Genotypic and phenotypic properties of coagulase-negative staphylococci causing dialysis catheter-related sepsis

Sixty coagulase-negative staphylococcus (CNS) isolates were recovered from the blood cultures or peritoneal dialysate effluent of 43 patients on renal dialysis. The patients had either renal dialysis catheter-related sepsis (CRS) or continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis. Isolates were characterized by biotyping, and genotyped by pulsed-field gel electrophoresis (PFGE). Phenotypic properties of the strains were also investigated. Several genotypes were identified with no one specific strain of CNS being associated with CRS. However, closely related strains were isolated from several patients within the units studied, suggesting horizontal transfer of micro-organisms. Genotypic macro-restriction profiles did not concur with phenotypic profiles or biotypes, confirming that genotyping is required for epidemiological studies. All staphylococcal strains were investigated for the production of phenotypic characteristics. Significant differences were predominantly seen in the production of lipase, esterase and elastase in strains isolated from the renal patients with CRS and CAPD-associated peritonitis, compared with a non-septic control group. These phenotypic characteristics may therefore have a role in the maintenance of CRS in renal patients. © 2003 The Hospital Infection Society. Published by Elsevier Science Ltd. All rights reserved.

Phenotypic and genotypic characterisation of Clostrum Difficile

Clostridium difficile is at present one of the most common nosocomial infections in the developed world. Hypervirulent strains (PCR ribotype 027) of C. difficile which produce enhanced levels of toxins have also been associated with other characteristics such as a greater rate of sporulation and resistance to fluoroquinolones. Infection due to C. difficile PCR ribotype 027 has also been associated with greater rates of morbidity and mortality. The aim of this thesis was to investigate both the phenotypic and genotypic characteristics of two populations of toxigenic clinical isolates of C. difficile which were recovered from two separate hospital trusts within the UK. Phenotypic characterisation of the isolates was undertaken using analytical profile indexes (APIs), minimum inhibitory concentrations(MICs) and S-layer protein typing. In addition to this, isolates were also investigated for the production of a range of extracellular enzymes as potential virulence factors. Genotypic characterisation was performed using a random amplification of polymorphic DNA(RAPD) PCR protocol which was fully optimised in this study, and the gold standard method, PCR ribotyping. The discriminatory power of both methods was compared and the similarity between the different isolates also analysed. Associations between the phenotypic and genotypic characteristics and the recovery location of the isolate were then investigated. Extracellular enzyme production and API testing revealed little variation between the isolates; with S-layer typing demonstrating low discrimination. Minimum inhibitory concentrations did not identify any resistance towards either vancomycin or metronidazole; there were however significant differences in the distribution of antibiogram profiles of isolates recovered from the two different trusts. The RAPD PCR protocol was successfully optimised and alongside PCR ribotyping, effectively typed all of the clinical isolates and also identified differences in the number of types defined between the two locations. Both PCR ribotyping and RAPD demonstrated similar discriminatory power; however, the two genotyping methods did not generate amplicons that mapped directly onto each other and therefore clearly characterised isolates based on different genomic markers. The RAPD protocol also identified different subtypes within PCR ribotypes, therefore demonstrating that all isolates defined as a particular PCR ribotype were not the same strain. No associations could be demonstrated between the phenotypic and genotypic characteristics observed; however, the location from which an isolate was recovered did appear to influence antibiotic resistance and genotypic characteristics. The phenotypic and genotypic characteristics observed amongst the C. difficile isolates in this study, may provide a basis for the identification of further targets which may be potentially incorporated into future methods for the characterisation of C. difficile isolates.

Transglutaminase 2 is involved in autophagosome maturation

Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g. nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. "Tissue" transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Phenotypic and genotypic analysis of intestinal spirochacies

