Question: Ethanol stress decreases the expression of IBP in bacteria.

Title: Complex physiology and compound stress responses during fermentation of alkali-pretreated corn stover hydrolysate by an Escherichia coli ethanologen. Content: The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

Title: Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes Content: Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression.

Title: Article Content: IBD is characterized by uncontrolled immune responses in inflamed mucosa, with dominance of IL-17-producing cells and deficiency of Treg cells. The aim of this study was to explore the effect and mechanisms of RA, the ligand of RARalpha, on immune responses in human and murine colitis. Colonic biopsies from patients with UC were cultured and treated with RA as the agonist of RARalpha or LE135 as the antagonist of RARalpha. Expressions of IL-17 and FOXP3 were detected by immunohistochemistry. Murine colitis was induced by intrarectal administration with TNBS at Day 1. Mice were then i.p.-treated with RA or LE135 daily for 7 days. Cytokine levels in the cultures of mouse LPMCs were measured. Expressions of FOXP3 and IL-17 in colon tissues or MLN were detected by immunohistological analysis. Body weight and colon inflammation were evaluated. RA treatment up-regulated FOXP3 expression and down-regulated IL-17 expression in colon biopsies of patients and in colon tissues and MLN of mice with colitis compared with controls. LPMCs from RA-treated mice produced lower levels of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-17) but more regulatory cytokines (IL-10, TGF-beta) compared with that of untreated mice. LE135 showed the opposite effect of RA. Furthermore, RA ameliorated TNBS-induced colitis in a dose-dependent manner, as seen by improved body weight and colon inflammation. RA down-regulates colon inflammatory responses in patients with IBD in vitro and in murine colitis in vivo, representing a potential therapeutic approach in IBD treatment.

Title: Effect of hyperosmolality on alkaline phosphatase and stress-response protein 27 of MCF-7 breast cancer cells Content: MCF-7, a continuous cell line derived from a human breast carcinoma, exhibits very low alkaline phosphatase (ALP) activity. The enzyme is heat-stable and is inhibited by L-phenylalanine and L-phenylalanylgly-cylglycine, but not by L-homoarginine, 1-bromotetramisole, or levamisole. These data indicate that MCF-7 produces term-placental ALP, the oncodevelopmental enzyme form inappropriately expressed by a variety of human tumors. In contrast to human cancer cells that produce this enzyme monophenotypically, ALP activity of MCF-7 cells is not significantly increased by glucocorticoids or sodium butyrate. By comparison, exposure to hyperosmolality causes a striking increase in enzyme activity. Cycloheximide blocks this effect. The results obtained with cell-free assays were confirmed by cytochemical and immunocytochemical assays on whole cells. Because some of the agents tested in the enzyme modulation experiments affect cell proliferation, their possible effect on two stress-response proteins (srp 27 and srp 72) was also examined; specific immunocytochemical assays were used. These tests revealed that neither protein is affected by glucocorticoids; that sodium butyrate has no effect on srp 27, but alters the intracellular distribution of srp 72; and that hyperosmolality, while not significantly affecting srp 72, causes an increase in srp 27.

Title: Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking. Content: Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the alpha4beta2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the alpha4beta2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

Title: Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms. Content: Nonreplicating and metabolically quiescent bacteria are implicated in latent tuberculosis infections and relapses following "sterilizing" chemotherapy. However, evidence linking bacterial dormancy and persistence in vivo is largely inconclusive. Here we measure the single-cell dynamics of Mycobacterium tuberculosis replication and ribosomal activity using quantitative time-lapse microscopy and a reporter of ribosomal RNA gene expression. Single-cell dynamics exhibit heterogeneity under standard growth conditions, which is amplified by stressful conditions such as nutrient limitation, stationary phase, intracellular replication, and growth in mouse lungs. Additionally, the lungs of chronically infected mice harbor a subpopulation of nongrowing but metabolically active bacteria, which are absent in mice lacking interferon-γ, a cytokine essential for antituberculosis immunity. These cryptic bacterial forms are prominent in mice treated with the antituberculosis drug isoniazid, suggesting a role in postchemotherapeutic relapses. Thus, amplification of bacterial phenotypic heterogeneity in response to host immunity and drug pressure may contribute to tuberculosis persistence.

Title: Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection Content: Antibiotics can have significant and long-lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including the primary bile acid taurocholate for germination, and carbon sources such as mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favours C. difficile germination and growth.

Title: Regulatory and metabolic rewiring during laboratory evolution of ethanol tolerance in E. coli Content: Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single-locus perturbations in the context of ethanol exposure. A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory-evolved ethanol-tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

Title: Ethanol extract of Allium fistulosum inhibits development of non-alcoholic fatty liver disease Content: BACKGROUND/OBJECTIVES Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease and is closely associated with metabolic syndrome. In the present study, we observed the effect of ethanol extract of Allium fistulosum (EAF) on NAFLD and have suggested the possibility of using EAF as a natural product for application in the development of a treatment for NAFLD. MATERIALS/METHODS The preventive effect on hepatic lipid accumulation was estimated by using an oleic acid (OA)-induced NAFLD model in vitro and a Western diet (high-fat high-sucrose; WD)-induced obese mouse model. Animals were divided into three groups (n = 7): normal diet group (ND), WD group, and WD plus 1% EAF group. RESULTS EAF reduced OA-stimulated lipid accumulation in HepG2 cells in the absence of cellular cytotoxicity and significantly blocked transcriptional activation of sterol regulatory element-binding protein 1 and fatty acid synthase genes. Subsequently, we investigated these effects in vivo in mice fed either ND or WD in the presence or absence of EAF supplementation. In comparison to the ND controls, the WD-fed mice exhibited increases in body weight, liver weight, epididymal fat weight, and accumulation of fat in hepatocytes, and these effects were significantly attenuated by EAF supplementation. CONCLUSIONS Allium fistulosum attenuates the development of NAFLD, and EAF elicits anti-lipogenic activity in liver. Therefore, EAF represents a promising candidate for use in the development of novel therapeutic drugs or drug combinations for the prevention and treatment of NAFLD.

Title: Leptin deficiency contributes to the pathogenesis of alcoholic fatty liver disease in mice. Content: White adipose tissue (WAT) secretes adipokines, which critically regulate lipid metabolism. The present study investigated the effects of alcohol on adipokines and the mechanistic link between adipokine dysregulation and alcoholic fatty liver disease. Mice were fed alcohol for 2, 4, or 8 weeks to document changes in adipokines over time. Alcohol exposure reduced WAT mass and body weight in association with hepatic lipid accumulation. The plasma adiponectin concentration was increased at 2 weeks, but declined to normal at 4 and 8 weeks. Alcohol exposure suppressed leptin gene expression in WAT and reduced the plasma leptin concentration at all times measured. There is a highly positive correlation between plasma leptin concentration and WAT mass or body weight. To determine whether leptin deficiency mediates alcohol-induced hepatic lipid dyshomeostasis, mice were fed alcohol for 8 weeks with or without leptin administration for the last 2 weeks. Leptin administration normalized the plasma leptin concentration and reversed alcoholic fatty liver. Alcohol-perturbed genes involved in fatty acid β-oxidation, very low-density lipoprotein secretion, and transcriptional regulation were attenuated by leptin. Leptin also normalized alcohol-reduced phosphorylation levels of signal transducer Stat3 and adenosine monophosphate-activated protein kinase. These data demonstrated for the first time that leptin deficiency in association with WAT mass reduction contributes to the pathogenesis of alcoholic fatty liver disease.

Title: Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene. Content: Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter.

Title: Evaluation of oxidative status and depression-like responses in Brown Norway rats with acute myeloid leukemia Content: It has been proved that oxidative stress increases when leukemia is accompanied by depression. This fact may indicate the role of oxidative stress in the development of depression in cancer patients. The aim of this study was to determine whether the acute myeloid leukemia of Brown Norway rats, which is accompanied by oxidative stress, evoked behavioral and receptor changes resembling alterations characteristic of rat models of depression. The rats were divided into two groups: leukemic rats and healthy control. Leukemia was induced through intraperitoneal injection of 10(7) promyelocytic leukemia cells to the Brown Norway rats. Depression-like behavior was evaluated in the forced swim test at 30 or 34 days after leukemic cells injection. The rats were killed after the evaluation and the spleen, brain cortex and hippocampus were excised. The red-ox state was assessed in homogenates of tissues by measuring total glutathione (GSH) content, the ferric ion reducing ability of plasma (FRAP) level, expression of heme oxygenase-1 (HO-1), biliverdin reductase (BvR) and ferritin mRNA, superoxide dismutase (SOD) activity, as well as malondialdehyde (MDA) concentration. Radioligand binding assay was used to assess of the effect of leukemia on cortical receptors. Leukemic cells were identified using RM-124 antibody by FACS Calibur flow cytometry. Leukemia influenced locomotory activity as well as forced swim test behavior in a 34-day series of experiments. Signs of oxidative stress in leukemic rats were observed in each examined stage of leukemia development. The FRAP values and glutathione contents, were significantly lowered whereas HO-1 mRNA expression, and malonodialdehyde concentrations were significantly increased in the spleen and brain structures of leukemic rats in comparison with the healthy controls. A significant increase in the potency of glycine to displace [(3)H]L-689,560 from the strychnine-insensitive glycine site of the N-methyl-D-aspartic (NMDA) receptors receptor complex in cortical homogenates of the leukemic rats in 30- and 34-day experimental series was observed in comparison with the control. Upregulation of 5-HT(2A) receptors was observed in rat cortex after 30 days of leukemia development but not in 34-days series compared with the control. It is concluded that disturbances in antioxidant system in brain cortex were accompanied by an activation of glycine sites of the NMDA receptor complex, regardless of stage of leukemia development, which are characteristic of model of depression. Findings of our study demonstrate the link between glutamatergic activity, oxidative stress and leukemia.

Title: C-reactive protein decreases interleukin-10 secretion in activated human monocyte-derived macrophages via inhibition of cyclic AMP production. Content: OBJECTIVE C-Reactive protein (CRP), a cardiovascular risk marker, could also participate in atherosclerosis. Atherosclerotic plaques express CRP and interleukin (IL)-10, a major antiinflammatory cytokine. IL-10 deficiency results in increased lesion formation, whereas IL-10 delivery attenuates lesions. We tested the effect of CRP on lipopolysaccharide (LPS)-induced IL-10 secretion in human monocyte-derived macrophages (HMDMs). METHODS AND RESULTS Incubation of HMDMs with CRP significantly decreased LPS-induced IL-10 mRNA and intracellular and secreted IL-10 protein and destabilized IL-10 mRNA. Also, CRP alone increased secretion of IL-8, IL-6, and tumor necrosis factor from HMDMs and did not inhibit LPS-induced secretion of these cytokines. Fc gamma receptor I antibodies significantly reversed CRP-mediated IL-10 inhibition. CRP significantly decreased intracellular cAMP, phospho-cAMP response element binding protein (pCREB), and adenyl cyclase activity. cAMP agonists reversed CRP-mediated IL-10 inhibition. Overexpression of wild-type and constitutively active CREB in THP-1 cells revealed attenuation of the inhibitory effect of CRP on LPS-induced IL-10 levels. CRP also inhibited hemoglobin:haptoglobin-induced IL-10 and heme oxygenase-1. Furthermore, administration of human CRP to rats significantly decreased IL-10 levels. CONCLUSIONS This study provides novel evidence that CRP, by decreasing IL-10 alters the antiinflammatory/proinflammatory balance, accentuating inflammation, which is pivotal in atherothrombosis.

