The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems, it appears to be PmrA-PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA-PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

Oncofetal gene SALL4 in aggressive hepatocellular carcinoma

Hepatocellular carcinoma is the third leading cause of cancer-related deaths worldwide. In the heterogeneous group of hepatocellular carcinomas, those with characteristics of embryonic stem-cell and progenitor-cell gene expression are associated with the worst prognosis. The oncofetal gene SALL4, a marker of a subtype of hepatocellular carcinoma with progenitor-like features, is associated with a poor prognosis and is a potential target for treatment.

Harnessing the complexity of gene expression data from cancer: from single gene to structural pathway methods

High-dimensional gene expression data provide a rich source of information because they capture the expression level of genes in dynamic states that reflect the biological functioning of a cell. For this reason, such data are suitable to reveal systems related properties inside a cell, e.g., in order to elucidate molecular mechanisms of complex diseases like breast or prostate cancer. However, this is not only strongly dependent on the sample size and the correlation structure of a data set, but also on the statistical hypotheses tested. Many different approaches have been developed over the years to analyze gene expression data to (I) identify changes in single genes, (II) identify changes in gene sets or pathways, and (III) identify changes in the correlation structure in pathways. In this paper, we review statistical methods for all three types of approaches, including subtypes, in the context of cancer data and provide links to software implementations and tools and address also the general problem of multiple hypotheses testing. Further, we provide recommendations for the selection of such analysis methods.

Neonatal Pain-Related Stress and NFKBIA Genotype Are Associated with Altered Cortisol Levels in Preterm Boys at School Age

Neonatal pain-related stress is associated with elevated salivary cortisol levels to age 18 months in children born very preterm, compared to full-term, suggesting early programming effects. Importantly, interactions between immune/inflammatory and neuroendocrine systems may underlie programming effects. We examined whether cortisol changes persist to school age, and if common genetic variants in the promoter region of the NFKBIA gene involved in regulation of immune and inflammatory responses, modify the association between early experience and later life stress as indexed by hair cortisol levels, which provide an integrated index of endogenous HPA axis activity. Cortisol was assayed in hair samples from 128 children (83 born preterm =32 weeks gestation and 45 born full-term) without major sensory, motor or cognitive impairments at age 7 years. We found that hair cortisol levels were lower in preterm compared to term-born children. Downregulation of the HPA axis in preterm children without major impairment, seen years after neonatal stress terminated, suggests persistent alteration of stress system programming. Importantly, the etiology was gender-specific such that in preterm boys but not girls, specifically those with the minor allele for NFKBIA rs2233409, lower hair cortisol was associated with greater neonatal pain (number of skin-breaking procedures from birth to term), independent of medical confounders. Moreover, the minor allele (CT or TT) of NFKBIA rs2233409 was associated with higher secretion of inflammatory cytokines, supporting the hypothesis that neonatal pain-related stress may act as a proinflammatory stimulus that induces long-term immune cell activation. These findings are the first evidence that a long-term association between early pain-related stress and cortisol may be mediated by a genetic variants that regulate the activity of NF-?B, suggesting possible involvement of stress/inflammatory mechanisms in HPA programming in boys born very preterm. © 2013 Grunau et al.

Bradykinin-related peptides (BRPs) from skin secretions of three genera of phyllomedusine leaf frogs and their comparative pharmacological effects on mammalian smooth muscles

While bradykinin has been identified in the skin secretions from several species of amphibian, bradykinin-related peptides (BRPs) are more common constituents. These peptides display a plethora of primary structural variations from the type peptide which include single or multiple amino acid substitutions, N- and/or C-terminal extensions and post-translational modifications such as proline hydroxylation and tyrosine sulfation. Such modified peptides have been reported in species from many families, including Bombinatoridae, Hylidae and Ranidae. The spectrum of these peptides in a given species is thought to be reflective of its predator profile from different vertebrate taxa. Here we report the isolation of BRPs and parallel molecular cloning of their respective biosynthetic precursor-encoding cDNAs from the skin secretions of the Mexican leaf frog (Pachymedusa dacnicolor), the Central American red-eyed leaf frog (Agalychnis callidryas) and the South American orange-legged leaf frog (Phyllomedusa hypochondrialis). Additionally, the eight different BRPs identified were chemically synthesized and screened for bioactivity using four different mammalian smooth muscle preparations and their effects and rank potencies were found to be radically different in these with some acting preferentially through bradykinin B1-type receptors and others through B2-type receptors.

