Background Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of ‘hypervirulent’ strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, R20291, two further CTns were described.ResultsCTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.ConclusionsThe genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host.

Our data provide important insights into the regulation of pro-inflammatory cytokine expression in human cardiac fibroblasts and suggest that the myocardial anti-inflammatory effects of statins and TZDs are not due to inhibition of TNFalpha-induced IL-1 or IL-6 expression by cardiac fibroblasts.

A model of the angiotensin AT 1 receptor and site-directed mutagenesis were used to identify key residues involved in ligand binding. We propose that substitution of these residues causes the loss of an interaction between transmembrane helices III and VII, which allows the AT 1 receptor to "relax" into its active conformation.The renin-angiotensin system plays a vital role in the regulation of cardiovascular function, and its activity may be abnormal in a number of disease states. The effector molecule of this system, the octapeptide angiotensin II (Ang II), 1 has a number of actions in a variety of cell types (1). Two distinct Ang II receptor subtypes, AT 1 and AT 2 have been identified (2). This characterization is based on the differential selectivity for these two receptors of the non-peptide Ang II receptor antagonists, DuP 753 (losartan; AT 1 selective) and PD123177 (AT 2 selective). In addition, the modified peptides CGP42112A (3) and [p-amino-Phe 6 ]angiotensin II (pNH 2 F 6 AII) (4), both display selectivity for the AT 2 receptor over the AT 1 receptor. Furthermore, several studies suggest that AT 1 and AT 2 receptors display subtle differences in their binding affinities for Ang II and angiotensin III (Ang III). AT 2 receptors bind Ang II and Ang III with equal affinity (5-8), while AT 1 receptors exhibit a slightly higher affinity for Ang II, between 3-and 20-fold greater than Ang III (9 -11). Many of the recognized actions of Ang II appear to be mediated through AT 1 receptors, predominantly through activation of the phospholipase C signal transduction pathway.AT 1 receptors have been cloned from a variety of species and tissues (9 -15). All these AT 1 receptors consist of a single polypeptide, 359 amino acids in length, arranged with a topography comprising seven ␣-helical transmembrane regions (TM I-VII), typical of the G-protein-coupled receptor family. They display a high degree of sequence identity at the amino acid level (over 94% identical between all mammalian species). Two closely related isoforms (AT 1A and AT 1B ) encoded by different genes have been identified in the rat and mouse (13, 16) Recently AT 2 receptors have been cloned (17-19), which are also members of the G-protein-coupled receptor family. They consist of 363 amino acids and share only 34% sequence identity at the amino acid level to the AT 1 receptors.Mutagenesis studies of the AT 1 receptor have recently identified residues important in the binding of the non-peptide antagonist, losartan, and the natural ligand Ang II. These residues appear to be mutually exclusive, since amino acid substitutions, which affect losartan binding, do not alter Ang II binding, and vice versa. The major determinants of losartan binding appear to be residues located within the transmembrane helices . In contrast, the major determinants of Ang II binding appear to be residues present in the extracellular regions, particularly in the N-terminal extension adjacent to the first transmembrane helix and in the C-terminal part of the third extracellula...

Cardiac myofibroblasts (CMF) play a key role in infarct repair and scar formation following myocardial infarction (MI) and are also an important source of proinflammatory cytokines. We postulated that interleukin-1alpha (IL-1alpha), a potential early trigger of acute inflammation post-MI, could stimulate human CMF to express additional proinflammatory cytokines. Furthermore, we hypothesized that these effects may be modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). Human CMF were cultured from atrial biopsies from multiple patients. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and cardiotrophin-1 (CT-1) mRNA expression and secretion were measured using quantitative real-time RT-PCR and enzyme-linked immunosorbent assay. IL-1alpha (0.001-10 ng/ml, 0-6 h) stimulated IL-1beta, TNF-alpha, and IL-6 mRNA expression with distinct temporal and concentration profiles, resulting in increased cytokine secretion. The response to IL-1alpha was much greater than with TNF-alpha. Neither IL-1alpha nor TNF-alpha treatment modulated CT-1 mRNA expression. Immunoblotting with phosphospecific antibodies revealed that IL-1alpha stimulated the extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and nuclear factor (NF)-kappaB signaling pathways. Pharmacological inhibitor studies indicated roles for PI 3-kinase/Akt and NF-kappaB pathways in mediating IL-1beta expression, and for NF-kappaB and p38 MAPK pathways in mediating TNF-alpha expression. IL-1alpha-induced IL-6 mRNA expression was reduced by p38 MAPK inhibition, but increased by ERK and JNK pathway inhibitors. IL-10 produced a consistent but modest reduction in IL-1alpha-induced IL-6 mRNA levels (not IL-1beta or TNF-alpha), but this was not reflected by reduced IL-6 protein secretion. In conclusion, IL-1alpha stimulates human CMF to express IL-1beta, TNF-alpha, and IL-6 via specific signaling pathways, responses that are unaffected by IL-10 exposure.