Title: Antioxidant Defenses in the Rat Placenta in Late Gestation: Increased Labyrinthine Expression of Superoxide Dismutases, Glutathione Peroxidase 3, and Uncoupling Protein 21 Content: Placental oxidative stress plays a key role in the pathophysiology of placenta-related disorders, most notably preeclampsia (PE) and intrauterine growth restriction (IUGR). Oxidative stress occurs when accumulation of reactive oxygen species (ROS) damages DNA, proteins and lipids, an outcome that is limited by antioxidant enzymes; mitochondrial uncoupling protein 2 (UCP2) may also limit oxidative stress by reducing ROS production. Here we characterized placental antioxidant defenses during normal gestation and following glucocorticoid-induced IUGR. Placentas were collected on Days 16 and 22 of normal rat pregnancy (term = Day 23) and at Day 22 after dexamethasone treatment from Day 13. Expression of several genes encoding antioxidant enzymes (Sod1, Sod2, Sod3, Cat, Gpx3, Txn1, Txnrd1, Txnrd2, and Txnrd3) and Ucp2 was measured by quantitative RT-PCR in the labyrinth (LZ) and junctional zones (JZ) of the placenta. Expression of Sod1 and Ucp2 mRNAs and the activity of xanthine oxidase, a source of ROS, all increased from Days 16 to 22 in both placental zones, whereas Sod2 and Gpx3 increased only in the rapidly growing LZ. In contrast, Sod3 and Txnrd1 expression fell in the LZ over this period, whereas total superoxide dismutase activity remained stable. Dexamethasone treatment reduced fetal-placental growth and LZ expression of Ucp2 but increased JZ expression of Txn1. Indices of placental oxidative damage (TBARS, F2-isoprostanes, and 8-OHdG) did not change with gestational age or dexamethasone, indicative of adequate antioxidant protection. Overall, our data suggest that the rat placenta is protected from oxidative stress by the dynamic zone- and stage-dependent expression of antioxidant defense genes.

Title: Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes Content: Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes. Expression of genes involved in ER stress was evaluated in insulin-secreting MIN6 cells exposed to elevated lipids, in islets isolated from db/db mice and in pancreas sections of humans with type 2 diabetes. Overproduction of the ER chaperone heat shock 70 kDa protein 5 (HSPA5, previously known as immunoglobulin heavy chain binding protein [BIP]) was performed to assess whether attenuation of ER stress affected lipid-induced apoptosis. We demonstrated that the pro-apoptotic fatty acid palmitate triggers a comprehensive ER stress response in MIN6 cells, which was virtually absent using non-apoptotic fatty acid oleate. Time-dependent increases in mRNA levels for activating transcription factor 4 (Atf4), DNA-damage inducible transcript 3 (Ddit3, previously known as C/EBP homologous protein [Chop]) and DnaJ homologue (HSP40) C3 (Dnajc3, previously known as p58) correlated with increased apoptosis in palmitate- but not in oleate-treated MIN6 cells. Attenuation of ER stress by overproduction of HSPA5 in MIN6 cells significantly protected against lipid-induced apoptosis. In islets of db/db mice, a variety of marker genes of ER stress were also upregulated. Increased processing (activation) of X-box binding protein 1 (Xbp1) mRNA was also observed, confirming the existence of ER stress. Finally, we observed increased islet protein production of HSPA5, DDIT3, DNAJC3 and BCL2-associated X protein in human pancreas sections of type 2 diabetes subjects. Our results provide evidence that ER stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure.

Title: Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species. Content: Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

Title: In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules Content: Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to Kb-, Db-, or Qa-1b–restricted CD8+ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8+ T cell responses against class I–presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Title: Genetic and biochemical interactions among Yar1, Ltv1 and Rps3 define novel links between environmental stress and ribosome biogenesis in Saccharomyces cerevisiae. Content: In the yeast S. cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis. In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production. We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle. We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits. Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions. Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants. We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production. The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity.

Title: Intestinal bacteria and the regulation of immune cell homeostasis. Content: The human intestine is colonized by an estimated 100 trillion bacteria. Some of these bacteria are essential for normal physiology, whereas others have been implicated in the pathogenesis of multiple inflammatory diseases including IBD and asthma. This review examines the influence of signals from intestinal bacteria on the homeostasis of the mammalian immune system in the context of health and disease. We review the bacterial composition of the mammalian intestine, known bacterial-derived immunoregulatory molecules, and the mammalian innate immune receptors that recognize them. We discuss the influence of bacterial-derived signals on immune cell function and the mechanisms by which these signals modulate the development and progression of inflammatory disease. We conclude with an examination of successes and future challenges in using bacterial communities or their products in the prevention or treatment of human disease.

Title: microRNAs: A Safeguard against Turmoil? Content: Emerging data suggest that microRNAs (miRNAs) are instrumental in a variety of stress responses in addition to their more recognized role in development. Surprisingly, miRNAs, which normally suppress expression of target transcripts, may become activators of expression during stress. This might be partially explained by new interactions of miRNA/Argonaute complexes with RNA-binding proteins that relocate from different subcellular compartments during stress.

Title: Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis Content: The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Here we identified 283 discrete chromosomal sites potentially bound by the Spx-RNA polymerase (Spx-RNAP) complex using chromatin immunoprecipitation of Spx. Three quarters of these sites were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx-RNAP complex increased in parallel with the number and occupancy of DNA sites. Correlation of Spx-RNAP-binding sites with gene differential expression in wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Spx regulation. Spx-controlled promoters exhibited an extended -35 box in which nucleotide composition at the -43/-44 positions strongly correlated with observed activation. In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by reduced Spx. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some genes and its complex interplay with other stress responses.

Title: IL-23–responsive innate lymphoid cells are increased in inflammatory bowel disease Content: Results of experimental and genetic studies have highlighted the role of the IL-23/IL-17 axis in the pathogenesis of inflammatory bowel disease (IBD). IL-23-driven inflammation has been primarily linked to Th17 cells; however, we have recently identified a novel population of innate lymphoid cells (ILCs) in mice that produces IL-17, IL-22, and IFN-γ in response to IL-23 and mediates innate colitis. The relevance of ILC populations in human health and disease is currently poorly understood. In this study, we have analyzed the role of IL-23-responsive ILCs in the human intestine in control and IBD patients. Our results show increased expression of the Th17-associated cytokine genes IL17A and IL17F among intestinal CD3⁻ cells in IBD. IL17A and IL17F expression is restricted to CD56⁻ ILCs, whereas IL-23 induces IL22 and IL26 in the CD56⁺ ILC compartment. Furthermore, we observed a significant and selective increase in CD127⁺CD56⁻ ILCs in the inflamed intestine in Crohn's disease (CD) patients but not in ulcerative colitis patients. These results indicate that IL-23-responsive ILCs are present in the human intestine and that intestinal inflammation in CD is associated with the selective accumulation of a phenotypically distinct ILC population characterized by inflammatory cytokine expression. ILCs may contribute to intestinal inflammation through cytokine production, lymphocyte recruitment, and organization of the inflammatory tissue and may represent a novel tissue-specific target for subtypes of IBD.

Title: RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever Content: Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

Title: Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli Content: Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation.

Title: Defective replication stress response inhibits lymphomagenesis and impairs lymphocyte reconstitution Content: DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

Title: Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting. Content: Ex-FABP, an extracellular fatty acid binding lipocalin, is physiologically expressed by differentiating chicken chondrocytes and myoblasts. Its expression is enhanced after cell treatment with inflammatory stimuli and repressed by anti-inflammatory agents, behaving as an acute phase protein. Chicken liver fragments in culture show enhanced protein expression after bacterial endotoxin treatment. To investigate the biological role of Ex-FABP, we stably transfected proliferating chondrocytes with an expression vector carrying antisense oriented Ex-FABP cDNA. We observed a dramatic loss of cell viability and a strong inhibition of cell proliferation and differentiation. When chondrocytes were transfected with the antisense oriented Ex-FABP cDNA we observed that Ex-FABP down-modulation increased apoptotic cell number. Myoblasts transfected with the same expression vector showed extensive cell death and impaired myotube formation. We suggest that Ex-FABP acts as a constitutive survival protein and that its expression and activation are fundamental to protect chondrocytes from cell death.

Title: Fine-tuning in regulation of Clp protein content in Bacillus subtilis. Content: Clp-controlled proteolysis in Bacillus subtilis seems to play a substantial role, particularly under stress conditions. Calibrated Western blot analyses were used to estimate the approximate numbers of heat-inducible Clp molecules within a single cell. According to these numbers, the different Clp ATPases do not seem to compete for the proteolytic subunit ClpP. Coimmunoprecipitation experiments revealed the predicted specific ClpX-ClpP, ClpC-ClpP, and ClpE-ClpP interactions. ClpE and ClpX are rapidly degraded in wild-type cells during permanent heat stress but remained almost stable in a clpP mutant, suggesting ClpP-dependent degradation. In particular, ClpCP appeared to be involved in the degradation of the short-lived ClpE ATPase, indicating a negative "autoregulatory" circuit for this particular Clp ATPase at the posttranslational level. Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B. subtilis.

Title: Direct Upregulation of STAT3 by MicroRNA-551b-3p Deregulates Growth and Metastasis of Ovarian Cancer. Content: 3q26.2 amplification in high-grade serous ovarian cancer leads to increased expression of mature microRNA miR551b-3p, which is associated with poor clinical outcome. Importantly, miR551b-3p contributes to resistance to apoptosis and increased survival and proliferation of cancer cells in vitro and in vivo. miR551b-3p upregulates STAT3 protein levels, and STAT3 is required for the effects of miR551b-3p on cell proliferation. Rather than decreasing levels of target mRNA as expected, we demonstrate that miR551b-3p binds a complementary sequence on the STAT3 promoter, recruiting RNA polymerase II and the TWIST1 transcription factor to activate STAT3 transcription, and thus directly upregulates STAT3 expression. Furthermore, anti-miR551b reduced STAT3 expression in ovarian cancer cells in vitro and in vivo and reduced ovarian cancer growth in vivo. Together, our data demonstrate a role for miR551b-3p in transcriptional activation. Thus, miR551b-3p represents a promising candidate biomarker and therapeutic target in ovarian cancer.

