Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. ...marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.
Observations of neutral-current nu interactions on deuterium in the Sudbury Neutrino Observatory are reported. Using the neutral current (NC), elastic scattering, and charged current reactions and ...assuming the standard 8B shape, the nu(e) component of the 8B solar flux is phis(e) = 1.76(+0.05)(-0.05)(stat)(+0.09)(-0.09)(syst) x 10(6) cm(-2) s(-1) for a kinetic energy threshold of 5 MeV. The non-nu(e) component is phi(mu)(tau) = 3.41(+0.45)(-0.45)(stat)(+0.48)(-0.45)(syst) x 10(6) cm(-2) s(-1), 5.3sigma greater than zero, providing strong evidence for solar nu(e) flavor transformation. The total flux measured with the NC reaction is phi(NC) = 5.09(+0.44)(-0.43)(stat)(+0.46)(-0.43)(syst) x 10(6) cm(-2) s(-1), consistent with solar models.
Normothermic ex vivo liver perfusion (NEVLP) offers the potential to optimize graft function prior to liver transplantation (LT). Hepatitis C virus (HCV) is dependent on the presence of ...miRNA(microRNA)‐122. Miravirsen, a locked‐nucleic acid oligonucleotide, sequesters miR‐122 and inhibits HCV replication. The aim of this study was to assess the efficacy of delivering miravirsen during NEVLP to inhibit miR‐122 function in a pig LT model. Pig livers were treated with miravirsen during NEVLP or cold storage (CS). Miravirsen absorption, miR‐122 sequestration, and miR‐122 target gene derepression were determined before and after LT. The effect of miravirsen treatment on HCV infection of hepatoma cells was also assessed. NEVLP improved miravirsen uptake versus CS. Significant miR‐122 sequestration and miR‐122 target gene derepression were seen with NEVLP but not with CS. In vitro data confirmed miravirsen suppression of HCV replication after established infection and prevented HCV infection with pretreatment of cells, analogous to the pretreatment of grafts in the transplant setting. In conclusion, miravirsen delivery during NEVLP is a potential strategy to prevent HCV reinfection after LT. This is the first large‐animal study to provide “proof of concept” for using NEVLP to modify and optimize liver grafts for transplantation.
Miravirsen delivery during normothermic ex vivo liver perfusion is a potential strategy to prevent hepatitis C reinfection after liver transplantation and, most importantly, provides a proof‐of‐concept for ex vivo liver graft modification.
Friction stir spot welding (FSSW) of Al alloy 6016-T4 sheet was evaluated using a conventional pin (CP) tool and off-center feature (OC) tool. Tool rotation speed and plunge depth were varied to ...determine the effect of individual process parameter on lap-shear separation load. Maximum separation load of about 3.3kN was obtained by using a 0.2mm shoulder penetration depth with 1500rpm tool rotation speed for the CP tool and 2500rpm for the OC tool. Three different weld separation modes under lap-shear loading were observed: interfacial separation, nugget fracture separation and upper sheet fracture separation. Microhardness profile for weld cross section indicated no direct relationship between microhardness distribution and separation locations.
The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse ...protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K+ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K+ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.
Synopsis
Systems biology has the potential to explain physiological processes as emergent properties of sets of genes and proteins. Beyond simple cellular systems, the challenge of delivering systems biology into the intact and freely behaving animal will require new methods. Currently, the most widely used approach is immunoprecipitation of the target protein and its associate binding proteins. This method suffers from the drawbacks of single step purification strategies that include a high level of non‐specific background proteins amongst other limitations. To overcome these limitations we demonstrate that the tandem affinity purification (TAP) technology originally developed in yeast (Rigaut et al, 1999), when combined with gene targeting, can be used to efficiently isolate highly specific complexes from mouse. The ‘targeted TAP tagging’ strategy combines the two major advantages of each system. The first advantage is that the insertion of two tags into the protein of interest allows two consecutive purification steps that facilitate the recovery of protein complexes with high confidence and decreases the recovery of non‐specific proteins or weak interactors. The second advantage, conferred by targeting the endogenous gene, is that the tagged protein is expressed under its natural regulatory mechanisms. We have designed an endogenous TAP targeting strategy to isolate complexes from mouse brain excitatory synapses. The brain is the most complex organ from a cellular and molecular perspective and thus an ideal model to explore the TAP method. Post Synaptic Density 95 (PSD‐95/Dlg4) is an adaptor protein comprised of PDZ, SH3 and GK domains and is expressed in the postsynaptic terminal of excitatory synapses where it organizes signaling from neurotransmitter receptors to downstream pathways (Kornau et al, 1995; Hunt et al, 1996; Tu et al, 1999; Husi et al, 2000; Nehring et al, 2000; Dosemeci et al, 2007; Carlisle et al, 2008). Mice carrying a knockout mutation in PSD‐95 show it is essential for synaptic plasticity and a range of important behaviours (Migaud et al, 1998; El‐Husseini et al, 2000; Beique et al, 2006). Here, a new TAP tag was fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function (Figure 2). As a result of four independent tandem purifications and mass spectrometry analysis, we were able to define PSD‐95 core complexes with high sensitivity and reproducibility. The four purifications show an average of 125±19 proteins, having 118 proteins (94%) common in at least three of four replicates. This reproducibility rate is among the highest rate reported for systematic protein complex isolation. To further validate this interaction data we compared it to information from public datasets. Of the 118 proteins, 22% were proteins that directly bind PSD‐95 and 18% were proteins not previously found in other PSD‐95 analysis. All together, these data show robust reproducibility and sensitivity of this method for purifying synaptic complexes. These PSD‐95 core complexes comprise key functional components of synapses including the glutamate