Effect of Energy Metabolism on NF-kB activity in Ovarian Cancer

NF-kB is a transcription factor involved in cancer cell growth and survival. The activation of NF-kB can be assessed by monitoring phosphorylation of RelA p65 at Ser-536, which is a surrogate of the NF-kB transcription factor activation. The objective of this study was to determine if the loss of ATP leads to NF-kB deficiency and thus, apoptotic cell death of “bad” cells in ovarian cancer cells. The independent variables were metformin (Met), an anti-diabetic medicine, another compound MinB functionally similar to Met and a glucose transporter inhibitor BAY-876. The dependent variables were the resulting effect of Met and MinB on phosphorylated AMPK at Thr-172 (marker of ATP loss) and RelA p65 at Ser-536 (marker of NF-kB activation). In each experiment, AMPK and RelA phosphorylation were tested by treatment of ovarian cancer cell lines with Met, MinB, BAY-876, Met+BAY-876, MinB+BAY-876. Western blotting was performed to determine the phosphorylation levels of AMPK and RelA p65. For two gels, the process was repeated. In each gel, Met or MinB treatment leads to thicker bands of AMPK-p, indicating decrease in cellular ATP levels following treatments. The effect of Met, MinB, or BAY-876 on RelA p65 was limited. However, co-treatment of Met or MinB with BAY-876 caused strong inhibition of NF-kB, as reflected by reduction in RelA p65-p. These results suggested that ATP deficiency together with inhibition of glucose transport cause inactivation of NK-kB. Future research will be conducted to study the effects of these compounds or their combinations on ovarian cancer cell growth and survival against from apoptosis.https://scholarscompass.vcu.edu/uresposters/1267/thumbnail.jp

The Role of Actin Depolymerizing Factor in Regulating Actin Dynamics in Toxoplasma gondii

Apicomplexan parasites utilize a unique process of rapid motility termed gliding, which is coupled to their invasion of host cells. Gliding and invasion are dependent on parasite actin filaments, yet parasite actin is mostly non-filamentous. Filaments have been detected only transiently during gliding, suggesting that parasite actin filaments are rapidly assembled and disassembled during gliding motility. Little is known about what regulates the turnover of parasite actin filaments. In higher eukaryotes the Actin Depolymerizing Factor: ADF)/Cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin binding proteins conserved in apicomplexan parasites. To investigate the role of ADF in regulating actin dynamics in apicomplexan parasites, Toxoplasma gondii was used as a model apicomplexan, and the activities of T. gondii ADF: TgADF) were analyzed in vitro and in vivo. We found that TgADF engaged in dual activities. In contrast to most ADF/Cofilin proteins, TgADF was found to be a potent actin monomer sequestering protein that strongly inhibited actin polymerization, suggesting that it likely functions to maintain high G-actin concentrations in the cytoplasm of non-motile parasites. This role was reflected in its molecular structure, in which conserved G-actin binding sites were maintained, while key F-actin binding residues were absent. Despite this, TgADF demonstrated the ability to promote actin filament disassembly via a severing mechanism. Using a conditional knockout system we examined the function of TgADF in the parasite. TgADF was found to be essential for controlling productive gliding motility, and its absence lead to defects in host cell invasion, parasite egress, and parasite dispersal. Detailed analysis of motility revealed that parasites were unable to engage in sustained helical gliding, and moved at markedly reduced speeds. These defects are predicted to arise from the presence of more stable actin filaments in the parasite. Overall both the monomer sequestering and filament severing activities of TgADF are predicted to serve important functions in vivo for maintaining high G-actin concentrations for rapid filament assembly, and disassembling actin filaments for rapid filament turnover, respectively. These studies demonstrated that ADF is essential for regulating actin dynamics in T. gondii

Exploring the genetics of irritable bowel syndrome: A GWA study in the general population and replication in multinational case-control cohorts