Title: Helicobacter pylori AlpA and AlpB bind host laminin and influence gastric inflammation in gerbils. Content: Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.

Title: Induction of the heat-shock response by carbon dioxide in Chironomus thummi. Content: The effects of a set of stress treatments on gene expression of Chironomus thummi salivary gland cells have been analyzed. Among the treatments assayed, only during recovery from carbon dioxide have we observed a response similar to that previously described after heat-shock treatment: induction of the heat-shock puffs and synthesis of the heat-shock polypeptides. In these conditions, puffing and transcription of telomeric regions were observed, which led to the appearance of the temperature-inducible telomeric Balbiani ring T-BR-III. Other treatments failed to induce the heat-shock response, despite promoting real stress conditions to C. thummi larvae or salivary gland cells.

Title: Retinal attenuates inflammatory arthritis by reciprocal regulation of IL-17-producing T cells and Foxp3(+) regulatory T cells and the inhibition of osteoclastogenesis. Content: Retinoids (e.g., vitamin A and its derivatives) can regulate immune responses. The aim of this study was to determine whether all-trans retinaldehyde (retinal), a vitamin A derivative, can inhibit inflammatory responses and joint destruction in DBA/1J mice with collagen-induced arthritis (CIA). The arthritis score and incidence of arthritis were lower in mice treated with retinal compared to those treated with cottonseed oil. Histopathologic evidence of joint damage was lower in mice treated with retinal, corresponding with a reduction in the infiltration of immune cells in mice treated with retinal type II collagen (CII)-stimulated spleen cells. In addition, the expression of proinflammatory cytokines, oxidative stress proteins, and osteoclast markers were significantly reduced in mice treated with retinal. In vitro, retinal induced increased Foxp3 expression and inhibited Th17 development. The proportion of Foxp3(+) Treg cells was increased in the spleens of mice treated with retinal, whereas the proportion of Th17 cells was reduced. In both mice and a human culture system, tartrate-resistant acid phosphatase (TRAP) positive mononuclear cells and multinucleated cells were significantly reduced after treatment with retinal. The expression of osteoclast differentiation markers was dramatically decreased upon addition of retinal. This is the first study to demonstrate the therapeutic effect of retinal on an autoimmune arthritis model in mice through reciprocal regulation of Th17 and regulatory T cells and protection of differentiation and activation of osteoclasts. Taken together, our findings indicate that retinal has profound immunoregulatory functions and potential value for the treatment of autoimmune inflammatory disorders.

Title: Regulation of the expression of alkaline phosphatase in a human breast-cancer cell line. Content: Treatment of the cultured human breast-cancer cells BC-M1 with dexamethasone induced a placental-type alkaline phosphatase (ALP). Both the ALP activity and the mRNA level in the cells were increased. The induction of ALP activity by dexamethasone was time- and dose-dependent. The accumulation of ALP mRNA was inhibited by both actinomycin D and cycloheximide, indicating that its induction is a complex event and may involve other regulatory proteins. Retinoic acid showed opposing effects with dexamethasone on the expression of alkaline phosphatase. Retinoic acid (RA) and phorbol 12-myristate 13-acetate also substantially reduced the dexamethasone-induced expression of ALP. Studies on thermostability and sensitivity to various amino acid inhibitors indicated that the BC-M1 ALP is most similar to the placental form. Northern hybridization analysis also revealed that ALP mRNA transcripts in BC-M1 and term placenta are similar in size and are distinct from that of the placental-like mRNA transcript in choriocarcinoma cells. Analysis of the degradation of BC-M1 ALP mRNA showed a similar half-life of 27 h in the untreated and in dexamethasone- or RA-treated cells. These findings demonstrated that the induction of ALP in BC-M1 cells by dexamethasone is mainly due to the increase in the transcription of the ALP gene.

Title: Increased gene expression of brown fat uncoupling protein (UCP)1 and skeletal muscle UCP2 and UCP3 in MAC16-induced cancer cachexia. Content: Weight loss in cancer cachexia is attributable to decreased food intake and/or enhanced energy expenditure. We investigated the roles of the uncoupling proteins (UCPs) UCPI, -2, and -3 in a murine model of cachexia, the MAC16 adenocarcinoma. Weight fell to 24% below that of non-tumor-bearing controls (P < 0.01) 18 days after MAC16 inoculation, with significant reductions in fat-pad mass (-67%; P < 0.01) and muscle mass (-20%; P < 0.01). Food intake was 26-60% lower (P < 0.01) than in controls on days 17-18. Non-tumor-bearing mice, pair-fed to match MAC16-induced hypophagia, showed less weight loss (10% below controls, P < 0.01; 16% above MAC-16, P < 0.01) and smaller decreases in fat-pad mass (21% below controls, P < 0.01). Core temperature in MAC16 mice was significantly lower (-2.4 degrees C, P < 0.01) than in controls, and pair-feeding had no effect. MAC16 mice showed significantly higher UCP1 mRNA levels in brown adipose tissue (BAT) than in controls (+63%, P < 0.01), and pair-feeding had no effect. UCP2 and -3 expression in BAT did not differ significantly between groups. By contrast, UCP2 mRNA levels in skeletal muscle were comparably increased in both MAC16 and pair-fed groups (respectively, 183 and 163% above controls; both, P < 0.05), with no significant difference between these two groups. Similarly, UCP3 mRNA was significantly higher than controls in both MAC16 (+163%, P < 0.05) and pair-fed (+253%, P < 0.01) groups, with no significant difference between the two experimental groups. Overexpression of UCP1 in BAT in MAC16-bearing mice may be an adaptive response to hypothermia, which is apparently induced by tumor products; increased thermogenesis in BAT could increase total energy expenditure and, thus, contribute to tissue wasting. Increased UCP2 and -3 expression in muscle are both attributable to reduced food intake and may be involved in lipid utilization during lipolysis in MAC16-induced cachexia.

Title: Hypoxia-induced energy stress regulates mRNA translation and cell growth. Content: Oxygen (O2) deprivation, or hypoxia, has profound effects on cell metabolism and growth. Cells can adapt to low O2 in part through activation of hypoxia-inducible factor (HIF). We report here that hypoxia inhibits mRNA translation by suppressing multiple key regulators, including eIF2alpha, eEF2, and the mammalian target of rapamycin (mTOR) effectors 4EBP1, p70S6K, and rpS6, independent of HIF. Hypoxia results in energy starvation and activation of the AMPK/TSC2/Rheb/mTOR pathway. Hypoxic AMP-activated protein kinase (AMPK) activation also leads to eEF2 inhibition. Moreover, hypoxic effects on cellular bioenergetics and mTOR inhibition increase over time. Mutation of the TSC2 tumor suppressor gene confers a growth advantage to cells by repressing hypoxic mTOR inhibition and hypoxia-induced G1 arrest. Together, eIF2alpha, eEF2, and mTOR inhibition represent important HIF-independent mechanisms of energy conservation that promote survival under low O2 conditions.

Title: Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Content: The Sir2 deacetylase modulates organismal life-span in various species. However, the molecular mechanisms by which Sir2 increases longevity are largely unknown. We show that in mammalian cells, the Sir2 homolog SIRT1 appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors, a family of proteins that function as sensors of the insulin signaling pathway and as regulators of organismal longevity. SIRT1 and the FOXO transcription factor FOXO3 formed a complex in cells in response to oxidative stress, and SIRT1 deacetylated FOXO3 in vitro and within cells. SIRT1 had a dual effect on FOXO3 function: SIRT1 increased FOXO3's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXO3's ability to induce cell death. Thus, one way in which members of the Sir2 family of proteins may increase organismal longevity is by tipping FOXO-dependent responses away from apoptosis and toward stress resistance.

Title: DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress Content: The stress-activated kinase JNK mediates key cellular responses to oxidative stress. Here we show that DAP kinase (DAPk), a cell death promoting Ser/Thr protein kinase, plays a main role in oxidative stress-induced JNK signaling. We identify protein kinase D (PKD) as a novel substrate of DAPk and demonstrate that DAPk physically interacts with PKD in response to oxidative stress. We further show that DAPk activates PKD in cells and that induction of JNK phosphorylation by ectopically expressed DAPk can be attenuated by knocking down PKD expression or by inhibiting its catalytic activity. Moreover, knockdown of DAPk expression caused a marked reduction in JNK activation under oxidative stress, indicating that DAPk is indispensable for the activation of JNK signaling under these conditions. Finally, DAPk is shown to be required for cell death under oxidative stress in a process that displays the characteristics of caspase-independent necrotic cell death. Taken together, these findings establish a major role for DAPk and its specific interaction with PKD in regulating the JNK signaling network under oxidative stress.

Title: Role of the novel Th17 cytokine IL-17F in inflammatory bowel disease (IBD): upregulated colonic IL-17F expression in active Crohn's disease and analysis of the IL17F p.His161Arg polymorphism in IBD. Content: BACKGROUND Interleukin (IL)-17F, produced in IL-23R-expressing Th17 cells, is a novel member of the IL-17 cytokine family. Given the association of IL23R with inflammatory bowel disease (IBD), we characterized the role of IL-17F in IBD including its intestinal gene expression and the effect of the IL17F p. His161Arg polymorphism on disease susceptibility and phenotype of Crohn's disease (CD) and ulcerative colitis (UC). In addition, we analyzed the IL17F p. His161Arg polymorphism for potential epistasis with IL23R and NOD2/CARD15 variants. METHODS Intestinal IL-17F mRNA expression was measured by quantitative polymerase chain reaction (PCR). Genomic DNA from 1682 individuals (CD: n = 499; UC: n = 216; controls: n = 967) was analyzed for the presence of the IL17F p. His161Arg polymorphism, the 3 NOD2 variants, p. Arg702Trp, p. Gly908Arg, and p. Leu1007fsX1008, and 10 CD-associated IL23R variants. RESULTS Intestinal IL-17F mRNA expression was 4.4-fold increased in inflamed colonic lesions compared to uninflamed biopsies in CD (P = 0.016) but not in UC. However, the mean intestinal IL-17F mRNA expression was higher in UC than in CD (P < 0.0001). The IL17F p. His161Arg substitution was observed with similar frequencies in IBD patients and controls and was not associated with a certain disease phenotype, but weakly associated with a low body mass index (BMI; P = 0.009) and an earlier age of disease onset (P = 0.039) in UC. There was no evidence for epistasis between the IL17F p. His161Arg polymorphism and IBD-associated single nucleotide polymorphisms within the IL23R gene. CONCLUSIONS Intestinal IL17F gene expression is increased in active CD. The IL17F p. His161Arg polymorphism is not associated with IBD susceptibility and has no epistatic interaction with CD-associated IL23R variants.

