Amplifying PCR productivity and environmental sustainability through shortened cycling protocols.

Since its inception in the 1980s, advancements in PCR technology using improved thermal cyclers, engineered DNA polymerases and commercial master mixes, have led to increased PCR productivity. Despite these advancements, PCR cycling protocols have largely remained unchanged over the same period. This study aimed to systemically evaluate the effect of reduced PCR cycling parameters on amplicon production.

Whole genomes of deep-sea sponge-associated bacteria exhibit high novel natural product potential.

Global antimicrobial resistance is a health crisis that can change the face of modern medicine. Exploring diverse natural habitats for bacterially-derived novel antimicrobial compounds has historically been a successful strategy. The deep-sea presents an exciting opportunity for the cultivation of taxonomically novel organisms and exploring potentially chemically novel spaces.

OXA-66 structure and oligomerisation of OXA enzymes.

The OXA β-lactamases are responsible for hydrolysing β-lactam antibiotics and contribute to the multidrug-resistant phenotype of several major human pathogens. The OXA enzymes are intrinsic to and can confer resistance to carbapenem antibiotics. Here we determined the structure of the most prevalent OXA enzyme, OXA-66.

Impact of growth media and pressure on the diversity and antimicrobial activity of isolates from two species of hexactinellid sponge.

Access to deep-sea sponges brings with it the potential to discover novel antimicrobial candidates, as well as novel cold- and pressure-adapted bacteria with further potential clinical or industrial applications. In this study, we implemented a combination of different growth media, increased pressure and high-throughput techniques to optimize recovery of isolates from two deep-sea hexactinellid sponges, and sp., in the first culture-based microbial analysis of these two sponges.

Variability in carbapenemase activity of intrinsic OxaAb (OXA-51-like) β-lactamase enzymes in Acinetobacter baumannii.

Objectives: To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC-MS/MS.

Methods: Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A.

Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S).

Identification of an antibacterial protein by functional screening of a human oral metagenomic library.

Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.

Aberrant phenotype in human endothelial cells of diabetic origin: implications for saphenous vein graft failure?

Type 2 diabetes (T2DM) confers increased risk of endothelial dysfunction, coronary heart disease, and vulnerability to vein graft failure after bypass grafting, despite glycaemic control. This study explored the concept that endothelial cells (EC) cultured from T2DM and nondiabetic (ND) patients are phenotypically and functionally distinct. Cultured human saphenous vein- (SV-) EC were compared between T2DM and ND patients in parallel.

Elevated expression levels of miR-143/5 in saphenous vein smooth muscle cells from patients with Type 2 diabetes drive persistent changes in phenotype and function.

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.

Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: possible role of differential RhoA activity.

Background/purpose: Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM.

Application of microarray and functional-based screening methods for the detection of antimicrobial resistance genes in the microbiomes of healthy humans.

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.

Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVβ3 and RhoA/ROCK-mediated mechanisms.

Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms.

TetAB46, a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity.

Objectives: To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient.

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition.

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals.

Cellular consequences of the expression of Alzheimer's disease-causing presenilin 1 mutations in human neuroblastoma (SH-SY5Y) cells.

Mutations in the presenilin 1 (PS1) gene lead to early-onset Alzheimer's disease with the S170F mutation causing the earliest reported age of onset. Expression of this, and other PS1 mutations, in SH-SY5Y cells resulted in significant loss of cellular viability compared to control cells. Basal Ca2+ concentrations in PS1 mutants were never lower than controls and prolonged incubation in Ca2+ -free solutions did not deplete Ca2+ stores, demonstrating there was no difference in Ca2+ leak from endoplasmic reticulum (ER) stores in PS1 mutants.

Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile.

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.

Isolation of bacterial extrachromosomal DNA from human dental plaque associated with periodontal disease, using transposon-aided capture (TRACA).

The human oral cavity is host to a complex microbial community estimated to comprise >700 bacterial species, of which at least half are thought to be not yet cultivable in vitro. To investigate the plasmids present in this community, we used a transposon-aided capture system, which allowed the isolation of plasmids from human oral supra- and subgingival plaque samples. Thirty-two novel plasmids and a circular molecule that could be an integrase-generated circular intermediate were isolated.

Modulatory effect of interleukin-1α on expression of structural matrix proteins, MMPs and TIMPs in human cardiac myofibroblasts: role of p38 MAP kinase.

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, 6h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin.

Porphyrin-nanosensor conjugates. New tools for the measurement of intracellular response to reactive oxygen species.

Reactive oxygen species (ROS) have for some time been implicated in the onset and progression of medical conditions including cancer, ageing, heart disease and Alzheimer's disease. Recently, it has been postulated that ROS play a much more subtle role in intracellular signalling mechanisms as second messengers. Given the importance of these species in influencing cellular processes, it is surprising that tools for studying intracellular levels of ROS are extremely limited and devices for studying the cells' response to internally generated ROS are virtually non-existent.

Inherent differences in morphology, proliferation, and migration in saphenous vein smooth muscle cells cultured from nondiabetic and Type 2 diabetic patients.

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.

Peroxisome proliferator-activated receptor gamma-independent effects of thiazolidinediones on human cardiac myofibroblast function.

1. Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor (PPAR) gamma agonists that are used to lower insulin resistance in Type 2 diabetic patients. Although TZDs exhibit beneficial effects on the vasculature, their effects on the heart are less clear and are the subject of current clinical debate.

Interleukin-1alpha stimulates proinflammatory cytokine expression in human cardiac myofibroblasts.

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).

Characterization of tet(32) genes from the oral metagenome.

tet(32) Was identified in three bacterial isolates and in metagenomic DNA from the human oral cavity. The regions immediately flanking the gene were found to have similarities to the mobile elements TnB1230 from Butyrivibrio fibrisolvens, ATE-3 from Arcanobacterium pyogenes, and CTn5 from Clostridium difficile.