Claudin-1 Is a p63 Target Gene with a Crucial Role in Epithelial Development

The epidermis of the skin is a self-renewing, stratified epithelium that functions as the interface between the human body and the outer environment, and acts as a barrier to water loss. Components of intercellular junctions, such as Claudins, are critical to maintain tissue integrity and water retention. p63 is a transcription factor essential for proliferation of stem cells and for stratification in epithelia, mutated in human hereditary syndromes characterized by ectodermal dysplasia. Both p63 and Claudin-1 null mice die within few hours from birth due to dehydration from severe skin abnormalities. These observations suggested the possibility that these two genes might be linked in one regulatory pathway with p63 possibly regulating Claudin-1 expression. Here we show that silencing of ΔNp63 in primary mouse keratinocytes results in a marked down-regulation of Claudin-1 expression (−80%). ΔNp63α binds in vivo to the Claudin-1 promoter and activates both the endogenous Claudin-1 gene and a reporter vector containing a –1.4 Kb promoter fragment of the Claudin-1 gene. Accordingly, Claudin-1 expression was absent in the skin of E15.5 p63 null mice and natural p63 mutant proteins, specifically those found in Ankyloblepharon–Ectodermal dysplasia–Clefting (AEC) patients, were indeed altered in their capacity to regulate Claudin-1 transcription. This correlates with deficient Claudin-1 expression in the epidermis of an AEC patient carrying the I537T p63 mutation. Notably, AEC patients display skin fragility similar to what observed in the epidermis of Claudin-1 and p63 null mice. These findings reinforce the hypothesis that these two genes might be linked in a common regulatory pathway and that Claudin-1 may is an important p63 target gene involved in the pathogenesis of ectodermal dysplasias

Molecular, microbiological and clinical characterization of Clostridium difficile isolates from tertiary care hospitals in Colombia

In Colombia, the epidemiology and circulating genotypes of Clostridium difficile have not yet been described. Therefore, we molecularly characterized clinical isolates of C.difficile from patients with suspicion of C.difficile infection (CDI) in three tertiary care hospitals. C.difficile was isolated from stool samples by culture, the presence of A/B toxins were detected by enzyme immunoassay, cytotoxicity was tested by cell culture and the antimicrobial susceptibility determined. After DNA extraction, tcdA, tcdB and binary toxin (CDTa/CDTb) genes were detected by PCR, and PCR-ribotyping performed. From a total of 913 stool samples collected during 2013–2014, 775 were included in the study. The frequency of A/B toxins-positive samples was 9.7% (75/775). A total of 143 isolates of C.difficile were recovered from culture, 110 (76.9%) produced cytotoxic effect in cell culture, 100 (69.9%) were tcdA+/tcdB+, 11 (7.7%) tcdA-/tcdB+, 32 (22.4%) tcdA-/tcdB- and 25 (17.5%) CDTa+/CDTb+. From 37 ribotypes identified, ribotypes 591 (20%), 106 (9%) and 002 (7.9%) were the most prevalent; only one isolate corresponded to ribotype 027, four to ribotype 078 and four were new ribotypes (794,795, 804,805). All isolates were susceptible to vancomycin and metronidazole, while 85% and 7.7% were resistant to clindamycin and moxifloxacin, respectively. By multivariate analysis, significant risk factors associated to CDI were, staying in orthopedic service, exposure to third-generation cephalosporins and staying in an ICU before CDI symptoms; moreover, steroids showed to be a protector factor. These results revealed new C. difficile ribotypes and a high diversity profile circulating in Colombia different from those reported in America and European countries

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection

The AEC mutations subvert p63 transcriptional potential.

<p>A) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for TAp63 mutants were cotransfected (0.025, 0.05, 0.1 µg) (striped, light grey and dark grey bars respectively). B) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for ΔNp63 mutants were cotransfected (0.025, 0.05, 0.1 µg) (striped, light grey and dark grey bars respectively). C) 0.05 µg of TAp63α or TA-AEC518 were transfected either alone or together in order to reproduce the heterozygous state found in AEC patients. D) 0.05 µg of ΔNp63α or ΔN-AEC518 were transfected either alone or together in order to reproduce the heterozygous state found in AEC patients. Cells were lysed after 24 hours and luciferase activity was determined. The basal activity of the reporter plasmids was set to 1. Data are presented as fold activation/repression relative to the sample without effectors. Each bar of the histogram represents the mean of three independent transfection duplicates. Standard deviations are indicated.</p

<i>Cldn-1</i> expression is down-regulated upon siRNA to <i>ΔNp63</i>.