Title: Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs. Content: Histone/protein deacetylases (HDACs) decrease histone and protein acetylation, typically leading to suppression of gene transcription and modulation of various protein functions. We found significant differences in expression of HDAC before and after stimulation of human T regulatory (Treg) and T effector cells, suggesting the potential for future selective targeting of Tregs with HDAC inhibitors (HDACi). Use of various HDACi small molecules enhanced, by up to 4.5-fold (average 2-fold), the suppressive functions of both freshly isolated and expanded human Tregs, consistent with our previous murine data. HDACi use increased Treg expression of CTLA-4, a key negative regulator of immune response, and we found a direct and significant correlation between CTLA-4 expression and Treg suppression. Hence, HDACi compounds are promising pharmacologic tools to increase Treg suppressive functions, and this action may potentially be of use in patients with autoimmunity or post-transplantation.

Title: NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions Content: Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

Title: Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function Content: Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.

Title: Diminished sympathetic nervous system activity in genetically obese (ob/ob) mouse. Content: The genetically obese (ob/ob) mouse exhibits defective thermoregulatory responses to cold exposure. Pathophysiological explanations for this phenomenon have focused on abnormalities in intracellular metabolism or insensitivity of peripheral tissues to the thermogenic effects of catecholamines. Because the sympathetic nervous system (SNS) is subject to feedback regulation, a peripheral impairment in thermogenesis should be associated with a compensatory increase in SNS activity. To examine SNS activity in the ob/ob mouse, norepinephrine (NE) turnover was measured in heart and interscapular brown adipose tissue (IBAT) of ob/ob and lean mice. The results from studies utilizing radiolabeled NE or inhibition of NE biosynthesis with alpha-methyl-p-tyrosine to measure NE turnover demonstrated reductions in SNS activity of 33-56% in heart and of 45-73% in IBAT in ob/ob mice at ambient temperature (22 degrees C) compared with measurements in lean controls. During cold exposure (4 degrees C) NE turnover increased in heart and IBAT to a similar extent in both ob/ob and lean mice, but NE turnover rates in heart, and probably in IBAT as well, remained lower in the obese mice than in the lean despite the gradual development of hypothermia in the ob/ob mice during this period. Administration of naltrexone, a long-acting opiate antagonist, failed to reverse the suppression of SNS activity observed in the ob/ob mice. These data indicate that diminished SNS activity in ob/ob mice may be an additional factor contributing to the defective thermogenesis characteristic of these animals.

Title: Increased short- and long-term risk of inflammatory bowel disease after salmonella or campylobacter gastroenteritis. Content: BACKGROUND & AIMS Various commensal enteric and potentially pathogenic bacteria may be involved in the pathogenesis of inflammatory bowel diseases (IBD). We compared the risk of IBD between a cohort of patients with documented Salmonella or Campylobacter gastroenteritis and an age- and gender-matched control group from the same population in Denmark. METHODS We identified 13,324 patients with Salmonella/Campylobacter gastroenteritis from laboratory registries in North Jutland and Aarhus counties, Denmark, from 1991 through 2003, and 26,648 unexposed controls from the same counties. Of these, 176 exposed patients with IBD before the infection, their 352 unexposed controls, and 80 unexposed individuals with IBD before the Salmonella/Campylobacter infection were excluded. The final study cohort of 13,148 exposed and 26,216 unexposed individuals were followed for up to 15 years (mean, 7.5 years). RESULTS A first-time diagnosis of IBD was reported in 107 exposed (1.2%) and 73 unexposed individuals (0.5%). By age, gender, and comorbidity adjusted Cox proportional hazards regression analysis, the hazard ratio (95% confidence interval) for IBD was 2.9 (2.2-3.9) for the whole period and 1.9 (1.4-2.6) if the first year after the Salmonella/Campylobacter infection was excluded. The increased risk in exposed subjects was observed throughout the 15-year observation period. The increased risk was similar for Salmonella (n = 6463) and Campylobacter (n = 6685) and for a first-time diagnosis of Crohn's disease (n = 47) and ulcerative colitis (n = 133). CONCLUSIONS In our population-based cohort study with complete follow-up, an increased risk of IBD was demonstrated in individuals notified in laboratory registries with an episode of Salmonella/Campylobacter gastroenteritis.

Title: Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress Content: During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.

Title: Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. Content: Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4- to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15- and 36-fold at shear stresses greater than approximately 5 dyne/cm2, whereas cyclic stretch had no effect. Prolonged LKLF induction in the absence of flow changed the expression of angiotensin-converting enzyme, endothelin-1, adrenomedullin, and endothelial nitric oxide synthase to levels similar to those observed under prolonged flow. LKLF repression by siRNA suppressed the flow response of endothelin-1, adrenomedullin, and endothelial nitric oxide synthase (P < 0.05). Thus, we demonstrate that endothelial LKLF is regulated by flow in vivo and is a transcriptional regulator of several endothelial genes that control vascular tone in response to flow.

Title: Involvement of ER Stress in Dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 Missense Mutations Shown by iPSC-Derived Oligodendrocytes Content: Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation.

Title: LncRNA-GAS5 induces PTEN expression through inhibiting miR-103 in endometrial cancer cells Content: BACKGROUND Growth arrest-specific 5 (GAS5) was reported to be implicated and aberrantly express in multiple cancers. However, the expression and mechanism of action of GAS5 were largely poor understood in endometrial carcinoma. RESULTS According to the result of real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and flow cytometry analysis, we identified that GAS5 was down-regulated in endometrial cancer cells and stimulated the apoptosis of endometrial cancer cells. To investigate the expression of GAS5, PTEN and miR-103, RT-PCR was performed. And we found that the expression of PTEN was up-regulated when endometrial cancer cells overexpressed GAS5. The prediction of bioinformatics online revealed that GAS5 could bind to miR-103, which was further found to be regulated by GAS5. Finally, we found that miR-103 mimic could decrease the mRNA and protein levels of PTEN through luciferase reporter assay and western blotting, and GAS5 plasmid may reverse this regulation effect in endometrial cancer cells. CONCLUSION In summary, we demonstrate that GAS5 acts as an tumor suppressor lncRNA in endometrial cancer. Through inhibiting the expression of miR-103, GAS5 significantly enhanced the expression of PTEN to promote cancer cell apoptosis, and, thus, could be an important mediator in the pathogenesis of endometrial cancer.

Title: 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress Content: Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.

Title: Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel. Content: The antibacterial peptide microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). Biochemical results indicate that inhibition of transcription occurs at the level of NTP uptake or NTP binding by RNAP. Genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the RNAP secondary channel (also known as the "NTP-uptake channel" or "pore"). Biophysical results indicate that inhibition of transcription involves binding of MccJ25 within the RNAP secondary channel. Molecular modeling indicates that binding of MccJ25 within the RNAP secondary channel obstructs the RNAP secondary channel. We conclude that MccJ25 inhibits transcription by binding within and obstructing the RNAP secondary channel--acting essentially as a "cork in a bottle. " Obstruction of the RNAP secondary channel represents an attractive target for drug discovery.

Title: Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p. Content: Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.

Title: Hmga2 Promotes Neural Stem Cell Self-Renewal in Young but Not Old Mice by Reducing p16Ink4a and p19Arf Expression Content: Stem cells persist throughout life in diverse tissues by undergoing self-renewing divisions. Self-renewal capacity declines with age, partly because of increasing expression of the tumor suppressor p16(Ink4a). We discovered that the Hmga2 transcriptional regulator is highly expressed in fetal neural stem cells but that expression declines with age. This decrease is partly caused by the increasing expression of let-7b microRNA, which is known to target HMGA2. Hmga2-deficient mice show reduced stem cell numbers and self-renewal throughout the central and peripheral nervous systems of fetal and young-adult mice but not old-adult mice. Furthermore, p16(Ink4a) and p19(Arf) expression were increased in Hmga2-deficient fetal and young-adult stem cells, and deletion of p16(Ink4a) and/or p19(Arf) partially restored self-renewal capacity. let-7b overexpression reduced Hmga2 and increased p16(Ink4a)/p19(Arf) expression. Hmga2 thus promotes fetal and young-adult stem cell self-renewal by decreasing p16(Ink4a)/p19(Arf) expression. Changes in let-7 and Hmga2 expression during aging contribute to the decline in neural stem cell function.

Title: Targeting A20 Decreases Glioma Stem Cell Survival and Tumor Growth Content: Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioblastoma stem cells (GSCs). GSCs are regulated by molecular pathways distinct from the bulk tumor that may be useful therapeutic targets. We determined that A20 (TNFAIP3), a regulator of cell survival and the NF-kappaB pathway, is overexpressed in GSCs relative to non-stem glioblastoma cells at both the mRNA and protein levels. To determine the functional significance of A20 in GSCs, we targeted A20 expression with lentiviral-mediated delivery of short hairpin RNA (shRNA). Inhibiting A20 expression decreased GSC growth and survival through mechanisms associated with decreased cell-cycle progression and decreased phosphorylation of p65/RelA. Elevated levels of A20 in GSCs contributed to apoptotic resistance: GSCs were less susceptible to TNFalpha-induced cell death than matched non-stem glioma cells, but A20 knockdown sensitized GSCs to TNFalpha-mediated apoptosis. The decreased survival of GSCs upon A20 knockdown contributed to the reduced ability of these cells to self-renew in primary and secondary neurosphere formation assays. The tumorigenic potential of GSCs was decreased with A20 targeting, resulting in increased survival of mice bearing human glioma xenografts. In silico analysis of a glioma patient genomic database indicates that A20 overexpression and amplification is inversely correlated with survival. Together these data indicate that A20 contributes to glioma maintenance through effects on the glioma stem cell subpopulation. Although inactivating mutations in A20 in lymphoma suggest A20 can act as a tumor suppressor, similar point mutations have not been identified through glioma genomic sequencing: in fact, our data suggest A20 may function as a tumor enhancer in glioma through promotion of GSC survival. A20 anticancer therapies should therefore be viewed with caution as effects will likely differ depending on the tumor type.

