Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ^E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S₄ dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.     

Prion Interaction with the 37-kDa/67-kDa Laminin Receptor on Enterocytes as a Cellular Model for Intestinal Uptake of Prions

Enterocytes, a major cell population of the intestinal epithelium, represent one possible barrier to the entry of prions after oral exposure. We established a cell culture system employing enterocytes from different species to study alimentary prion interaction with the 37-kDa/67-kDa laminin receptor LRP/LR. Human, bovine, porcine, ovine, and cervid enterocytes were cocultured with brain homogenates from cervid, sheep, and cattle suffering from chronic wasting disease (CWD), scrapie, and bovine spongiform encephalopathy (BSE), respectively. PrP^CWD, ovine PrP^Sc, and PrP^BSE all colocalized with LRP/LR on human enterocytes. PrP^CWD failed to colocalize with LRP/LR on bovine, porcine, and ovine enterocytes. Ovine PrP^Sc colocalized with the receptor on bovine enterocytes, but failed to colocalize with LRP/LR on cervid and porcine enterocytes. PrP^BSE failed to colocalize with the receptor on cervid and ovine enterocytes. These data suggest possible oral transmissibility of CWD and sheep scrapie to humans and may confirm the oral transmissibility of BSE to humans, resulting in zoonotic variant Creutzfeldt-Jakob disease. CWD might not be transmissible to cattle, pigs, and sheep. Sheep scrapie might have caused BSE, but may not cause transmissible spongiform encephalopathy in cervids and pigs. BSE may not be transmissible to cervids. Our data recommend the enterocyte model system for further investigations of the intestinal pathophysiology of alimentary prion infections.     

Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.     

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo.     

Regulating Wnt signaling： a strategy to prevent neurodegeneration and induce regeneration

During the past three decades, Wingtess/Int （Wnt）signaling has emerged as an essential regu{ator crucial for neuronal development and maintenance （Inestrosa and Arenas, 201_0）. In addition, Wnt signal- ing was recently shown to be involved in the regula- tion of synaptic function and plasticity, which is critical for learning and memory （Oliva et aL, 2013）. Deregulation of Wnt signaling has been proposed as a key contributor to the pathogenesis of neurode- generative disorders including Alzheimer＇s disease （AD） and Parkinson＇s disease （PD）. This increasing knowledge of the specific roles of Wnt signaling cascades during different stages of life has suggested innovative therapeutic strategies for the treatment of neurodegenerative diseases.     

Loss of phylogenetic and functional originalities of woody cerrado species in simulated extinction scenarios

Originality measures how different a given species is from all other co‐occurring species regarding either their phylogenetic history or functional traits. Since it is important to preserve the various aspects of diversity and original species carry more phylogenetic or functional information, originality may be used to assign conservation priorities. Our goal was to evaluate the relationships between phylogenetic and functional originalities, and their simulated losses under extinction scenarios based on abundance, fire tolerance and habitat preference. We placed 100 plots in a cerrado reserve located in central Brazil, sampled all woody plants species within the plots, measured 14 functional traits and measured fire history. We assembled a phylogenetic tree and a functional dendrogram, with which we calculated the originalities. Phylogenetic‐ and functional‐based originalities were correlated. However, the loss of functional originality was different from random extinctions on the abundance and fire tolerance scenarios, whereas the loss of phylogenetic originality was not. When compared with phylogenetic originality, functional originality brought more information to be used in conservation strategies because it was sensitive to differences in species abundance and fire tolerance. Thus, the extinction of rare or fire‐sensitive species would result in important functional changes due to loss of distinctive traits.     

Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.     

Accurate genome relative abundance estimation based on shotgun metagenomic reads

Accurate estimation of microbial community composition based on metagenomic sequencing data is fundamental for subsequent metagenomics analysis. Prevalent estimation methods are mainly based on directly summarizing alignment results or its variants; often result in biased and/or unstable estimates. We have developed a unified probabilistic framework (named GRAMMy) by explicitly modeling read assignment ambiguities, genome size biases and read distributions along the genomes. Maximum likelihood method is employed to compute Genome Relative Abundance of microbial communities using the Mixture Model theory (GRAMMy). GRAMMy has been demonstrated to give estimates that are accurate and robust across both simulated and real read benchmark datasets. We applied GRAMMy to a collection of 34 metagenomic read sets from four metagenomics projects and identified 99 frequent species (minimally 0.5% abundant in at least 50% of the data-sets) in the human gut samples. Our results show substantial improvements over previous studies, such as adjusting the over-estimated abundance for Bacteroides species for human gut samples, by providing a new reference-based strategy for metagenomic sample comparisons. GRAMMy can be used flexibly with many read assignment tools (mapping, alignment or composition-based) even with low-sensitivity mapping results from huge short-read datasets. It will be increasingly useful as an accurate and robust tool for abundance estimation with the growing size of read sets and the expanding database of reference genomes.     

Pretreatment Antibiotic Resistance in Helicobacter pylori Infection: Results of Three Randomized Controlled Studies

Background. Although combinations of antibiotics and antisecretory drugs are useful for treatment of Helicobacter pylori infection, treatment failure is common. The aim of this study was to evaluate the relation between pretreatment antibiotic resistance and outcome by using six different treatment regimens for H. pylori infection. Patients and Methods. Three hundred sixty-nine consecutive H. pylori–infected patients with dyspeptic symptoms were enrolled in three consecutive randomized, controlled, single-center clinical trials: trial A, 128 patients; trial B, 125 patients; trial C, 116 patients. Treatments consisted of (A) a 15-day course of dual therapy (omeprazole, 20 mg bid, and amoxicillin, 1 gm bid, or clarithromycin, 500 mg tid) (OA vs OC); (B) a 7-day triple therapy of omeprazole, 20 mg bid, plus metronidazole, 500 mg bid, and amoxicillin, 1,000 mg bid, or clarithromycin, 500 mg tid (OMA vs OMC); or (C) omeprazole, 20 mg bid, plus metronidazole, 500 mg bid, plus tetracycline, 500 mg qid, or doxycycline, 100 mg tid (OMT vs OMD). Diagnostic endoscopy was made in all patients before and 5 to 6 weeks after therapy. Six biopsies were taken from each patient for histology, rapid urease test, and H. pylori culture; antibiotic susceptibility testing was performed using the E-test method. Results. Overall cure rates were poor for both dual therapies OA and OC (38% and 37%, respectively) and for triple therapies OMA, OMC, and OMD (57%, 55%, and 58%, respectively). The OMT combination was successful in 91% (95% confidence interval [CI], 80.4%–97%). Metronidazole resistance was present in 29.7% (95% CI, 24%–35%), amoxicillin resistance was present in 26% (95% CI, 21%–32%), clarithromycin resistance was present in 23.1% (95% CI, 18%–29%), tetracycline resistance was present in 14% (95% CI, 10%–20%), and doxycycline resistance was present in 33.3% (95% CI, 21%–47%). Antibiotic resistance markedly reduced the cure rates and accounted for most of the poor results with the triple therapies: 89% versus 23%; 77% versus 26%; 100% versus 60%; and 67% versus 23% for OMC, OMA, OMT, and OMD, respectively. OMT appeared to be the best because of the high success rate with metronidazole-resistant H. pylori (71%) and in low-level tetracycline resistance. Conclusions. Pretreatment antibiotic-resistant H. pylori can, in part, explain the low cure rate of the infection and the variability in outcome in reported trials.