<p>A) Primary mouse keratinocytes were transfected by control siRNA (siGFP), scrambled siRNA (Dharmacon) or <i>ΔNp63</i> specific siRNA (siΔNp63). 24 hours after transfection total RNA was extracted and subjected to RealTime PCR to verify <i>ΔNp63</i> and <i>p53</i> downregulation. Results are expressed as mean expression of three different samples. Standard Deviations (SD) are indicated. B) <i>Cldn-1</i>, <i>3</i> and <i>-10</i> expressions in the same samples as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002715#pone-0002715-g001" target="_blank">Figure 1A</a>. Expression of the three genes was normalized to <i>CyclophilinA</i> expression. SD from three different samples is indicated.</p

The <i>Cldn-1</i> promoter is regulated by the ΔNp63_ isoforms.

<p>A) Schematic representation of the <i>Cldn-1</i> promoter constructs used. White boxes: <i>Sp1</i> binding sites. Black boxes: <i>p53</i> binding sites. B) U2OS cells were transfected with the −1.4 Kb <i>Cldn-1</i> reporter plasmid (0.3 µg) (white bar). Different quantities of expression plasmids for the p63 isoforms and p53 were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). C) U2OS cells were transfected with the deletion constructs of the <i>Cldn-1</i> reporter plasmid (0.3 µg). Different quantities of expression plasmids for the ΔNp63α were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). D) U2OS cells were transfected with the wild- type or the mutated −61 bp promoter constructs (0.3 µg). Different quantities of expression plasmids for the ΔNp63α were cotransfected (0.025, 0.05 and 0.1 µg) (striped, light grey and black bars respectively). Cells were lysed after 24 hours and luciferase activity was determined. The basal activity of the reporter plasmids was set to 1. Data are presented as fold activation/repression relative to the sample without effectors. Each bar of the histogram represents the mean of three independent transfection duplicates. Standard deviations are indicated.</p

ΔΝp63_α functionally interacts <i>in vivo</i> with the <i>Cldn-1</i> gene.

<p>A) Schematic representation of the human, rat and mouse <i>Cldn-1</i> promoter regions. Two regions of high interspecies homology were identified (R1 and R2, black and striped boxes respectively). The degree of homology within R1 and R2 is shown on the right. R3, mapping at −3.5 Kb from the ATG of the <i>Cldn-1</i> gene, did not show any homology and was used as negative control. B) Three amplicons centered on R1, R2 and R3 of the <i>Cldn-1</i> gene were used in semiquantitative PCR amplifications with chromatin extracted from mouse primary keratinocytes, induced with Ca²⁺ for 24 hours, and immunoprecipitated with anti-p63 antibodies (4A4, Santa Cruz Biotech). R1 and R2 were positives with the anti-p63 antibodies while R3 was negative. As positive control, oligonucleotides annealing to the <i>IKKα</i> mouse promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002715#pone.0002715-Marinari1" target="_blank">[25]</a> were used. C) H1299 cells were treated with 20 µM Doxicycline in order to induce ΔNp63α expression. Thirty hours after induction mRNA was extracted from uninduced (-Dox) and induced (+Dox) cells and levels of endogenous <i>Cldn-1</i> and <i>GAPDH</i> were assessed by semiquantitative RT-PCR. D) Expression of the Cldn-1 protein is clearly detected upon ΔNp63α expression in H1299 cells. E) RNA extracted from mouse HL at E10.5, E11.5 and E12.5 (white, grey and black bars respectively) were used to verify <i>ΔNp63</i> and <i>Cldn-1</i> levels of expression.</p

FOCUS 1: a randomized, double-blinded, multicentre, Phase III trial of the efficacy and safety of ceftaroline fosamil versus ceftriaxone in community-acquired pneumonia

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