OBJECTIVE: IBS shows genetic predisposition, but adequately powered gene-hunting efforts have been scarce so far. We sought to identify true IBS genetic risk factors by means of genome-wide association (GWA) and independent replication studies. DESIGN: We conducted a GWA study (GWAS) of IBS in a general population sample of 11\u2005326 Swedish twins. IBS cases (N=534) and asymptomatic controls (N=4932) were identified based on questionnaire data. Suggestive association signals were followed-up in 3511 individuals from six case-control cohorts. We sought genotype-gene expression correlations through single nucleotide polymorphism (SNP)-expression quantitative trait loci interactions testing, and performed in silico prediction of gene function. We compared candidate gene expression by real-time qPCR in rectal mucosal biopsies of patients with IBS and controls. RESULTS: One locus at 7p22.1, which includes the genes KDELR2 (KDEL endoplasmic reticulum protein retention receptor 2) and GRID2IP (glutamate receptor, ionotropic, delta 2 (Grid2) interacting protein), showed consistent IBS risk effects in the index GWAS and all replication cohorts and reached p=9.31 710(-6) in a meta-analysis of all datasets. Several SNPs in this region are associated with cis effects on KDELR2 expression, and a trend for increased mucosal KDLER2 mRNA expression was observed in IBS cases compared with controls. CONCLUSIONS: Our results demonstrate that general population-based studies combined with analyses of patient cohorts provide good opportunities for gene discovery in IBS. The 7p22.1 and other risk signals detected in this study constitute a good starting platform for hypothesis testing in future functional investigations. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions

Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose

Background Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection. Objectives To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship. Methods Human airway epithelial cells grown at air–liquid interface (±18 h pre-treatment, 30 μM–1 mM metformin) were inoculated with 5×105 colony-forming units (CFU)/cm2 S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6–10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×107 CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected. Results Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia–bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas. Conclusions Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection

Effect of a prebiotic galactooligosaccharide mixture (B-GOS®) on gastrointestinal symptoms in adults selected from a general population who suffer with bloating, abdominal pain, or flatulence

Background Prebiotics exert beneficial effects upon gastrointestinal (GI) environment, but this is not always accompanied with a positive effect on GI symptoms. B‐GOS® is a prebiotic with high selectivity toward bifidobacteria and a variety of other beneficial effects in humans. Here, we investigated its effect on GI symptoms in adults who suffer with bloating, abdominal pain, and flatulence. Methods In a double‐blind, placebo‐controlled, crossover study, 83 subjects from the general population who presented with GI symptoms during screening period and had a predicted probability of functional bowel disorder of more than 75% were randomized to receive either a placebo or the B‐GOS® treatment (2.75 g/d). Subjects were screened for the presence of GI symptoms for 1 week, they consumed the treatments for 2 weeks, and then went through a 2‐week washout period, before switching to the other treatment for the final 2 weeks. GI symptoms, bowel movements, and stool consistency were assessed in daily and weekly questionnaires. Quality of life was assessed weekly and depression and anxiety at the end of each treatment period. Results B‐GOS® resulted in significantly (P < 0.001) lower scores for bloating, flatulence, and abdominal pain both from baseline and placebo at the end of first week. The effect was sustained at the end of second week. It had no effect on the number of bowel movements, consistency of stools, quality of life, or mood throughout the study. Conclusion Results suggest that B‐GOS® could possibly be used in the management of bloating, flatulence, or abdominal pain and warrant further investigation

Irritable bowel syndrome, inflammatory bowel disease and the microbiome

PURPOSE OF REVIEW: The review aims to update the reader on current developments in our understanding of how the gut microbiota impact on inflammatory bowel disease and the irritable bowel syndrome. It will also consider current efforts to modulate the microbiota for therapeutic effect. RECENT FINDINGS: Gene polymorphisms associated with inflammatory bowel disease increasingly suggest that interaction with the microbiota drives pathogenesis. This may be through modulation of the immune response, mucosal permeability or the products of microbial metabolism. Similar findings in irritable bowel syndrome have reinforced the role of gut-specific factors in this ‘functional’ disorder. Metagenomic analysis has identified alterations in pathways and interactions with the ecosystem of the microbiome that may not be recognized by taxonomic description alone, particularly in carbohydrate metabolism. Treatments targeted at the microbial stimulus with antibiotics, probiotics or prebiotics have all progressed in the past year. Studies on the long-term effects of treatment on the microbiome suggest that dietary intervention may be needed for prolonged efficacy. SUMMARY: The microbiome represents ‘the other genome’, and to appreciate its role in health and disease will be as challenging as with our own genome. Intestinal diseases occur at the front line of our interaction with the microbiome and their future treatment will be shaped as we unravel our relationship with it

Genetic diversity and gene expression analysis of Phytophthora pluvialis, a foliar pathogen of conifers : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics, Massey University, Manawatū, New Zealand