Pulsed field gel electrophoresis of 82 intestinal spirochaete isolates showed specific differentiation of Serpulina pilosicoli and Serpulina hyodysenteriae although considerable heterogeneity was observed, especially amongst S. pilosicoli isolates. In several cases genotypically similar isolates originated from different animals suggesting that cross-species transmission may have occurred. The Caco-2 and Caco-21HT29 cell models have been proposed as potentially realistic models of intestinal infection. Quantitation of adhesion to the cells showed isolate 3 82/91 (from a bacteraemia) to adhere at significantly greater numbers than any other isolate tested. This isolate produced a PFGE profile which differed from other S. pilosicoli isolates and so would be of interest for further study. Comparison of bacteraemic and other S. pilosicoli isolates suggested that bacteraemic isolates were not more specifically adapted for adhesion to, or invasion of the epithelial cell layer than other S. pilosicoli isolates. Genotypically similar isolates from differing animal origins adhered to the Caco-2 model at similar levels. Generation of a random genomic library of S. pilosicoli and screening with species specific monoclonal antibody has enabled the identification of a gene sequence encoding a protein which showed significant homology with an ancestral form of the enzyme pyruvate oxidoreductase. Immunoscreening with polyclonal serum identified the sequences of two gene clusters and a probable arylsulphatase. One gene cluster represented a ribosomal gene cluster which has a similar molecular arrangement to Borrelia burgdorjeri, Treponema pallidum and Thermatoga maritima. The other gene cluster contained an ABC transporter protein, sorbitol dehydrogenase and phosphomannose isomerase. An ELISA type assay was used to demonstrate that isolates of S. pilosicoli could adhere to components of the extracellular matrix such as collagen (type 1), fibronectin, laminin, and porcine gastric mucin.

Meta-analysis of the population and phenotypic expression of CYP2C9/2C19 polymorphism on drug metabolism in different ethnicities

The term "pharmacogenetics" has been defined as the scientific study of inherited factors that affect the human drug response. Many pharmacogenetie studies have been published since 1995 and have focussed on the principal enzyme family involved in drug metabolism, the cytochrome P450 family, particularly cytochrome P4502C9 and 2C19. In order to investigate the pharmacogenetic aspect of pharmacotherapy, the relevant studies describing the association of pharmacogenetic factor(s) in drug responses must be retrieved from existing literature using a systematic review approach. In addition, the estimation of variant allele prevalence for the gene under study between different ethnic populations is important for pharmacogenetic studies. In this thesis, the prevalence of CYP2C9/2C19 alleles between different ethnicities has been estimated through meta-analysis and the population genetic principle. The clinical outcome of CYP2C9/2C19 allelic variation on the pharmacotherapy of epilepsy has been investigated; although many new antiepileptic drugs have been launched into the market, carbamazepine, phenobarbital and phenytoin are still the major agents in the pharmacotherapy of epilepsy. Therefore, phenytoin was chosen as a model AED and the effect of CYP2C9/2C19 genetic polymorphism on phenytoin metabolism was further examined.An estimation of the allele prevalence was undertaken for three CYP2C9/2C19 alleles respectively using a meta-analysis of studies that fit the Hardy-Weinberg equilibrium. The prevalence of CYP2C9*1 is approximately 81%, 96%, 97% and 94% in Caucasian, Chinese, Japanese, African populations respectively; the pooled prevalence of CYP2C19*1 is about 86%, 57%, 58% and 85% in these ethnic populations respectively. However, the studies of association between CYP2C9/2C19 polymorphism and phenytoin metabolism failed to achieve any qualitative or quantitative conclusion. Therefore, mephenytoin metabolism was examined as a probe drug for association between CYP2C19 polymorphism and mephenytoin metabolic ratio. Similarly, analysis of association between CYP2C9 polymorphism and warfarin dose requirement was undertaken.It was confirmed that subjects carrying two mutated CYP2C19 alleles have higher S/R mephenytoin ratio due to deficient CYP2C19 enzyme activity. The studies of warfarin and CYP2C9 polymorphism did not provide a conclusive result due to poor comparability between studies.The genetic polymorphism of drug metabolism enzymes has been studied extensively, however other genetic factors, such as multiple drug resistance genes (MDR) and genes encoding ion channels, which may contribute to variability in function of drug transporters and targets, require more attention in future pharmacogenetic studies of antiepileptic drugs.