Cationic Antimicrobial Peptide Cytotoxicity

Fluorescence microscopy serves as a valuable tool for assessing the structural integrity and viability of eukaryotic cells. Through the use of calcein AM and the DNA stain 4,6-diamidino-2 phenylindole (DAPI), cell viability and membrane integrity can be qualified. Our group has previously shown the ultra-short cationic antimicrobial peptide H-OOWW-NH₂; the amphibian derived 27-mer peptide Maximin-4and the ultra-short lipopeptide C₁₂-OOWW-NH₂ to be effective against a range of bacterial biofilms [1], displaying potential for use in the prevention of medical device-related infections [2]. Analysis of fluorescence micrographs, after staining with calcein AM and DAPI, shows the likely mode of cytotoxic action of cationic antimicrobial peptides and lipopeptides are via directmembrane disruption in eukaryotic cells. Selectivity is towards cidal action against prokaryotic cells, whose membranes are anionic in composition, such as those of bacteria, rather than for neutral zwitterionic membranes of eukaryotic cells. Membrane selectivity is determined by a multitude of physical parameters, particularly charge and hydrophobicity. The charge of the antimicrobial determines the extent of the initial electrostatic interactions with both prokaryotic and eukaryotic membranes, with a larger cationic charge favoring antimicrobial action. Tailoring of these properties is likely to be the key in successfully transferring antimicrobial peptides from laboratory experiments into clinical practice as safe pharmaceutical formulations.

Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog, Odorrana versabilis

One of the most widespread and abundant families of pharmacologically active peptides in amphibian defensive skin secretions is the bradykinins and related peptides. Despite retaining certain primary structural attributes that assign them to this peptide family, bradykinins and related peptides are unique among amphibian skin peptides in that they exhibit a wide range of primary structural variations, post-translational modifications and/or N-terminal or C-terminal extensions. Initially it was believed that their high degree of primary structural heterogeneity was reflective of random gene mutations within species, but latterly, there is an increasing body of evidence that the spectrum of structural modifications found within this peptide family is reflective of the vertebrate predator spectrum of individual species. Here we report the discovery of ornithokinin (avian bradykinin – Thr6, Leu8-bradykinin) in the skin secretion of the Chinese bamboo odorous frog, Odorrana versabilis. Molecular cloning of its biosynthetic precursor-encoding cDNA from a skin secretion-derived cDNA library revealed a deduced open-reading frame of 86 amino acid residues, encoding a single copy of ornithokinin towards its C-terminus. The domain architecture of this ornithokinin precursor protein was consistent with that of a typical amphibian skin peptide and quite different to that of the ornithokininogen from chicken plasma. Ornithokinin was reported to induce hypotension in the chicken and to contract the chicken oviduct but to have no obvious effect on the rat uterus. However, in this study, synthetic ornithokinin was found to contract the rat ileum (EC50 = 539 nM) and to increase contraction frequency in the rat uterus (EC50 = 1.87 μM).

A Burkholderia cenocepacia gene encoding a non-functional tyrosine phosphatase is required for the delayed maturation of the bacteria-containing vacuoles in macrophages