Title: Angiopoietin-2 is increased in sepsis and inversely associated with nitric oxide-dependent microvascular reactivity Content: INTRODUCTION Angiopoietin-2 (ang-2), an angiogenic peptide released by endothelial cell Weibel-Palade bodies (WPBs), increases endothelial activation and vascular permeability. Ang-2 is raised in severe sepsis but the mechanisms underlying this are not known. Nitric oxide (NO) inhibits WPB exocytosis, and bioavailability of endothelial NO is decreased in sepsis. We hypothesized that endothelial NO bioavailability would be inversely correlated with ang-2 concentrations in sepsis. METHODS Plasma ang-2, vascular endothelial growth factor (VEGF) and endothelial-active cytokines were assessed in 83 patients with early sepsis and 41 hospital controls, and related to reactive hyperaemia-peripheral arterial tonometry, RH-PAT, a measure of endothelial NO bioavailability. RESULTS Plasma Ang-2 was elevated in sepsis (median [interquartile range (IQR)], ng/ml: severe sepsis 12.4 [8.5-33.4], sepsis without organ failure 6.1 [5.0-10.4], controls 2.7 [2.2-3.6], P < 0.0001). It correlated inversely with RH-PAT (r = -0.38, P < 0.0001) and positively with IL-6 (r = 0.57, P < 0.0001) and degree of organ failure (sequential organ function assessment score) (r = 0.58, P < 0.0001). The correlation of ang-2 with RH-PAT persisted after controlling for sepsis severity. In a longitudinal mixed-effects model, recovery of RH-PAT over time was associated with decline in ang-2. CONCLUSIONS Ang-2 is elevated in proportion to sepsis severity, and inversely correlated with NO-dependent microvascular reactivity. Impaired endothelial NO bioavailability may contribute to increased endothelial cell release of ang-2, endothelial activation and capillary leak. Agents that increase endothelial NO bioavailability or inhibit WPB exocytosis and/or Ang-2 activity may have therapeutic potential in sepsis.

Title: Activator protein-1 (AP-1) DNA binding activity is induced by hydroxyurea in organogenesis stage mouse embryos Content: Hydroxyurea is a potent teratogen; free radical scavengers or antioxidants reduce its teratogenicity. Activator Protein-1 (AP-1) and NF-kappaB are redox-sensitive transcription factors with important roles in normal development and the stress response. This study was designed to determine if exposure to teratogenic doses of hydroxyurea induces oxidative stress and alters gene expression by activating these transcription factors. Pregnant mice were treated with saline or hydroxyurea (400, 500, or 600 mg/kg) on gestation day 9 (GD 9) and killed either on GD 9, 0.5, 3, or 6 h after treatment, to assess oxidative stress and transcription factor activities, or on GD 18, to assess fetal development. Exposure to 400 mg/kg hydroxyurea did not affect the progeny, whereas exposure to 500 or 600 mg/kg resulted in dose-dependent increases in fetal resorptions and malformations, including curly tails, abnormal limbs (oligodactyly, hemimelia, and amelia), and short ribs. Hydroxyurea did not induce oxidative stress, as assessed by the ratio of oxidized to reduced glutathione, nor did it alter NF-kappaB DNA binding activity in the GD 9 conceptus. In contrast, exposure to hydroxyurea at any dose increased AP-1 DNA binding activity in embryos and yolk sacs 0.5 or 3 h after treatment. Hydroxyurea-induced c-Fos heterodimer activity in the embryo peaked 3-4-fold above control at 3 h and remained elevated by 6 h; in contrast, the activity of c-Jun dimers was not altered by drug exposure. A dramatic and region-specific increase in c-Fos immunoreactivity was found in hydroxyurea-treated embryos. The induction of AP-1 DNA binding activity by hydroxyurea represents an early, sensitive marker of the embryonic response to insult.

Title: Effect of lipid restriction on mitochondrial free radical production and oxidative DNA damage. Content: Many studies have shown that caloric restriction (40%) decreases mitochondrial reactive oxygen species (ROS) generation in rodents. Moreover, we have recently found that 7 weeks of 40% protein restriction without strong caloric restriction also decreases ROS production in rat liver. This is interesting since it has been reported that protein restriction can also extend longevity in rodents. In the present study we have investigated the possible role of dietary lipids in the effects of caloric restriction on mitochondrial oxidative stress. Using semipurified diets, the ingestion of lipids in male Wistar rats was decreased by 40% below controls, while the other dietary components were ingested at exactly the same level as in animals fed ad libitum. After 7 weeks of treatment the liver mitochondria of lipid-restricted animals showed significant increases in oxygen consumption with complex I-linked substrates (pyruvate/malate and glutamate/malate). Neither mitochondrial H(2)O(2) production nor oxidative damage to mitochondrial or nuclear DNA was modified in lipid-restricted animals. Oxidative damage to mitochondrial DNA was one order of magnitude higher than that of nuclear DNA in both dietary groups. These results deny a role for lipids and reinforce the possible role of dietary proteins as being responsible for the decrease in mitochondrial ROS production and DNA damage in caloric restriction.

Title: The human long non-coding RNA-RoR is a p53 repressor in response to DNA damage Content: It is well known that upon stress, the level of the tumor suppressor p53 is remarkably elevated. However, despite extensive studies, the underlying mechanism involving important inter-players for stress-induced p53 regulation is still not fully understood. We present evidence that the human lincRNA-RoR (RoR) is a strong negative regulator of p53. Unlike MDM2 that causes p53 degradation through the ubiquitin-proteasome pathway, RoR suppresses p53 translation through direct interaction with the heterogeneous nuclear ribonucleoprotein I (hnRNP I). Importantly, a 28-base RoR sequence carrying hnRNP I binding motifs is essential and sufficient for p53 repression. We further show that RoR inhibits p53-mediated cell cycle arrest and apoptosis. Finally, we demonstrate a RoR-p53 autoregulatory feedback loop where p53 transcriptionally induces RoR expression. Together, these results suggest that the RoR-hnRNP I-p53 axis may constitute an additional surveillance network for the cell to better respond to various stresses.

Title: A myeloid hypoxia-inducible factor 1α-Krüppel-like factor 2 pathway regulates gram-positive endotoxin-mediated sepsis. Content: Although gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of gram-positive endotoxin-mediated sepsis.

Title: Mechanisms of Renal Cell Apoptosis Induced by Cyclosporine A: A Systematic Review of in vitro Studies Content: Background:Chronic cyclosporine A (CsA) nephrotoxicity (CCN) is a major cause of chronic renal dysfunction and has no effective clinical interventions yet. Objective: To reveal the mechanisms of renal cell apoptosis in CCN, we analyzed all in vitro studies of such mechanisms. Methods: We collected all in vitro studies about the mechanisms of renal cell apoptosis induced by CsA in Medline (1966 to July 2010), Embase (1980 to July 2010) and ISI (1986 to July 2010), evaluated their quality according to in vitro standards and extracted data following the PICOS principles and synthesized the data. Results: First,CsA could upregulate Fas and Fas-L expression, increase FADD and apoptosis enzymes (caspase-2, -3, -4, -7, -8, -9 and -10) and downregulate the Bcl-2 and Bcl-xL. Second, CsA could induce oxidative stress and damage the antioxidant defense system. Third, CsA could increase the expression of HERP, GRP78 and CHOP. Fourth, CsA could induce renal cell apoptosis and increase their iNOS and p53 expression in cultured cells. Conclusions: At least four pathways are involved in renal cell apoptosis induced by CsA in different cell species. Caspases might be their final common pathway in vitro. They might all provide potential points for interventions, but these need to be confirmed in vivo.

Title: Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis. Content: Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2. Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

Title: Noxa controls Mule-dependent Mcl-1 ubiquitination through the regulation of the Mcl-1/USP9X interaction. Content: The level of the Mcl-1 pro-survival protein is highly regulated, and the down-regulation of Mcl-1 expression favors the apoptotic process. Mcl-1 physically interacts with different BH3-only proteins; particularly, Noxa is involved in the modulation of Mcl-1 expression. In this study, we demonstrated that Noxa triggers the degradation of Mcl-1 at the mitochondria according to the exclusive location of Noxa at this compartment. The Noxa-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Because the USP9X deubiquitinase was recently demonstrated to be involved in Mcl-1 protein turnover by preventing its degradation through the removal of conjugated ubiquitin, we investigated whether Noxa affected the deubiquitination process. Interestingly, Noxa over-expression caused a decrease in the USP9X/Mcl-1 interaction associated with an increase in the Mcl-1 polyubiquitinated forms. Additionally, Noxa over-expression triggered an increase in the Mule/Mcl-1 interaction in parallel with the decrease in Mule/USP9X complex formation. Taken together, these modifications result in the degradation of Mcl-1 by the proteasome machinery. The implication of Noxa in the regulation of Mcl-1 proteasomal degradation adds complexity to this process, which is governed by multiple interactions.

Title: Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Content: Increases in the intracellular levels of reactive oxygen species (ROS), frequently referred to as oxidative stress, represents a potentially toxic insult which if not counteracted will lead to membrane dysfunction, DNA damage and inactivation of proteins. Chronic oxidative stress has numerous pathological consequences including cancer, arthritis and neurodegenerative disease. Glutathione-associated metabolism is a major mechanism for cellular protection against agents which generate oxidative stress. It is becoming increasingly apparent that the glutathione tripeptide is central to a complex multifaceted detoxification system, where there is substantial inter-dependence between separate component members. Glutathione participates in detoxification at several different levels, and may scavenge free radicals, reduce peroxides or be conjugated with electrophilic compounds. Thus, glutathione provides the cell with multiple defences not only against ROS but also against their toxic products. This article discusses how glutathione biosynthesis, glutathione peroxidases, glutathione S-transferases and glutathione S-conjugate efflux pumps function in an integrated fashion to allow cellular adaption to oxidative stress. Co-ordination of this response is achieved, at least in part, through the antioxidant responsive element (ARE) which is found in the promoters of many of the genes that are inducible by oxidative and chemical stress. Transcriptional activation through this enhancer appears to be mediated by basic leucine zipper transcription factors such as Nrf and small Maf proteins. The nature of the intracellular sensor(s) for ROS and thiol-active chemicals which induce genes through the ARE is described. Gene activation through the ARE appears to account for the enhanced antioxidant and detoxification capacity of normal cells effected by many cancer chemopreventive agents. In certain instances it may also account for acquired resistance of tumours to cancer chemotherapeutic drugs. It is therefore clear that determining the mechanisms involved in regulation of ARE-driven gene expression has enormous medical implications.

Title: Specific interference by ingested dsRNA. Content: A genetic interference phenomenon in the nematode Caenorhabditis elegans has been described in which expression of an individual gene can be specifically reduced by microinjecting a corresponding fragment of double-stranded (ds) RNA. One striking feature of this process is a spreading effect: interference in a broad region of the animal is observed following the injection of dsRNA into the extracellular body cavity. Here we show that C. elegans can respond in a gene-specific manner to dsRNA encountered in the environment. C. elegans normally feed on bacteria, ingesting and grinding them in the pharynx and subsequently absorbing bacterial contents in the gut. We find that Escherichia coli bacteria expressing dsRNAs can confer specific interference effects on the nematode larvae that feed on them.