Circannual rhythms of maturation in the female rainbow trout and their entrainment by changes in photoperiod

The importance of endogenous rhythms in the photoperiodic control of the annual reproduction cycle in female rainbow trout was investigated. The effect of photoperiod regimes on the different stages of maturation was assessed by recording the timing of ovulation and from quantifying associated changes in serum oestradiol-17,testosterone and total calcium. Maintained under constant 6L:18D and constant temperature for up to four years, rainbow trout exhibited an endogenous rhythm of maturation with a periodicity of approximately one year. This rhythm of maturation appears to be driven by an autonomous circannual oscillator or clock which can be dissociated from the neuroendocrine mechanisms controlling gonadal maturation. Under conditions of constant 18L:6D or LL the periodicity of the maturation rhythm was 5.5-6 months; it is suggested that this periodicity may be caused by a splitting or uncoupling of at least two circannual clocks involved in the control of maturation. Abrupt changes in the length of the photoperiod act as a zeitgeber to entrain the endogenous rhythm of maturation. Whether the timing of maturation is advanced or delayed depends primarily on the direction of the change in photoperiod and its timing in relation to the phase of the rhythm, with the magnitude of the alteration in photoperiod having only a supplementary effect. The effect of specific changes in photoperiod on the entrainment of the maturation cycle can be described in terms of a phase-response curve. Photic information is transduced, probably by the pineal gland, into a daily rhythm of melatonin; exposure of rainbow trout to skeleton and resonance photoperiod regimes indicated that daylength measurement is effected by endogenous circadian clock(s) rather than by hour-glass mechanisms. A gating mechanism is closely associated with the circannual clock which determines the timing of onset of maturation in virgin female rainbow trout, only allowing fish that have attained a threshold stage of development to undergo gonadal maturation. Collectively the results support the hypothesis that the female rainbow trout exhibits an endogenous circannual rhythm of maturation which can be entrained by changes in photoperiod.

Effects of lithium and valproic acid on gene expression and phenotypic markers in an NT2 neurosphere model of neural development

Mood stabilising drugs such as lithium (LiCl) and valproic acid (VPA) are the first line agents for treating conditions such as Bipolar disorder and Epilepsy. However, these drugs have potential developmental effects that are not fully understood. This study explores the use of a simple human neurosphere-based in vitro model to characterise the pharmacological and toxicological effects of LiCl and VPA using gene expression changes linked to phenotypic alterations in cells. Treatment with VPA and LiCl resulted in the differential expression of 331 and 164 genes respectively. In the subset of VPA targeted genes, 114 were downregulated whilst 217 genes were upregulated. In the subset of LiCl targeted genes, 73 were downregulated and 91 were upregulated. Gene ontology (GO) term enrichment analysis was used to highlight the most relevant GO terms associated with a given gene list following toxin exposure. In addition, in order to phenotypically anchor the gene expression data, changes in the heterogeneity of cell subtype populations and cell cycle phase were monitored using flow cytometry. Whilst LiCl exposure did not significantly alter the proportion of cells expressing markers for stem cells/undifferentiated cells (Oct4, SSEA4), neurons (Neurofilament M), astrocytes (GFAP) or cell cycle phase, the drug caused a 1.4-fold increase in total cell number. In contrast, exposure to VPA resulted in significant upregulation of Oct4, SSEA, Neurofilament M and GFAP with significant decreases in both G2/M phase cells and cell number. This neurosphere model might provide the basis of a human-based cellular approach for the regulatory exploration of developmental impact of potential toxic chemicals.

Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis

Objective-We previously demonstrated that upregulation of intermediate-conductance Ca2+ -activated K+ channels (KCa 3.1) is necessary for mitogen-induced phenotypic modulation in isolated porcine coronary smooth muscle cells (SMCs). The objective of the present study was to determine the role of KCa3.1 in the regulation of coronary SMC phenotypic modulation in vivo using a swine model of postangioplasty restenosis. Methods and Results-Balloon angioplasty was performed on coronary arteries of swine using either noncoated or balloons coated with the specific KCa3.1 blocker TRAM-34. Expression of KCa3.1, c-jun, c-fos, repressor element-1 silencing transcription factor (REST), smooth muscle myosin heavy chain (SMMHC), and myocardin was measured using qRT-PCR in isolated medial cells 2 hours and 2 days postangioplasty. KCa3.1, c-jun, and c-fos mRNA levels were increased 2 hours postangioplasty, whereas REST expression decreased. SMMHC expression was unchanged at 2 hours, but decreased 2 days postangioplasty. Use of TRAM-34 coated balloons prevented KCa3.1 upregulation and REST downregulation at 2 hours, SMMHC and myocardin downregulation at 2 days, and attenuated subsequent restenosis 14 and 28 days postangioplasty. Immunohistochemical analysis demonstrated corresponding changes at the protein level. Conclusion-Blockade of KCa3.1 by delivery of TRAM-34 via balloon catheter prevented smooth muscle phenotypic modulation and limited subsequent restenosis. © 2008 American Heart Association, Inc.

