Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbe Enterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) from E. faecalis V583 and E. faecalis lacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutant E. faecalis strains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2^−/−) mice were used to investigate the role of this receptor in E. faecalis-induced permeability. Gelatinase (GelE) purified from E. faecalis V583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2^−/− mice by fecal supernatants from ulcerative colitis patients was assessed. Secreted E. faecalis proteins induced permeability in epithelial cell monolayers, which was reduced in the absence of gelE or by blocking PAR2 activity. Secreted E. faecalis proteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2^−/− mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2^−/− mice. Our investigations demonstrate that GelE from E. faecalis can regulate enteric epithelial permeability via PAR2.     

Microinflammation in patients with Crohn's disease in clinical remission

Background and aimsIt is not clear whether Crohn's disease patients in clinical remission (Crohn's disease activity index < 150) display normal concentrations of inflammation sensitive biomarkers. Our goal in this work was to explore the intensity of the microinflammatory response in a group of Crohn's disease patients in clinical remission.MethodsHigh sensitivity C-reactive protein, quantitative fibrinogen, erythrocyte sedimentation rate as well as platelet and leukocyte counts were examined in a group of 76 patients with Crohn's disease in remission and in 228 matched controls.ResultsCrohn's disease patients in clinical remission displayed a statistically significant (p < 0.001) elevated concentration of hs-CRP (4.83 ± 3.8 mg/l) compared to controls (1.05 ± 2.9 mg/l). All other bio-markers were also significantly higher in Crohn's disease patients in remission compared to controls. Similar results were obtained in a subgroup of Crohn's disease patients with very low disease activity — CDAI < 75.ConclusionsClinical remission is not equivalent to biochemical remission raising a question concerning the true definition of remission in Crohn's disease.     

Effectiveness and safety of vedolizumab for maintenance treatment in inflammatory bowel disease—The Israeli real world experience

Introduction

Several real-world experience (RWE) studies with vedolizumab (VDZ) for induction of remission in inflammatory bowel diseases (IBD) have been published; however, long-term RWE data is scarce.

Tyrosine phosphorylation-dependent localization of TmaR that controls activity of a major bacterial sugar regulator by polar sequestration

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

The central dogma describes the flow of genetic information from DNA to RNA to protein. However, for this process to be successful, the final product—the protein—needs to be in the right place and at the right time. The consequences of mislocalization can be harmful to any cell type, let alone to the unicellular bacterial cell, whose survival depends on fast and efficient response to environmental changes. Hence, protein localization is an important posttranslational regulatory step. Thus far, most examples of protein targeting were reported in eukaryotic cells, usually in the context of transport from one organelle to another (1). In recent years, it became evident that localization of proteins and RNAs to specific subcellular domains occurs also in prokaryotic cells and is vital for many cellular processes (2–5). However, the mechanisms underlying macromolecules targeting to specific subcellular domains in bacterial cells, with the exception of membrane and cell division proteins, remain largely unknown.The bacterial cell poles are emerging as important domains that accommodate protein and RNA assemblies (5, 6). Pole-localized proteins are involved in a wide range of cellular functions, including motility, regulation of cell cycle, metabolism, differentiation, pathogenesis, and secretion (7). Several proteins were reported to be kept as inactive at the Escherichia coli cell pole until needed, e.g., MurG (8), and FtsZ (9). The phosphotransferase system (PTS), which controls sugar utilization and metabolism in most bacteria, provides an example for regulation via polar cluster formation. Execution of the PTS functions depends on a phosphorylation cascade that initiates with EI and HPr—the general PTS proteins—that deliver the phosphate to the PTS sugar permeases, which import and phosphorylate the incoming sugars (10). The PTS proteins also exert different effects on non-PTS proteins depending on their phosphorylation on histidine residues, thus modulating the hierarchy in sugar utilization (10). The PTS-imported sugars enter glycolysis, whose product, phosphoenolpyruvate (PEP), phosphorylates EI, making EI an important link between glycolysis and sugar uptake (11). We have previously shown that the general PTS proteins localize to the E. coli cell poles (12), although their localization depends on yet-unknown factors (13), that during growth EI polar clusters form stochastically from preexisting dispersed molecules, and that EI clustering negatively correlates with EI function (14). Still, conclusive proof for polar localization as an inhibitory mechanism of EI function is lacking and the identity of the factor that captures EI at the pole remained unknown.Polar clusters offer additional benefits, such as communication between signal transduction systems in order to generate an optimal response, e.g., the chemotaxis and the PTS system (15), or the establishment of cellular asymmetry to coordinate developmental programs with cell cycle progression (7). Polar proteins that recruit other proteins to the poles, thus regulating cell cycle progression, were discovered in some bacteria, e.g., DivIVA in Bacillus subtilis, PopZ and TipN in Caulobacter crescentus, and HubP in Vibrio cholerae (7). In E. coli, the three Min proteins, MinCDE, cooperate to position the cell division site through pole-to-pole oscillation (16).In some bacteria, e.g., C. crescentus, specific localization of proteins is linked to their phosphorylation or to the phosphorylation of factors regulating them (17, 18). In most cases, these proteins are members of the two-component systems, which mediate sensing and regulation by phosphorylation on histidine and aspartic acid residues (19), events considered most prevalent in bacteria. Only in recent years, improved methodologies revealed numerous previously unknown Ser/Thr/Tyr phosphorylation sites, once thought to be hallmarks of eukaryotes, in bacterial and archaeal proteins. The degree to which these putative sites are phosphorylated is still unclear and proofs for their importance in vivo are just beginning to emerge. Also, the linkage between phosphorylation on Ser/Thr/TyR and localization of the phosphorylated bacterial proteins remained unknown.In this study, we show that a previously uncharacterized E. coli protein, YeeX, which is prevalent among Gram-negative bacteria, clusters at the pole in a tyrosine phosphorylation-dependent manner and recruits the major sugar utilization regulator EI. We, therefore, renamed this protein TmaR for targeting of sugar metabolism-associated regulator. TmaR and EI are shown to physically interact and to colocalize. TmaR is necessary for EI polar clustering, but the opposite is not true. Only phosphorylated, TmaR can release EI from the poles, since the diffuse nonphosphorylated TmaR binds to EI quite irreversibly. Notably, tyrosine phosphorylation of EI is also required for its polar localization. We further show that TmaR-mediated EI clustering inversely correlates with EI-mediated sugar uptake, implying that the polar clusters serve as a reservoir for ready-to-act EI molecules. Cells lacking TmaR have detrimental effects on cell survival, which is affected by EI and sugar concentration, when challenged with mildly acidic conditions that are typical to various E. coli habitats. Taken together, our study identifies TmaR as a spatial regulator of sugar metabolism and bacterial survival.     