Title: Increased expression of CYP1A1 and CYP1B1 in ovarian/peritoneal endometriotic lesions. Content: Endometriosis is an estrogen-dependent disease affecting up to 10% of all premenopausal women. There is evidence that different endometriosis sites show distinct local estrogen concentration, which, in turn, might be due to a unique local estrogen metabolism. We aimed to investigate whether there was a site-specific regulation of selected enzymes responsible for the oxidative metabolism of estrogens in biopsy samples and endometrial and endometriotic stromal cells. Cytochrome P450 (CYP) 1A1 and CYP1B1 mRNA and protein expressions in deep-infiltrating (rectal, retossigmoidal, and uterossacral) lesions, superficial (ovarian and peritoneal) lesions, and eutopic and healthy (control) endometrium were evaluated by real-time PCR and western blot. Using a cross-sectional study design with 58 premenopausal women who were not under hormonal treatment, we were able to identify an overall increased CYP1A1 and CYP1B1 mRNA expression in superficial lesions compared with the healthy endometrium. CYP1A1 mRNA expression in superficial lesions was also greater than in the eutopic endometrium. Interestingly, we found a similar pattern of CYP1A1 and CYP1B1 expression in in vitro stromal cells isolated from ovarian lesions (n=3) when compared with stromal cells isolated from either rectum lesions or eutopic endometrium. In contradiction, there was an increased half-life of estradiol (measured by HPLC-MS-MS) in ovarian endometriotic stromal cells compared with paired eutopic stromal endometrial cells. Our results indicate that there is a site-dependent regulation of CYP1A1 and CYP1B1 in ovarian/peritoneal lesions and ovarian endometriotic stromal cells, whereas a slower metabolism is taking place in these cells.

Title: Control of Nutrient Stress-Induced Metabolic Reprogramming by PKCζ in Tumorigenesis Content: Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.

Title: Quercetin attenuates vascular calcification by inhibiting oxidative stress and mitochondrial fission. Content: Vascular calcification is a strong independent predictor of increased cardiovascular morbidity and mortality and has a high prevalence among patients with chronic kidney disease. The present study investigated the effects of quercetin on vascular calcification caused by oxidative stress and abnormal mitochondrial dynamics both in vitro and in vivo. Calcifying vascular smooth muscle cells (VSMCs) treated with inorganic phosphate (Pi) exhibited mitochondrial dysfunction, as demonstrated by decreased mitochondrial potential and ATP production. Disruption of mitochondrial structural integrity was also observed in a rat model of adenine-induced aortic calcification. Increased production of reactive oxygen species, enhanced expression and phosphorylation of Drp1, and excessive mitochondrial fragmentation were also observed in Pi-treated VSMCs. These effects were accompanied by mitochondria-dependent apoptotic events, including release of cytochrome c from the mitochondria into the cytosol and subsequent activation of caspase-3. Quercetin was shown to block Pi-induced apoptosis and calcification of VSMCs by inhibiting oxidative stress and decreasing mitochondrial fission by inhibiting the expression and phosphorylation of Drp1. Quercetin also significantly ameliorated adenine-induced aortic calcification in rats. In summary, our findings suggest that quercetin attenuates calcification by reducing apoptosis of VSMCs by blocking oxidative stress and inhibiting mitochondrial fission.

Title: SATB1 Expression Governs Epigenetic Repression of PD‐1 in Tumor‐Reactive T Cells Content: &NA; Despite the importance of programmed cell death‐1 (PD‐1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT‐rich sequence‐binding protein‐1 (Satb1) restrains PD‐1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40‐fold increase in PD‐1 expression. Tumor‐derived transforming growth factor &bgr; (Tgf‐&bgr;) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1‐NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1‐mediated repression, Satb1‐deficient tumor‐reactive T cells lost effector activity more rapidly than wild‐type lymphocytes at tumor beds expressing PD‐1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor‐infiltrating T cells results in diminished anti‐tumor immunity. Graphical Abstract Figure. No caption available. HighlightsT cell activation increased the expression of Satb1 in mature CD8+ and CD4+ T cellsRecruitment of the NuRD repression complex by Satb1 inhibits expression of Pdcd1In tumors, TGF‐&bgr; inhibits Satb1 expression in T cells, increasing Pdcd1 expressionSatb1−/− T cells express high amounts of PD‐1 and have decreased anti‐tumor activity &NA; Stephen et al. show that the chromatin organizer Satb1 controls expression levels of PD‐1 upon T cell activation through the recruitment of a de‐acetylase complex to regulatory regions of the Pdcd1 gene. Tumor‐derived TGF‐&bgr; dysregulates this pathway, unleashing PD‐1 expression in tumor‐infiltrating T cells and decreasing anti‐tumor immunity.

Title: Enhanced monocyte Fc phagocytosis by a homologue of interleukin-10 encoded by human cytomegalovirus. Content: Human cytomegalovirus (HCMV) expresses several homologues of human interleukin 10 (hIL-10) possessing immunomodulatory properties which may promote viral infection by modulating the function of myeloid cells. We examined the phenotype and phagocytic capability of human monocytes exposed to hIL-10, an HCMV-encoded hIL-10 homologue expressed during the productive phase of infection (cmvIL-10), and a differentially spliced form of cmvIL-10 expressed during latent and productive phases of infection, (LAcmvIL-10). hIL-10 and cmvIL-10 upregulated expression of Fcgamma receptors, stimulated phagocytosis of IgG-opsonised erythrocytes and decreased MHC class II (HLA-DR) expression on purified monocytes within 24 h. In contrast, LAcmvIL-10 decreased HLA-DR expression at later times (48 h and 72 h) but did not increase Fcgamma receptor expression. We conclude that cmvIL-10 promotes differentiation of monocytes towards a pro-phagocytic phenotype and that LAcmvIL-10 does not affect monocytes by the same mechanism as cmvIL-10. The significance of these properties to cytomegalovirus pathogenesis is discussed.

Title: The first gene of the Bacillus subtilis clpC operon, ctsR, encodes a negative regulator of its own operon and other class III heat shock genes. Content: The Bacillus subtilis clpC operon is regulated by two stress induction pathways relying on either sigmaB or a class III stress induction mechanism acting at a sigmaA-like promoter. When the clpC operon was placed under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter, dramatic repression of the natural clpC promoters fused to a lacZ reporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of the clpC operon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of the clpC operon, ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion of ctsR abolished the negative regulation and resulted in high expression of both the clpC operon and the clpP gene under nonstressed conditions. Nevertheless, a further increase in clpC and clpP mRNA levels was observed after heat shock, even in the absence of sigmaB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by the ctsR deletion. Studies with different clpC promoter fragments either fused to the reporter gene bgaB or used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of the clpC operon, as well as other class III heat shock genes, by preventing unstressed transcription from either the sigmaB- or sigmaA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.

Title: MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors. Content: Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-alpha 1 and -2 (Col1a1 and -2). Transforming growth factor beta1 (TGF-beta), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-beta increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, deltaEF1. TGF-beta treatment or short hairpin RNAs targeting deltaEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-beta also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to deltaEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-beta in MMC. TGF-beta treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3' UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with deltaEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-beta and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-beta-induced Col1a2 expression by down-regulating E-box repressors.

Title: Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Content: Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Title: Teratogen-Induced Oxidative Stress Targets Glyceraldehyde-3-Phosphate Dehydrogenase in the Organogenesis Stage Mouse Embryo Content: Exposure during the organogenesis stage of the mouse embryo to the model teratogen, hydroxyurea (HU), induces curly tail and limb malformations. Oxidative stress contributes to the developmental toxicity of HU. Reactive oxygen species (ROS) interact with polyunsaturated bilipid membranes to form α,β-unsaturated reactive aldehydes; 4-hydroxy-2-nonenal (4-HNE), one of the most cytotoxic of these aldehydes, covalently adducts with proteins, lipids, and nucleic acids. The goal of the current study is to determine if HU exposure of CD1 mice on gestation day 9 generates region-specific 4-HNE-protein adducts in the embryo and to identify the proteins targeted. The formation of 4-HNE-protein adducts was elevated in the caudal region of control embryos; HU exposure further increased 4-HNE-protein adduct formation in this area. Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism. The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%. Furthermore, HU exposure induced the nuclear translocation of GAPDH in the caudal region of exposed embryos; this nuclear translocation may be associated with the reactivation of oxidized proteins involved in DNA repair, such as apurinic/apyrimidinic endonuclease-1, and the stimulation of E1A-associated P300 protein/creb-binding protein (p300/CBP) activity, initiating cell death in a p53-dependent pathway. We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

Title: Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice Content: Endogenous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b+ cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b+ cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.

Title: Functional and molecular defects of pancreatic islets in human type 2 diabetes. Content: To shed further light on the primary alterations of insulin secretion in type 2 diabetes and the possible mechanisms involved, we studied several functional and molecular properties of islets isolated from the pancreata of 13 type 2 diabetic and 13 matched nondiabetic cadaveric organ donors. Glucose-stimulated insulin secretion from type 2 diabetic islets was significantly lower than from control islets, whereas arginine- and glibenclamide-stimulated insulin release was less markedly affected. The defects were accompanied by reduced mRNA expression of GLUT1 and -2 and glucokinase and by diminished glucose oxidation. In addition, AMP-activated protein kinase activation was reduced. Furthermore, the expression of insulin was decreased, and that of pancreatic duodenal homeobox-1 (PDX-1) and forkhead box O1 (Foxo-1) was increased. Nitrotyrosine and 8-hydroxy-2'-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment. Accordingly, 24-h exposure to glutathione significantly improved glucose-stimulated insulin release and decreased nitrotyrosine concentration, with partial recovery of insulin mRNA expression. These results provide direct evidence that the defects of insulin secretion in type 2 diabetic islets are associated with multiple islet cell alterations. Most importantly, the current study shows that the functional impairment of type 2 diabetic islets can be, at least in part, reversible. In this regard, it is suggested that reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.