Phenotypic effect of mutations in evolving populations of RNA molecules

Background. The secondary structure of folded RNA sequences is a good model to map phenotype onto genotype, as represented by the RNA sequence. Computational studies of the evolution of ensembles of RNA molecules towards target secondary structures yield valuable clues to the mechanisms behind adaptation of complex populations. The relationship between the space of sequences and structures, the organization of RNA ensembles at mutation-selection equilibrium, the time of adaptation as a function of the population parameters, the presence of collective effects in quasispecies, or the optimal mutation rates to promote adaptation all are issues that can be explored within this framework. Results. We investigate the effect of microscopic mutations on the phenotype of RNA molecules during their in silico evolution and adaptation. We calculate the distribution of the effects of mutations on fitness, the relative fractions of beneficial and deleterious mutations and the corresponding selection coefficients for populations evolving under different mutation rates. Three different situations are explored: the mutation-selection equilibrium (optimized population) in three different fitness landscapes, the dynamics during adaptation towards a goal structure (adapting population), and the behavior under periodic population bottlenecks (perturbed population). Conclusions. The ratio between the number of beneficial and deleterious mutations experienced by a population of RNA sequences increases with the value of the mutation rate µ at which evolution proceeds. In contrast, the selective value of mutations remains almost constant, independent of µ, indicating that adaptation occurs through an increase in the amount of beneficial mutations, with little variations in the average effect they have on fitness. Statistical analyses of the distribution of fitness effects reveal that small effects, either beneficial or deleterious, are well described by a Pareto distribution. These results are robust under changes in the fitness landscape, remarkably when, in addition to selecting a target secondary structure, specific subsequences or low-energy folds are required. A population perturbed by bottlenecks behaves similarly to an adapting population, struggling to return to the optimized state. Whether it can survive in the long run or whether it goes extinct depends critically on the length of the time interval between bottlenecks. © 2010 Stich et al; licensee BioMed Central Ltd.

GP130/OSMR is the only LIF/IL-6 family receptor complex to promote osteoblast differentiation of calvaria progenitors

Leukemia inhibitory factor (LIF) and its receptor (LIFR) are "twins" of Oncostatin M (OSM) and OSMR, respectively, likely having arisen through gene duplications. We compared their effects in a bone nodule-forming model of in vitro osteogenesis, rat calvaria (RC) cell cultures. Using a dominant-negative LIF mutant (hLIF-05), we showed that in RC cell cultures mouse OSM (mOSM) activates exclusively glycoprotein 130 (gp130)/OSMR. In treatments starting at early nodule formation stage, LIF, mOSM, IL-11, and IL-6 + sIL-6R inhibit bone nodule formation, that is, osteoprogenitor differentiation. Treatment with mOSM, and no other cytokine of the family, in early cultures (day 1-3 or 1-4) increases bone colony numbers. hLIF-05 also dose dependently stimulates bone nodule formation, confirming the inhibitory action of gp130/LIFR on osteogenesis. In pulse treatments at successive stages of bone nodule formation and maturation, LIF blocks osteocalcin (OCN) expression by differentiated osteoblasts, but has no effect on bonesialoprotein (BSP) expression. Mouse OSM inhibits OCN and BSP expression in preconfluent cultures with no or progressively reduced effects at later stages, reflecting the disruption of early nodules, possibly due to the strong apoptotic action of mOSM in RC cell cultures. In summary, LIFR and OSMR display differential effects on differentiation and phenotypic expression of osteogenic cells, most likely through different signal transduction pathways. In particular, gp130/OSMR is the only receptor complex of the family to stimulate osteoprogenitor differentiation in the RC cell culture model. © 2005 Wiley-Liss, Inc.

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease-related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF-1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety-related behaviour in open field activities in male and female offspring (P <0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P <0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P <0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P <0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P <0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post-fertilization preimplantation development which may reflect the relative contribution of the paternal genome. © Journal compilation © 2008 The Physiological Society.