contents were higher in ISO-treated hearts than that in controls. ISO-treated rats showed lower +/-LV dp/dt(max) values and higher LVEDP value (all P<0.01), which suggested severe heart failure. As well, the apelin content in plasma, atrial and ventricular myocardium was decreased by 27%, 30% and 25% (P<0.01), respectively. The mRNA levels of apelin and APJ in myocardia were also markedly reduced; but the APJ protein level in myocardia was increased. However, administration of apelin significantly ameliorated myocardial injury and ISO-induced heart failure. Compared with the ISO-alone group, the group given low-dosage apelin (5 nmol/kg/day) had 39% and 66% higher +LV dp/dt(max) and -LV dp/dt(max) values, and 40.7% lower LVEDP value (P<0.01), and the leakage of myocardial LDH and increased MDA content were attenuated (all P<0.01). Interestingly, bolus injections of apelin (10 nmol/kg/day) resulted in potent inotropic effects in ISO-treated rats. ISO-induced myocardial injury resulted in hypoexpression of apelin and its receptor APJ, and the administration of exogenous apelin ameliorated heart failure and myocardial injury. Apelin could have a cardioprotective effect, and the apelin-APJ system may be a new therapeutic target in myocardial injury and heart failure.     

Current and future approaches using genetically modified mice in endocrine research

Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre-loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism.     

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.     

Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit

In higher eukaryotic organisms, the checkpoint kinase 1 (Chk1) contributes essential functions to both cell cycle and checkpoint control. Chk1 executes these functions, in part, by targeting the Cdc25A protein phosphatase for ubiquitin-mediated proteolysis. In response to genotoxic stress, Chk1 is phosphorylated on serines 317 (S317) and 345 (S345) by the ataxia-telangiectasia-related (ATR) protein kinase. Phosphorylation of Chk1 on these C-terminal serine residues is used as an indicator of Chk1 activation in vivo. Here, we report that inhibition of Chk1 kinase activity paradoxically leads to the accumulation of S317- and S345-phosphorylated Chk1 in vivo and that ATR catalyzes Chk1 phosphorylation under these conditions. We demonstrate that Chk1 phosphorylation by ATR is antagonized by protein phosphatase 2A (PP2A). Importantly, dephosphorylation of Chk1 by PP2A is regulated, in part, by the kinase activity of Chk1. We propose that the ATR-Chk1-PP2A regulatory circuit functions to keep Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 primed to respond rapidly in the event that cells encounter genotoxic stress.