Title: Replication stress activates DNA repair synthesis in mitosis Content: Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress. The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN+) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

Title: Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency III-infected cells. Content: Epstein-Barr virus (EBV) latency III infection converts B lymphocytes into lymphoblastoid cell lines (LCLs) by expressing EBV nuclear and membrane proteins, EBNAs, and latent membrane proteins (LMPs), which regulate transcription through Notch and tumor necrosis factor receptor pathways. The role of NF-kappa B in LMP1 and overall EBV latency III transcriptional effects was investigated by treating LCLs with BAY11-7082 (BAY11). BAY11 rapidly and irreversibly inhibited NF-kappa B, decreased mitochondrial membrane potential, induced apoptosis, and altered LCL gene expression. BAY11 effects were similar to those of an NF-kappa B inhibitor, Delta N-I kappa B alpha, in effecting decreased JNK1 expression and in microarray analyses. More than 80% of array elements that decreased with Delta N-I kappa B alpha expression decreased with BAY11 treatment. Newly identified NF-kappa B-induced, LMP1-induced, and EBV-induced genes included pleckstrin, Jun-B, c-FLIP, CIP4, and I kappa B epsilon. Of 776 significantly changed array elements, 134 were fourfold upregulated in EBV latency III, and 74 were fourfold upregulated with LMP1 expression alone, whereas only 28 were more than fourfold downregulated by EBV latency III. EBV latency III-regulated gene products mediate cell migration (EBI2, CCR7, RGS1, RANTES, MIP1 alpha, MIP1 beta, CXCR5, and RGS13), antigen presentation (major histocompatibility complex proteins and JAW1), mitogen-activated protein kinase pathway (DUSP5 and p62Dok), and interferon (IFN) signaling (IFN-gamma R alpha, IRF-4, and STAT1). Comparison of EBV latency III LCL gene expression to immunoglobulin M (IgM)-stimulated B cells, germinal-center B cells, and germinal-center-derived lymphomas clustered LCLs with IgM-stimulated B cells separately from germinal-center cells or germinal-center lymphoma cells. Expression of IRF-2, AIM1, ASK1, SNF2L2, and components of IFN signaling pathways further distinguished EBV latency III-infected B cells from IgM-stimulated or germinal-center B cells.

Title: β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. Content: Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

Title: Effects of repeated social stress on leukocyte distribution in bone marrow, peripheral blood and spleen Content: Leukocyte trafficking between the various body compartments has an important surveillance function that ensures the detection of antigen and enables the immune system to initiate a rapid and effective response. Repeated social defeat of group-housed male mice induced by daily, acute encounters with an aggressive conspecific substantially altered leukocyte trafficking and led to a gradual redistribution of immune cells in bone marrow, peripheral blood and spleen. Recurrent exposure to the stressor over a period of 2, 4 or 6 consecutive days was associated with cell mobilization and increased myelopoiesis in the bone marrow that was paralleled by an accumulation of neutrophils and monocytes in circulation and spleen. Substantial depletion of B cells in bone marrow and blood was associated with an increase in splenic B cells indicating a redirection of this cell type to the spleen. In contrast, T cells were markedly reduced in these immune compartments. The recruitment of CD11b+ leukocytes (i.e., monocytes/macrophages and neutrophils) from the bone marrow to the spleen might play a critical role in the development of functional glucocorticoid resistance in the murine spleen that was reported in context with repeated social defeat.

Title: Polyphosphate kinase 1, a central node in the stress response network of Mycobacterium tuberculosis, connects the two-component systems MprAB and SenX3-RegX3 and the extracytoplasmic function sigma factor, sigma E. Content: Polyphosphate (poly P) metabolism regulates the stress response in mycobacteria. Here we describe the regulatory architecture of a signal transduction system involving the two-component system (TCS) SenX3-RegX3, the extracytoplasmic function sigma factor sigma E (SigE) and the poly P-synthesizing enzyme polyphosphate kinase 1 (PPK1). The ppk1 promoter of Mycobacterium tuberculosis is activated under phosphate starvation. This is attenuated upon deletion of an imperfect palindrome likely representing a binding site for the response regulator RegX3, a component of the two-component system SenX3-RegX3 that responds to phosphate starvation. Binding of phosphorylated RegX3 to this site was confirmed by electrophoretic mobility shift assay. The activity of the ppk1 promoter was abrogated upon deletion of a putative SigE binding site. Pull-down of SigE from M. tuberculosis lysates of phosphate-starved cells with a biotinylated DNA harbouring the SigE binding site confirmed the likely binding of SigE to the ppk1 promoter. In vitro transcription corroborated the involvement of SigE in ppk1 transcription. Finally, the overexpression of RseA (anti-SigE) attenuated ppk1 expression under phosphate starvation, supporting the role of SigE in ppk1 transcription. The regulatory elements identified in ppk1 transcription in this study, combined with our earlier observation that PPK1 is itself capable of regulating sigE expression via the MprAB TCS, suggest the presence of multiple positive-feedback loops in this signalling circuit. In combination with the sequestering effect of RseA, we hypothesize that this architecture could be linked to bistability in the system that, in turn, could be a key element of persistence in M. tuberculosis.

Title: The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. Content: The differential transcriptional response of Mycobacterium tuberculosis to drugs and growth-inhibitory conditions was monitored to generate a data set of 430 microarray profiles. Unbiased grouping of these profiles independently clustered agents of known mechanism of action accurately and was successful at predicting the mechanism of action of several unknown agents. These predictions were validated biochemically for two agents of previously uncategorized mechanism, pyridoacridones and phenothiazines. Analysis of this data set further revealed 150 underlying clusters of coordinately regulated genes offering the first glimpse at the full metabolic potential of this organism. A signature subset of these gene clusters was sufficient to classify all known agents as to mechanism of action. Transcriptional profiling of both crude and purified natural products can provide critical information on both mechanism and detoxification prior to purification that can be used to guide the drug discovery process. Thus, the transcriptional profile generated by a crude marine natural product recapitulated the mechanistic prediction from the pure active component. The underlying gene clusters further provide fundamental insights into the metabolic response of bacteria to drug-induced stress and provide a rational basis for the selection of critical metabolic targets for screening for new agents with improved activity against this important human pathogen.

Title: Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine Content: Despite growing interest and a recent surge in papers, the role of autophagy in glucose and lipid metabolism is unclear. We produced mice with skeletal muscle–specific deletion of Atg7 (encoding autophagy-related 7). Unexpectedly, these mice showed decreased fat mass and were protected from diet-induced obesity and insulin resistance; this phenotype was accompanied by increased fatty acid oxidation and browning of white adipose tissue (WAT) owing to induction of fibroblast growth factor 21 (Fgf21). Mitochondrial dysfunction induced by autophagy deficiency increased Fgf21 expression through induction of Atf4, a master regulator of the integrated stress response. Mitochondrial respiratory chain inhibitors also induced Fgf21 in an Atf4-dependent manner. We also observed induction of Fgf21, resistance to diet-induced obesity and amelioration of insulin resistance in mice with autophagy deficiency in the liver, another insulin target tissue. These findings suggest that autophagy deficiency and subsequent mitochondrial dysfunction promote Fgf21 expression, a hormone we consequently term a 'mitokine', and together these processes promote protection from diet-induced obesity and insulin resistance.

Title: Flow-Dependent Regulation of Kruppel-Like Factor 2 Is Mediated by MicroRNA-92a. Content: BACKGROUND Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2. METHODS AND RESULTS Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3' untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow. CONCLUSIONS Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

Title: Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells. Content: Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a "lethal tumor micro-environment. " Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a "metabolic" and "mutagenic" motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the "Reverse Warburg Effect"). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use "oxidative stress" in adjacent fibroblasts (i) as an "engine" to fuel their own survival via the stromal production of nutrients and (ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the "field effect" in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to downregulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified and are effectively "contagious"--spread from cell-to-cell like a virus--creating an "oncogenic/mutagenic" field promoting widespread DNA damage.

Title: Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. Content: Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell-wall-associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII-mediated inhibition of translation and degradation of the stable spa mRNA by the double-strand-specific endoribonuclease III (RNase III). The 3' end domain of RNAIII, partially complementary to the 5' part of spa mRNA, efficiently anneals to spa mRNA through an initial loop-loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

Title: Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories Content: Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

Title: Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone. Content: Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 nM rotenone increased mitochondrial superoxide (O2*-) levels and potentiated glutamate-induced cytoplasmic Ca2+ deregulation, the first irreversible stage of necrotic cell death. However, the potent cell-permeant O2*- trap manganese tetrakis (N-ethylpyridinium-2yl) porphyrin failed to prevent the effects of the inhibitor. The bioenergetic consequences of rotenone addition were quantified by monitoring cell respiration. Glutamate activation of NMDA receptors used the full respiratory capacity of the in situ mitochondria, and >80% of the glutamate-stimulated respiration was attributable to increased cellular ATP demand. Rotenone at 20 nM inhibited basal and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated cell respiration and caused respiratory failure in the presence of glutamate. ATP synthase inhibition by oligomycin was also toxic in the presence of glutamate. We conclude that the cell vulnerability in the rotenone model of partial complex I deficiency under these specific conditions is primarily determined by spare respiratory capacity rather than oxidative stress.

Title: Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis. Content: Cu/Zn-superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER-Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER-Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER-Golgi transport by over-expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER-Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

Title: Epigenetic-induced repression of microRNA-205 is associated with MED1 activation and a poorer prognosis in localized prostate cancer Content: Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n=14), when compared with matched normal prostate (n=7). An expanded patient cohort (tumor n=149, matched normal n=30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio=2.005, 95% confidence interval (1.109, 3.625), P=0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.

Title: The effect of cigarette smoking, alcohol consumption and fruit and vegetable consumption on IVF outcomes: a review and presentation of original data Content: BACKGROUND Lifestyle factors including cigarette smoking, alcohol consumption and nutritional habits impact on health, wellness, and the risk of chronic diseases. In the areas of in-vitro fertilization (IVF) and pregnancy, lifestyle factors influence oocyte production, fertilization rates, pregnancy and pregnancy loss, while chronic, low-grade oxidative stress may underlie poor outcomes for some IVF cases. METHODS Here, we review the current literature and present some original, previously unpublished data, obtained from couples attending the PIVET Medical Centre in Western Australia. RESULTS During the study, 80 % of females and 70 % of male partners completed a 1-week diary documenting their smoking, alcohol and fruit and vegetable intake. The subsequent clinical outcomes of their IVF treatment such as quantity of oocytes collected, fertilization rates, pregnancy and pregnancy loss were submitted to multiple regression analysis, in order to investigate the relationship between patients, treatment and the recorded lifestyle factors. Of significance, it was found that male smoking caused an increased risk of pregnancy loss (p = 0.029), while female smoking caused an adverse effect on ovarian reserve. Both alcohol consumption (β = 0.074, p < 0.001) and fruit and vegetable consumption (β = 0.034, p < 0.001) had positive effects on fertilization. CONCLUSION Based on our results and the current literature, there is an important impact of lifestyle factors on IVF clinical outcomes. Currently, there are conflicting results regarding other lifestyle factors such as nutritional habits and alcohol consumption, but it is apparent that chronic oxidative stress induced by lifestyle factors and poor nutritional habits associate with a lower rate of IVF success.