Burkholderia cenocepacia infects patients with cystic fibrosis. We have previously shown that B. cenocepacia can survive in macrophages within membrane vacuoles (BcCVs) that preclude fusion with the lysosome. The bacterial factors involved in B. cenocepacia intracellular survival are not fully elucidated. We report here that deletion of BCAM0628, encoding a predicted low-molecular weight protein tyrosine phosphatase (LMW-PTP) that is restricted to B. cenocepacia strains of the transmissible ET-12 clone, accelerates the maturation of the BcCVs. Compared to parental strain and deletion mutants in other LMW-PTPs that are widely conserved in Burkholderia species, a greater proportion of BcCVs containing the BCAM0628 mutant were targeted to the lysosome. Accelerated BcCV maturation was not due to reduced intracellular viability since BCAM0628 survived and replicated in macrophages similarly to the parental strain. Therefore, BCAM0628 was referred to as dpm (delayed phagosome maturation). We provide evidence that the Dpm protein is secreted during growth in vitro and upon macrophage infection. Dpm secretion requires an N-terminal signal peptide. Heterologous expression of Dpm in B. multivorans confers to this bacterium a similar phagosomal maturation delay as found with B. cenocepacia. We demonstrate that Dpm is an inactive phosphatase, suggesting that its contribution to phagosomal maturation arrest must be unrelated to tyrosine phosphatase activity.

Identification of a collagen type I adhesin of Bacteroides fragilis

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼31 and ∼34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼31 kDa and the ∼34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein. © 2014 Galvão et al.

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10,337 cases and 11,174 controls (OR=1.10; p-value=3.79×10(-5)). Thus, it appears that rare and common variants in a single gene - FBN2 - can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

Development of a method to quantify the DNA content in DNA- cationic peptide nanoparticles

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.

VDR and MEK-ERK dependent induction of the antimicrobial peptide cathelicidin in keratinocytes by lithocholic acid

Cathelicidin is an antimicrobial peptide (AMP) and signaling molecule in innate immunity and a direct target of 1,25-dihydroxyvitamin D3 (1,25D3) in primary human keratinocytes (NHEK). The expression of cathelicidin is dysregulated in various skin diseases and its regulation differs depending on the epithelial cell type. The secondary bile acid lithocholic acid (LCA) is a ligand of the vitamin D receptor (VDR) and can carry out in vivo functions of vitamin D3. Therefore we analyzed cathelicidin mRNA- and peptide expression levels in NHEK and colonic epithelial cells (Caco-2) after stimulation with LCA. We found increased expression of cathelicidin mRNA and peptide in NHEK, in Caco-2 colon cells no effect was observed after LCA stimulation. The VDR as well as MEK-ERK signaled the upregulation of cathelicidin in NHEK induced by LCA. Collectively, our data indicate that cathelicidin induction upon LCA treatment differs in keratinocytes and colonic epithelial cells. Based on these observations LCA-like molecules targeting cathelicidin could be designed for the treatment of cutaneous diseases that are characterized by disturbed cathelicidin expression.

Vitamin D analogs differentially control antimicrobial peptide/"alarmin" expression in psoriasis

Antimicrobial peptides (AMPs) are strongly expressed in lesional skin in psoriasis and play an important role as proinflammatory "alarmins" in this chronic skin disease. Vitamin D analogs like calcipotriol have antipsoriatic effects and might mediate this effect by changing AMP expression. In this study, keratinocytes in lesional psoriatic plaques showed decreased expression of the AMPs beta-defensin (HBD) 2 and HBD3 after topical treatment with calcipotriol. At the same time, calcipotriol normalized the proinflammatory cytokine milieu and decreased interleukin (IL)-17A, IL-17F and IL-8 transcript abundance in lesional psoriatic skin. In contrast, cathelicidin antimicrobial peptide expression was increased by calcipotriol while psoriasin expression remained unchanged. In cultured human epidermal keratinocytes the effect of different vitamin D analogs on the expression of AMPs was further analyzed. All vitamin D analogs tested blocked IL-17A induced HBD2 expression by increasing IkappaB-alpha protein and inhibition of NF-kappaB signaling. At the same time vitamin D analogs induced cathelicidin through activation of the vitamin D receptor and MEK/ERK signaling. These studies suggest that vitamin D analogs differentially alter AMP expression in lesional psoriatic skin and cultured keratinocytes. Balancing AMP "alarmin" expression might be a novel goal in treatment of chronic inflammatory skin diseases.