neurotransmitter receptors, K+ channels, scaffolding and signaling proteins. These complexes contain ionotropic glutamate receptors of the NMDA, AMPA and kainate subtypes as well as major K+ channels that together are the major postsynaptic constituents responsible for synaptic transmission and shaping the postsynaptic electrophysiological response to presynaptic input (Watanabe et al, 2002; Chen et al, 2006; Kim et al, 2007). We believe that this is the first method that has allowed the robust copurification of these proteins. To explore functional organization using network models, we manually curated interactions (Pocklington et al, 2006) and the UniHi database (http://www.mdc‐berlin.de/unihi) to identify 119 interactions between 50 proteins (excluding self‐interactions) of the PSD‐95 core complexes. Network clustering of the interacting proteins showed 40 out of the 50 proteins formed a large connected component (major connected component, MCC) and a modular structure that was segregated into 5 clusters referred to as cluster a (Cla) to cluster e (Cle) (Figure 5A). In addition to the 5 MCC clusters, 2 further disconnected clusters (‘Clf’ and ‘Clg’) were found. Of great interest is the location and proximity of the receptors and channels responsible for the postsynaptic depolarization and subsequent action potential generation. All NMDA, AMPA and kainate glutamate receptors were restricted to Cla and Clb and the voltage‐dependent K+ channels were found in Cla and Clc (entirely comprised of K+ channels). It therefore appears that Cla, Clb and Clc are enriched with membrane proteins responsible for electrical properties of the postsynaptic terminal. The central role of PSD‐95 was supported by calculation of the shortest path from each protein to every other protein and PSD‐95 showed the lowest. Annotation of clusters with human disease associations revealed that multiple disorders map onto the network with a highly significant correlation of schizophrenia within the glutamate receptor clusters (P<10−6). 20 genes involved in schizophrenia were significantly associated with the clusters Cla and Clb that contains all the glutamate receptors and MAGUK/Dlg proteins (Figure 5B). Mapping the primary interactors of these schizophrenia proteins recruited many other proteins found in the other modules of the network. This suggests that the overall network and its different modules are a substrate for schizophrenia, and not simply the glutamate receptors, as was generally considered in the ‘glutamate hypothesis’ of schizophrenia (Greene, 2001; Coyle, 2006; Lisman et al, 2008). This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease. TAP tagged mice are a valuable resource and useful for a wide range of physiological studies and whole animal studies.
A novel approach for isolating native protein complexes from mouse tissues using gene targeting of tandem affinity tags is presented.
A protein core complex from brain synapses comprising principal electrophysiological and signalling components for synaptic transmission and synaptic plasticity was isolated.
The protein interaction network shows clusters of functionally distinct proteins and schizophrenia susceptibility genes.
This targeted TAP tagging method has general application to all types of protein complexes in the mouse and will be particularly useful for analysing molecular networks and systems biology in the intact animal.
Lay Summary
We assessed the impact of a dedicated inflammatory bowel disease flare clinic. Attenders had more changes to maintenance medications made and less steroid use than inflammatory bowel ...disease patients reviewed at the Acute Receiving Unit. This model of care may potentially reduce hospital admissions.
Outcomes of living versus deceased donor liver transplantation in patients with chronic liver disease and hepatorenal syndrome (HRS) was compared using a matched pair study design. Thirty patients ...with HRS receiving a live donor liver transplantation (LDLT) and 90 HRS patients receiving a full graft deceased donor liver transplantation (DDLT) were compared. LDLT versus DDLT of patients with HRS was associated with decreased peak aspartate aminotransferase levels (339 ± 214 vs. 935 ± 1253 U/L; p = 0.0001), and similar 7‐day bilirubin (8.42 ± 7.89 vs. 6.95 ± 7.13 mg/dL; p = 0.35), and international normalized ratio levels (1.93 ± 0.62 vs. 1.78 ± 0.78; p = 0.314). LDLT vs. DDLT had a decreased intensive care unit (2 1–39 vs. 4 0–93 days; p = 0.004), and hospital stay (17 4–313 vs. 26 0–126 days; p = 0.016) and a similar incidence of overall postoperative complications (20% vs. 27%; p = 0.62). No difference was detected between LDLT and DDLT patients regarding graft survival at 1 (80% vs. 82%), at 3 (69% vs. 76%) and 5 years (65% vs. 76%) (p = 0.63), as well as patient survival at 1 (83% vs. 82%), 3 (72% vs. 77%) and 5 years (72% vs. 77%) (p = 0.93). The incidence of chronic kidney disease post‐LT (10% vs. 6%; p = 0.4) was similar between both groups. LDLT results in identical long‐term outcome when compared with DDLT in patients with HRS.
Using a matched case‐control study, the authors find that live donor liver transplantation when compared to deceased donation provides similar outcome in patients suffering from hepatorenal syndrome, and they discuss live donation as a strategy to provide immediate access to transplantation for this patient population.
The tumor microenvironment is the cellular and molecular environment in which the tumor exists and with which it continuously interacts. In B-cell lymphomas, this microenvironment is intriguing in ...that it plays critical roles in the regulation of tumor cell survival and proliferation, fostering immune escape as well as the development of treatment resistance. The purpose of this review is to summarize the proceedings of the Second Annual Summit on the Immune Microenvironment in Hematologic Malignancies that took place on September 11-12, 2014 in Dublin, Ireland. We provide a timely overview of the composition and biological relevance of the cellular and molecular microenvironment interface and discuss the role of interactions between the microenvironment and neoplastic cells in a variety of B-cell lymphomas. In addition, we focus on various novel therapeutic strategies that target the tumor microenvironment, including agents that modulate B-cell receptor pathways and immune-checkpoints, chimeric antigen receptor T cells and immunomodulatory agents.
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