Emergent Strain of Human Adenovirus Endemic in Iowa

We evaluated 76 adenovirus type 7 (Ad7) isolates collected in Iowa from 1992 to 2002 and found that genome type Ad7d2 became increasingly prevalent. By 2002, it had supplanted all other Ad7 genome types. The association of Ad7d2 with severe illness and death calls for heightened public health concern

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

ATP8B1 Gene Expression Is Driven by a Housekeeping-Like Promoter Independent of Bile Acids and Farnesoid X Receptor

BACKGROUND: Mutations in ATP8B1 gene were identified as a cause of low γ-glutamyltranspeptidase cholestasis with variable phenotype, ranging from Progressive Familial Intrahepatic Cholestasis to Benign Recurrent Intrahepatic Cholestasis. However, only the coding region of ATP8B1 has been described. The aim of this research was to explore the regulatory regions, promoter and 5'untranslated region, of the ATP8B1 gene. METHODOLOGY/PRINCIPAL FINDINGS: 5'Rapid Amplification of cDNA Ends using human liver and intestinal tissue was performed to identify the presence of 5' untranslated exons. Expression levels of ATP8B1 transcripts were determined by quantitative reverse-transcription PCR and compared with the non-variable part of ATP8B1. Three putative promoters were examined in vitro using a reporter gene assay and the main promoter was stimulated with chenodeoxycholic acid. Four novel untranslated exons located up to 71 kb upstream of the previously published exon 1 and twelve different splicing variants were found both in the liver and the intestine. Multiple transcription start sites were identified within exon -3 and the proximal promoter upstream of this transcription start site cluster was proven to be an essential regulatory element responsible for 70% of total ATP8B1 transcriptional activity. In vitro analysis demonstrated that the main promoter drives constitutive ATP8B1 gene expression independent of bile acids. CONCLUSIONS/SIGNIFICANCE: The structure of the ATP8B1 gene is complex and the previously published transcription start site is not significant. The basal expression of ATP8B1 is driven by a housekeeping-like promoter located 71 kb upstream of the first protein coding exon

Functional analysis of <i>ATP8B1</i> promoter regions.

<p>HepG2 cells were transiently transfected with luciferase reporter gene constructs containing 12 different fragments of putative <i>ATP8B1</i> promoters. Nine luciferase constructs (Prom 1 to Prom 9) were designed to comprise the putative dominant promoter P3, two constructs covered promoter P1 (Proms 11 and 12) and one covered promoter P2 (Prom 10). The position of the tested fragments are indicated by horizontal double arrow lines. The number in brackets next to the construct name represents its size (bp). Prom 3 and Prom 4 were designed to include/exclude a putative FXR/RXR binding site indicated by black oval. Antisense construct encodes the same region as Prom 5, but in antisense orientation. Putative promoters (P1–P3) are depicted as horizontal thick arrows. Transcriptional activity for each construct was measured in relative light units per second (RLU/s) and corrected for the transfection efficiency using the internal control <i>Renilla</i> pRL-TK expression plasmid. The data shown are calculated from 3–5 independent experiments and related to the pGL3 Basic activity.</p

Putative transcription factor binding sites and conservation of <i>ATP8B1</i> 5′UTR.

<p>(A) Sequence alignment using ClustalW algorithm (<a href="http://www.ebi.ac.uk/Tools/clustalw/" target="_blank">http://www.ebi.ac.uk/Tools/clustalw/</a>). High level of conservation among mouse (M), rat (R) and human (H) genome was detected for Ex −3 and Ex −4 (conserved nucleotides indicated by stars). Putative Sp-1, Ap-2, NFκB transcription factor binding sites were predicted in exonic/promoter region P3. Putative CREB and HNF-4 binding sites were identified within a distal part of promoter P3 corresponding to Ex −4 sequence. Initiator element sequence encompasses the main TSS cluster in Ex −3. Exonic regions are underlined, transcription start sites are indicated by arrows and bold letters and putative transcription factor binding sites by grey boxes. Two upstream ATG are in bold. (B) Sequence of Ex −2 and (C) Ex −1. Exonic region is highlighted in bold. Alu consensus sequences are underlined.</p

<i>ATP8B1</i> promoter activity in cells stimulated with bile acids.

<p>No significant change in activity was detected after stimulation with CDCA. (A) HepG2 cells were transiently transfected with three previously characterised (Fig. 4) <i>ATP8B1</i> promoter gene constructs (Prom 3, 4 and 6) and stimulated with 0, 10, 50 and 100 µM CDCA for 24 hours. All constructs comprise proximal 434 bp-promoter P3, Prom 4 includes putative FXR binding site identified by MatInspector computer analysis software, and Prom 6 represents the largest construct containing 3379 bp of 5′flanking region. (B) HepG2 cells stably expressing rat sodium-taurocholate co-transporting polypeptide (rNtcp) were transiently transfected with constructs Prom 3, 4 and 6 together with 50 ng of pCI_hRXRα and 50 ng pCI_hFXR plasmids and treated with 0, 10 and 25 µM CDCA for 24 hours.</p

Relative expression levels of different splicing forms assessed by qRT-PCR.

<p>Diagram of individually designed probes (for probe and primer sequences see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051650#pone.0051650.s003" target="_blank">Table S1</a>) used to evaluate the expression levels of twelve identified splicing variants of <i>ATP8B1</i> 5′UTR. Tested splicing variants are indicated by Latin numbers on the left, average expression levels for each transcript from normal liver tissues (n = 7) are shown on the right. The expression levels are presented as a relative value normalised to the expression of the protein coding region represented by Ex +1/Ex +2 boundary.</p

Thermodynamic properties of identified 5′UTR isoforms.

<p>The comparison of the 5′UTR length and RNA secondary structure free energy and percentage of minimal free energy (MFE) for all identified <i>ATP8B1</i> 5′UTR isoforms, schematically depicted on the left. Prediction for the most frequent isoforms initiating at Ex −3 was calculated using TSS at position −125 from 3′end of Ex −3. Data in brackets (row 3 and 4) represent data for TSS at position −509. Putative secondary RNA structures predicted using RNAfold web tool (<a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi" target="_blank">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</a>) are summarised in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051650#pone.0051650.s002" target="_blank">Fig. S2</a>.</p

The heterogeneity of 5′UTR of <i>ATP8B1</i> gene.

<p>(A) Length and position of novel untranslated exons of <i>ATP8B1</i> gene. (B) Six identified alternatively spliced variants are indicated by diagonal lines. (C) The existence of two acceptor splice sites CAGCAG (tandem acceptors) at the 5′ boundary of the first translated exon (Ex +1) of <i>ATP8B1</i> allows the generation of two different splice forms for each combination of upstream exons with Ex +1. Thus generated splice forms differ from each other by only three nucleotides CAG and give rise to twelve <i>ATP8B1</i> isoforms in total (D).</p