Effects of diets high in simple sugars on urinary chromium losses

Thirty-seven subjects, 19 men and 18 women, consumed reference diets for 12 weeks formulated by nutritionists to contain optimal levels of protein, fat, carbohydrate, and other nutrients; the following 6 weeks, subjects consumed high sugar diets. The reference diets contained 35% of total calories from complex carbohydrates and 15% from simple sugars while the high sugar diets contained 15% complex carbohydrates and 35% simple sugars. Chromium contents of the reference and high sugar diets were both approximately 16 micrograms per 1000 calories. Compared to the reference diets, consumption of the high sugar diets increased urinary Cr losses from 10% to 300% for 27 of 37 subjects. Urinary Cr excretion of males and females was similar, and there was no significant difference in Cr absorption (calculated from urinary excretion divided by intake times 100) between sexes when adjusted for the increased caloric intake of males. These data demonstrate that consumption of diets high in simple sugars stimulates Cr losses; this coupled with marginal intake of dietary Cr may lead to marginal Cr deficiency, which is associated with impaired glucose and lipid metabolism.     

Healing of sites within the dog periodontal ligament after application of cold to the periodontal attachment apparatus

The potential of periodontal ligament-derived tissues to regenerate periodontal attachment after cryosurgical trauma to the PDL in dogs was evaluated. The buccal alveolar plate of each canine tooth was exposed by a semi-lunar excision. A 3 mm thick cryoprobe, cooled to -81 degrees C, was placed on the bone 5 mm apical to the crest for 10 s. This induced cellular devitalization in the bone directly in contact with the probe and the PDL under it. The freezing-thawing cycle was repeated 3 times. Control sites were sham-operated at room temperature. Histologic sections from the center of the lesions were obtained from 1 h, 48 h and 30 d specimens. 1-h control and experimental histologic sections were similar. At 48 h post-surgery, the cellular component of the frozen PDL could not be identified and inflammatory response was minimal. The collagenous framework, however, appeared to form a continuum between the alveolar bone and cementum. Lacunae in the bone at the frozen segment were empty. The injured PDL was surrounded by normal PDL. Control specimens appeared normal. At 30 d, the PDL space in the frozen segments was populated by PDL-like tissue which did not differ significantly from the PDL coronal or apical to it. Collagen fibers appeared to be attached to the cementum on one side and to the alveolar bone on the other. Bone resorption or ankylosis was not observed in the experimental sites. It is suggested that the extracellular matrix in the devitalized area was preserved, supporting regeneration of the cryolesion.     