Phytophthora pluvialis is the causal agent of red needle cast on Pinus radiata in New Zealand. It was first isolated in 2008 but had previously been recovered from tanoak (Notholithocarpus densiflorus) and Douglas fir (Pseudotsuga menziesii) trees in Oregon, USA in 2002. Phytophthora pluvialis was subsequently described as a new species in 2013 and classified as a clade 3 Phytophthora species. The aims of this study were to (1) gain a better understanding of the genetic diversity and population structure of P. pluvialis and (2) examine gene expression profiles of P. pluvialis from naturally infected P. radiata seedlings. Studying the genetic diversity and population structure of P. pluvialis provided insight into the mode of reproduction of this pathogen and helped determine if P. pluvialis was introduced into New Zealand. This information is also important for the development of management strategies for P. pluvialis. Twenty-seven single nucleotide polymorphism (SNP) markers were designed to genotype a total of 360 isolates of P. pluvialis collected from New Zealand and the USA. The genotypic data showed that the population in New Zealand has lower diversity than the USA population. A minimum spanning network (MSN) showed two unique clusters in the New Zealand population, suggesting there may have been two separate introductions of P. pluvialis. For the second study, samples were collected from 45 P. radiata grafted plants that were part of a field trial, with the aim of identifying genes that are highly expressed and may be important for virulence. Interestingly, Phytophthora kernoviae was found in more of the samples than P. pluvialis. Needle samples were collected, RNA was extracted and sequenced, and the normalised reads that mapped to the genome of P. pluvialis were compared to those from P. pluvialis grown in culture. Differentially expressed genes (DEGs) of P. pluvialis that showed higher expression in the field trial included potential orthologs of sugar transporter, GH12 and effector genes with known pathogenicity functions in other species. This is the first study to examine the genetic diversity of P. pluvialis in New Zealand and the USA., and to examine the gene expression of a Phytophthora forest pathogen in the field. The results from these studies provide useful tools for forest disease management. The SNP markers can be used to monitor the population of P. pluvialis in New Zealand. The highly expressed genes can be used to help identify resistance genes in P. radiata that can be incorporated into future breeding programs

European consensus conference on faecal microbiota transplantation in clinical practice

Faecal microbiota transplantation (FMT) is an important therapeutic option for Clostridium difficile infection. Promising findings suggest that FMT may play a role also in the management of other disorders associated with the alteration of gut microbiota. Although the health community is assessing FMT with renewed interest and patients are becoming more aware, there are technical and logistical issues in establishing such a non-standardised treatment into the clinical practice with safety and proper governance. In view of this, an evidence-based recommendation is needed to drive the practical implementation of FMT. In this European Consensus Conference, 28 experts from 10 countries collaborated, in separate working groups and through an evidence-based process, to provide statements on the following key issues: FMT indications; donor selection; preparation of faecal material; clinical management and faecal delivery and basic requirements for implementing an FMT centre. Statements developed by each working group were evaluated and voted by all members, first through an electronic Delphi process, and then in a plenary consensus conference. The recommendations were released according to best available evidence, in order to act as guidance for physicians who plan to implement FMT, aiming at supporting the broad availability of the procedure, discussing other issues relevant to FMT and promoting future clinical research in the area of gut microbiota manipulation. This consensus report strongly recommends the implementation of FMT centres for the treatment of C. difficile infection as well as traces the guidelines of technicality, regulatory, administrative and laboratory requirements.Peer reviewe

Plausibility criteria for putative pathophysiological mechanisms in functional gastrointestinal disorders: a consensus of experts

The functional gastrointestinal disorders (FGIDs), are extremely common conditions associated with a considerable personal, social and health economic burden. Managing FGIDs in clinical practice is challenging because of the uncertainty of symptom-based diagnosis, the high frequency of overlap between these conditions and the limited efficacy of available therapies. It has often been argued that successful drug development and management of FGIDs requires knowledge of the underlying pathophysiology. Numerous and highly variable candidate pathophysiological mechanisms have been implicated in the generation of FGID symptoms, but there is no current consensus on how to best define the relevance of these disturbances. Methods: A group of international experts on FGIDs developed plausibility criteria that should be fulfilled by relevant pathophysiological mechanisms in FGIDs. Results: Five criteria are proposed: 1) presence of the abnormality in a subset of patients; 2) temporal association between proposed mechanism and symptom(s); 3) correlation between the level of impairment of the mechanism and symptom(s); 4) induction of the symptom(s) by provoking the pathophysiological abnormality in healthy subjects and 5) treatment response by a therapy specifically correcting the underlying disorder, or congruent natural history of symptoms and dysfunction in the absence of specific therapy. Based on strength of evidence for these 5 criteria, a plausibility score is proposed. Conclusion: Evaluation of the strength of evidence for candidate pathophysiological abnormalities fulfilling these 5 plausibility criteria will help to identify the most relevant mechanisms to target for novel diagnostic approaches and for the development of new therapies