The proinflammatory cytokine interleukin-1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac myofibroblasts (CMF), or the role of p38 mitogen-activated protein kinase (MAPK). We used a focused RT-PCR microarray to investigate the effects of IL-1α on expression of 41 ECM genes in CMF cultured from different patients, and explored regulation by p38 MAPK.IL-1α (10 ng/ml, 6 h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin. However, it induced marked increases in expression of specific ECM proteases, including matrix metalloproteinases MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B) and MMP-10 (stromelysin-2). Conversely, IL-1α reduced mRNA and protein expression of ADAMTS1, a metalloproteinase that suppresses neovascularization. IL-1α increased expression of TIMP-1 slightly, but not TIMP-2. Data for MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and ADAMTS1 were confirmed by quantitative real-time RT-PCR. Tumor necrosis factor-alpha (TNFα), another important myocardial proinflammatory cytokine, did not alter expression of these metalloproteinases. IL-1α strongly activated the p38 MAPK pathway in human CMF. Pharmacological inhibitors of p38-α/β (SB203580) or p38-α/β/γ/δ (BIRB-0796) reduced MMP-3 and ADAMTS1 mRNA expression, but neither inhibitor affected MMP-9 levels. MMP-1 and MMP-10 expression were inhibited by BIRB-0796 but not SB203580, suggesting roles for p38-γ/δ.In summary, IL-1α induces a distinct pattern of ECM protein and protease expression in human CMF, in part regulated by distinct p38 MAPK subtypes, affirming the key role of IL-1α and CMF in post-infarction cardiac remodeling.

Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFβ) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.

The aim of this study was to screen for the presence of antimicrobial resistance genes within the saliva and faecal microbiomes of healthy adult human volunteers from five European countries. Two non-culture based approaches were employed to obviate potential bias associated with difficult to culture members of the microbiota. In a gene target-based approach, a microarray was employed to screen for the presence of over 70 clinically important resistance genes in the saliva and faecal microbiomes. A total of 14 different resistance genes were detected encoding resistances to six antibiotic classes (aminoglycosides, β-lactams, macrolides, sulphonamides, tetracyclines and trimethoprim). The most commonly detected genes were erm(B), bla TEM, and sul2. In a functional-based approach, DNA prepared from pooled saliva samples was cloned into Escherichia coli and screened for expression of resistance to ampicillin or sulphonamide, two of the most common resistances found by array. The functional ampicillin resistance screen recovered genes encoding components of a predicted AcrRAB efflux pump. In the functional sulphonamide resistance screen, folP genes were recovered encoding mutant dihydropteroate synthase, the target of sulphonamide action. The genes recovered from the functional screens were from the chromosomes of commensal species that are opportunistically pathogenic and capable of exchanging DNA with related pathogenic species. Genes identified by microarray were not recovered in the activity-based screen, indicating that these two methods can be complementary in facilitating the identification of a range of resistance mechanisms present within the human microbiome. It also provides further evidence of the diverse reservoir of resistance mechanisms present in bacterial populations in the human gut and saliva. In future the methods described in this study can be used to monitor changes in the resistome in response to antibiotic therapy.

Madi HA, Riches K, Warburton P, O'Regan DJ, Turner NA, Porter KE. Inherent differences in morphology, proliferation, and migration in saphenous vein smooth muscle cells cultured from nondiabetic and Type 2 diabetic patients. Am J Physiol Cell Physiol 297: C1307-C1317, 2009. First published September 9, 2009 doi:10.1152/ajpcell.00608.2008.-Individuals with Type 2 diabetes mellitus (T2DM) are at increased risk of saphenous vein (SV) graft stenosis following coronary artery bypass. Graft stenosis is caused by intimal hyperplasia, a pathology characterized by smooth muscle cell (SMC) proliferation and migration. We hypothesized that SV-SMC from T2DM patients were intrinsically more proliferative and migratory than those from nondiabetic individuals. SV-SMC were cultured from nondiabetic and T2DM patients. Cell morphology (light microscopy, immunocytochemistry), S100A4 expression (real-time RT-PCR, immunoblotting), proliferation (cell counting), migration (Boyden chamber assay), and cell signaling (immunoblotting with phosphorylation state-specific antibodies) were studied. SV-SMC from T2DM patients were morphologically distinct from nondiabetic patients and exhibited a predominantly rhomboid phenotype, accompanied by disrupted F-actin cytoskeleton, disorganized ␣-smooth muscle actin network, and increased focal adhesion formation. However, no differences were observed in expression of the calcium-binding protein S100A4, a marker of rhomboid SMC phenotype, between the two cell populations. T2DM cells were less proliferative in response to fetal calf serum than nondiabetic cells, but both populations had similar proliferative responses to insulin plus PDGF. Under high glucose concentration conditions in the presence of insulin, migration of diabetic SV-SMC was greater than nondiabetic cells. Glucose concentration did not affect SV-SMC proliferation. No differences in insulin or PDGF-induced phosphorylation of ERK-1/2 or components of the Akt pathway (Akt-Ser473, Akt-Thr308, and GSK-3␤) were apparent between the two populations. In conclusion, SV-SMC from T2DM patients differ from nondiabetic SV-SMC in that they exhibit a rhomboid phenotype and are more migratory, but less proliferative, in response to serum. diabetes mellitus; vein graft stenosis; metabolic memory DIABETES MELLITUS IS A SERIOUS and escalating health problem worldwide (3) that currently affects more than 2