Screening for 137Cs body burden due to the Chernobyl accident in Korosten City, Zhitomir, Ukraine: 1996-2008

During the Chernobyl Nuclear Power Plant (CNPP) accident on 26 April 1986, large amounts of radionuclides were released and spread to vast areas. Inhabitants residing around CNPP have been exposed to external and internal irradiation due to the long half-life of (137)Cs (30 years). In this study, we screened for internal whole-body (137)Cs concentration using a whole-body counter in the Zhitomir state of Ukraine. The total number of participants was 144,972 (96,149 females and 48,823 males). The median body burden of (137)Cs per body weight decreased from 1996 to 2008. In particular, after 2003, more than half of subjects had internal exposure doses below the detectable level. A weak seasonal effect was found in measurement data from 1997 to 1999, but no such effects were observed in later years. We also calculated annual dose for each year and confirmed that doses have been decreasing gradually. In particular, after 2003, the annual effective dose decreased to 0.1 mSv y(-1) for 95% of the participants. Only two persons were found to have received more than 5 mSv y(-1) since 2007. Although the health effects of (137)Cs body burden due to the Chernobyl accident remain uncertain, further screening is needed to monitor the health status and to allay the anxiety of inhabitants in the contaminated areas around CNPP.     

Interaction of chlorhexidine with smooth and rough types of titanium surfaces

BACKGROUND: Chlorhexidine (CHX) digluconate exerts plaque inhibitory efficacy in the natural dentition environment due to a superior degree of persistence at the tooth surface. The purpose of the present study was to assess the interaction of CHX with titanium surfaces to estimate its antiplaque potential in the peri-implant environment. METHODS: Saliva-coated machined smooth (S) and sand-blasted acid-etched rough (R) titanium disks were soaked in either 0.1% or 0.2% CHX solution. After 24 hours, CHX amounts that were adsorbed, washed out, and desorbed from the titanium surfaces were determined spectrophotometrically at 230 nm. The antibacterial activity of CHX-treated titanium disks was assessed by measuring bacterial inhibition zones on Streptococcus mutans lawns. RESULTS: Titanium disks adsorbed 3% to 8% of the available CHX, which was significantly higher with 0.2% CHX (P<0.001) than with 0.1% CHX and two-fold higher on the R titanium disks compared to S titanium surface (P<0.001). After rinsing with water, 2.2% of the adsorbed CHX was washed out. Over 24 hours, S- and R-type disks released 1.1% and 0.6% of the adsorbed agent, respectively. Larger bacterial inhibition zones were obtained with 0.2% CHX and in R disks compared to S disks. CONCLUSIONS: CHX displayed persistence at the titanium surface. The adsorption level and bacterial growth inhibition were affected by CHX concentration and titanium surface characteristics, with higher levels of adsorption and antibacterial activity with 0.2% CHX and rough titanium surface. The slow CHX release rate suggests persistence of this agent at the titanium-pellicle surface, which can provide a long-term antiplaque effect.     

Lack of effect of acute,subchronic, or chronic stress on glycogen synthase kinase-3beta protein levels in rat frontal cortex

Glycogen synthase kinase (GSK)-3β is a conserved serine/threonine protein kinase highly abundant in brain tissue. A dominant mechanism by which cells react to stress involves GSK-3β. We studied the effect of stress on GSK-3β levels ex vivo. We have previously found reduced GSK-3β protein levels and GSK-3 activity in postmortem prefrontal cortex of schizophrenic patients. Since schizophrenic patients experience stress more severely than healthy people, we questioned whether their GSK-3β reduction is stress-related using a rat model. Rats were exposed to acute, subchronic, or chronic stress using brief cold restraint. No effect was found on frontal cortex GSK-3β protein levels. These results suggest that reduction in GSK-3β levels in schizophrenic patients is not affected by cold restraint stress and supports the possibility that the changes observed in postmortem brains may be related to the disease.     

GSK-3 parameters in lymphocytes of schizophrenic patients

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase that is involved in the signal transduction cascades of multiple cellular processes. GSK-3 has two isoforms, designated alpha and beta. GSK-3beta protein levels and GSK-3 enzyme activity have been reported to be reduced by over 40% in postmortem frontal cortex of schizophrenic patients. GSK-3 is also present in peripheral tissue such as lymphocytes. In this study we aimed to find whether the reduction in brain GSK-3beta measures is reflected in peripheral tissue of schizophrenic patients. Fresh lymphocytes from schizophrenic patients showed no difference in GSK-3 alpha and GSK-3beta mRNA levels, GSK-3beta protein levels, or total GSK-3 (alpha+beta) enzyme activity compared with findings in control subjects. In addition, lymphocyte-derived cell lines from schizophrenic patients did not differ in their GSK-3beta protein levels from levels in normal control subjects. The results rule out the use of lymphocyte GSK-3 as a marker for central GSK-3 abnormalities in schizophrenia.