Title: WK175, a novel antitumor agent, decreases the intracellular nicotinamide adenine dinucleotide concentration and induces the apoptotic cascade in human leukemia cells. Content: We recently developed a class of novel antitumor agents that elicit a potent growth-inhibitory response in many tumor cells cultured in vitro. WK175, a member of this class, was chosen as a model compound that showed strong in vitro efficacy. WK175 interferes with the intracellular steady-state level of NAD(+), resulting in a decreased cellular NAD(+) concentration. We found that WK175 induces apoptotic cell death without any DNA-damaging effect. The apoptotic death signaling pathway initiated by WK175 was examined in detail: mitochondrial membrane potential, cytochrome c release, caspase 3 activation, caspase 3 and poly(ADP-ribose) polymerase cleavage, and the appearance of a sub-G(1) cell cycle population were determined in time course studies in THP-1 (a human monocytic leukemia cell line) cells. We found activation of this cascade after 24 h of treatment with 10 nM WK175. Induction of apoptosis was prevented by bongkrekic acid, Z-Asp-Glu-Val-Asp-fluoromethylketone, and Z-Leu-Glu-His-Asp-fluoromethylketone, inhibitors of the mitochondrial permeability transition and of caspase 3 and 9, respectively, but not by Ac-Tyr-Val-Ala-Asp-CHO, a specific caspase 1 inhibitor, suggesting the involvement of the permeability transition pore, caspase 3, and caspase 9 in the WK175-induced apoptotic cascade. These results imply that decreased NAD(+) concentration initiates the apoptotic cascade, resulting in the antitumor effect of WK175.

Title: On the origins of ultra-fine anaphase bridges. Content: Comment on: Chan KL, Palmai-Pallag T, Ying S, Hickson ID. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol 2009; 11:753-60.

Title: A Nexus for Gene Expression—Molecular Mechanisms of Spt5 and NusG in the Three Domains of Life Content: Evolutionary related multisubunit RNA polymerases (RNAPs) transcribe the genomes of all living organisms. Whereas the core subunits of RNAPs are universally conserved in all three domains of life-indicative of a common evolutionary descent-this only applies to one RNAP-associated transcription factor-Spt5, also known as NusG in bacteria. All other factors that aid RNAP during the transcription cycle are specific for the individual domain or only conserved between archaea and eukaryotes. Spt5 and its bacterial homologue NusG regulate gene expression in several ways by (i) modulating transcription processivity and promoter proximal pausing, (ii) coupling transcription and RNA processing or translation, and (iii) recruiting termination factors and thereby silencing laterally transferred DNA and protecting the genome against double-stranded DNA breaks. This review discusses recent discoveries that identify Spt5-like factors as evolutionary conserved nexus for the regulation and coordination of the machineries responsible for information processing in the cell.

Title: The DHR96 nuclear receptor regulates xenobiotic responses in Drosophila. Content: Exposure to xenobiotics such as plant toxins, pollutants, or prescription drugs triggers a defense response, inducing genes that encode key detoxification enzymes. Although xenobiotic responses have been studied in vertebrates, little effort has been made to exploit a simple genetic system for characterizing the molecular basis of this coordinated transcriptional response. We show here that approximately 1000 transcripts are significantly affected by phenobarbital treatment in Drosophila. We also demonstrate that the Drosophila ortholog of the human SXR and CAR xenobiotic receptors, DHR96, plays a role in this response. A DHR96 null mutant displays increased sensitivity to the sedative effects of phenobarbital and the pesticide DDT as well as defects in the expression of many phenobarbital-regulated genes. Metabolic and stress-response genes are also controlled by DHR96, implicating its role in coordinating multiple response pathways. This work establishes a new model system for defining the genetic control of xenobiotic stress responses.

Title: Identification of short-lived long non-coding RNAs as surrogate indicators for chemical stress response. Content: Abiotic and biotic stressors in human cells are often a result of sudden and/or frequent changes in environmental factors. The molecular response to stress involves elaborate modulation of gene expression and is of homeostatic, ecological, and evolutionary importance. Although attention has primarily focused on signaling pathways and protein networks, long non-coding RNAs (ncRNAs) are increasingly involved in the molecular mechanisms associated with responses to cellular stresses. We identified six novel short-lived long ncRNAs (MIR22HG, GABPB-AS1, LINC00152, IDI2-AS1, SNHG15, and FLJ33630) that responded to chemical stressors (cisplatin, cycloheximide, and mercury (II) oxide) in HeLa Tet-off cells. Our results indicate that short-lived long ncRNAs respond to general and specific chemical stressors. The expression levels of the short-lived long ncRNAs were elevated because of prolonged decay rates in response to chemical stressors and interruption of RNA degradation pathways. We propose that these long ncRNAs have the potential to be surrogate indicators of cellular stress responses.

Title: Microarray analysis of pneumococcal gene expression during invasive disease. Content: Streptococcus pneumoniae is a leading cause of invasive bacterial disease. This is the first study to examine the expression of S. pneumoniae genes in vivo by using whole-genome microarrays available from The Institute for Genomic Research. Total RNA was collected from pneumococci isolated from infected blood, infected cerebrospinal fluid, and bacteria attached to a pharyngeal epithelial cell line in vitro. Microarray analysis of pneumococcal genes expressed in these models identified body site-specific patterns of expression for virulence factors, transporters, transcription factors, translation-associated proteins, metabolism, and genes with unknown function. Contributions to virulence predicted for several unknown genes with enhanced expression in vivo were confirmed by insertion duplication mutagenesis and challenge of mice with the mutants. Finally, we cross-referenced our results with previous studies that used signature-tagged mutagenesis and differential fluorescence induction to identify genes that are potentially required by a broad range of pneumococcal strains for invasive disease.

Title: Wedelolactone protects human bronchial epithelial cell injury against cigarette smoke extract-induced oxidant stress and inflammation responses through Nrf2 pathway. Content: Cigarette smoke is the leading cause of the development of various lung diseases including lung cancer through triggering oxidant stress and inflammatory responses which contributed to the lesions of normal human bronchial epithelial (NHBE) cell. Wedelolactone (WEL), a natural compound from Eclipta prostrata L., has been found to possess the inhibitive effects on the proliferation and growth of cancers. In the present study, we investigated the effects of WEL on NHBE cell injury induced by cigarette smoke extract (CSE) in vitro. It showed that the pretreatment WEL (2.5-20μM) resulted in a significant protective effect on 10% CSE-induced cell death in NHBE cells. The pretreatment with WEL dose-dependently and significantly reversed the activities of SOD, CAT, GSH and the level of MDA to normal level. We also found that the protein expression levels of COX-2 and ICAM-1 which are related to inflammatory response were remarkably reduced by WEL compared with 10% CSE treatment. Additionally, WEL also reduced the expressions of antioxidases including NAD(P)H dehydrogenase:Quinone 1 (NQO1) and heme oxygenase-1 (HO-1). Moreover, Nrf2 inhibitor all-trans-retinoic acid (ATRA) decreased remarkably their expressions. These results suggest that WEL protects NHBE cell against CSE-induced injury through modulating Nrf2 pathway. Our study indicates that WEL may be a new potential protective agent against CSE-induced lung injury.

Title: Regulation of autophagy by cytoplasmic p53 Content: Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53−/− cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Title: Regulation of arabinose and xylose metabolism in Escherichia coli. Content: Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.

Title: Blimp-1 attenuates Th1 differentiation by repression of ifng, tbx21, and bcl6 gene expression. Content: T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.

Title: Macrophages induce COX-2 expression in breast cancer cells: role of IL-1β autoamplification. Content: Tumor-associated macrophages and high levels of cyclooxygenase-2 (COX-2) are associated with poor prognosis in breast cancer patients, but their potential interdependence has not been evaluated. The objective of this study was to determine whether macrophages regulate COX-2 expression in breast cancer cells. For this purpose, THP-1 cells were cocultured with HCC1954 breast cancer cells. Coculture led to increased COX-2 expression in the HCC1954 cells and elevated prostaglandin E(2) levels in conditioned media. Similar results were observed when THP-1 cells were incubated with HCC1937 breast cancer cells or when human monocyte-derived macrophages were cocultured with HCC1954 cells. Coculture triggered production of reactive oxygen species (ROS) in HCC1954 cells. COX-2 induction was blocked in cells preincubated with an reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or by silencing p67PHOX, a subunit of NADPH oxidase. ROS production triggered activation of Src and mitogen-activated protein kinases (MAPKs). Blocking Src or MAPK activities or antagonizing the activator protein-1 (AP-1) transcription factor attenuated COX-2 induction in HCC1954 cells. Coculture caused rapid induction of interleukin-1β (IL-1β) in both breast cancer cells and macrophages. Increased IL-1β expression was blocked by an interleukin-1 receptor antagonist (IL-1Ra), suggesting autocrine and paracrine effects. Importantly, macrophage-induced COX-2 expression was blocked in HCC1954 cells preincubated with IL-1Ra or anti-IL-1β IgG. Together, these results indicate that macrophage-mediated induction of COX-2 in breast cancer cells is a consequence of IL-1β-mediated stimulation of ROS→Src→MAPK→AP-1 signaling. IL-1β-dependent induction of COX-2 in breast cancer cells provides a mechanism whereby macrophages contribute to tumor progression and potential therapeutic targets in breast cancer.

Title: The RNA Polymerase “Switch Region” Is a Target for Inhibitors Content: The alpha-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP "switch region"--the hinge that mediates opening and closing of the RNAP active center cleft--to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related alpha-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

Title: Cystine/glutamate exchange regulates metabotropic glutamate receptor presynaptic inhibition of excitatory transmission and vulnerability to cocaine seeking. Content: Withdrawal from chronic cocaine reduces extracellular glutamate levels in the nucleus accumbens by decreasing cystine/glutamate exchange (xc-). Activating xc- with N-acetylcysteine restores extracellular glutamate and prevents cocaine-induced drug seeking. It was hypothesized that the activation of xc- prevents drug seeking by increasing glutamatergic tone on presynaptic group II metabotropic glutamate receptors (mGluR2/3) and thereby inhibiting excitatory transmission. In the first experiment, the capacity of glutamate derived from xc- to regulate excitatory transmission via mGluR2/3 was determined. Physiological levels of cystine (100-300 nm) were restored to acute tissue slices from the nucleus accumbens or prefrontal cortex. Cystine increased glutamate efflux and decreased miniature EPSC (mEPSC) and spontaneous EPSC (sEPSC) frequency as well as evoked EPSC amplitude. These effects of cystine were presynaptic, because there was no change in mEPSC or sEPSC amplitude, and an increase in the evoked EPSC paired-pulse facilitation ratio. The cystine-induced reduction in EPSCs was reversed by blocking either xc- or mGluR2/3. In the second experiment, blocking mGluR2/3 prevented the ability of N-acetylcystine to inhibit the reinstatement of drug seeking in rats trained to self-administer cocaine. These data demonstrate that nonsynaptic glutamate derived from xc- modulates synaptic glutamate release and thereby regulates cocaine-induced drug seeking.