Identification of circular RNAs with host gene-independent expression in human model systems for cardiac differentiation and disease.

AIMS: Cardiovascular disease, one of the most common causes of death in western populations, is characterized by changes in RNA splicing and expression. Circular RNAs (circRNA) originate from back-splicing events, which link a downstream 5' splice site to an upstream 3' splice site. Several back-splicing junctions (BSJ) have been described in heart biopsies from human, rat and mouse hearts (Werfel et al., 2016; Jakobi et al., 2016 ). Here, we use human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to identify circRNA and host gene dynamics in cardiac development and disease. In parallel, we explore candidate interactions of selected homologs in mouse and rat via RIP-seq experiments. METHODS AND RESULTS: Deep RNA sequencing of cardiomyocyte development and ß-adrenergic stimulation uncovered 4518 circRNAs. The set of circular RNA host genes is enriched for chromatin modifiers and GTPase activity regulators. RNA-seq and qRT-PCR data showed that circular RNA expression is highly dynamic in the hiPSC-CM model with 320 circRNAs showing significant expression changes. Intriguingly, 82 circRNAs are independently regulated to their host genes. We validated the same circRNA dynamics for circRNAs from ATXN10, CHD7, DNAJC6 and SLC8A1 in biopsy material from human dilated cardiomyopathy (DCM) and control patients. Finally, we could show that rodent homologs of circMYOD, circSLC8A1, circATXN7 and circPHF21A interact with either the ribosome or Argonaute2 protein complexes. CONCLUSION: CircRNAs are dynamically expressed in a hiPSC-CM model of cardiac development and stress response. Some circRNAs show similar, host-gene independent expression dynamics in patient samples and may interact with the ribosome and RISC complex. In summary, the hiPSC-CM model uncovered a new signature of potentially disease relevant circRNAs which may serve as novel therapeutic targets.

Detection of novel skeletogenesis target genes by comprehensive analysis of a Runx2(-/-) mouse model.

Runx2 is an essential factor for skeletogenesis and heterozygous loss causes cleidocranial dysplasia in humans and a corresponding phenotype in the mouse. Homozygous Runx2-deficient mice lack hypertrophic cartilage and bone. We compared the expression profiles of E14.5 wildtype and Runx2(-/-) murine embryonal humeri to identify new transcripts potentially involved in cartilage and bone development. Seventy-one differentially expressed genes were identified by two independent oligonucleotide-microarray hybridizations and quantitative RT-PCR experiments. Gene Ontology analysis demonstrated an enrichment of the differentially regulated genes in annotations to terms such as extracellular, skeletal development, and ossification. In situ hybridization on E15.5 limb sections was performed for all 71 differentially regulated genes. For 54 genes conclusive in situ hybridization results were obtained and all of them showed skeletal expression. Co-expression with Runx2 was demonstrated for 44 genes. While 41 of the 71 differentially expressed genes have a known role in bone and cartilage, we identified 21 known genes that have not yet been implicated in skeletal development and 9 entirely new transcripts. Expression in the developing skeleton was demonstrated for 21 of these genes.

Developments in CORG: a gene-centric comparative genomics resource.

The CORG resource (Comparative Regulatory Genomics, http://corg.eb.tuebingen.mpg.de) provides extensive cross-species comparisons of promoter regions in particular and whole gene loci in general. Pairwise as well as multiple alignments of 10 vertebrate species form the key component of CORG. We implemented a rapid alignment approach based on weight matrix motif anchors to ensure efficient computation and biologically informative alignments. All CORG workbench components have been enhanced towards more flexibility and interactivity. Reference sequence based data presentation and analysis was put into the well-known and modular Generic Genome Browser framework. Herein, various plugins facilitate online data analysis and integration with static conservation data. Main emphasis was put on the design of a new JAVA WebStart application for comparative data display. Flexible data import and export options for standard formats complete the provided services.

Ab initio identification of putative human transcription factor binding sites by comparative genomics.

BACKGROUND: Understanding transcriptional regulation of gene expression is one of the greatest challenges of modern molecular biology. A central role in this mechanism is played by transcription factors, which typically bind to specific, short DNA sequence motifs usually located in the upstream region of the regulated genes. We discuss here a simple and powerful approach for the ab initio identification of these cis-regulatory motifs. The method we present integrates several elements: human-mouse comparison, statistical analysis of genomic sequences and the concept of coregulation. We apply it to a complete scan of the human genome. RESULTS: By using the catalogue of conserved upstream sequences collected in the CORG database we construct sets of genes sharing the same overrepresented motif (short DNA sequence) in their upstream regions both in human and in mouse. We perform this construction for all possible motifs from 5 to 8 nucleotides in length and then filter the resulting sets looking for two types of evidence of coregulation: first, we analyze the Gene Ontology annotation of the genes in the set, searching for statistically significant common annotations; second, we analyze the expression profiles of the genes in the set as measured by microarray experiments, searching for evidence of coexpression. The sets which pass one or both filters are conjectured to contain a significant fraction of coregulated genes, and the upstream motifs characterizing the sets are thus good candidates to be the binding sites of the TF's involved in such regulation. In this way we find various known motifs and also some new candidate binding sites. CONCLUSION: We have discussed a new integrated algorithm for the "ab initio" identification of transcription factor binding sites in the human genome. The method is based on three ingredients: comparative genomics, overrepresentation, different types of coregulation. The method is applied to a full-scan of the human genome, giving satisfactory results.

Exploring potential target genes of signaling pathways by predicting conserved transcription factor binding sites.

Many cellular signaling pathways induce gene expression by activating specific transcription factor complexes. Conventional approaches to the prediction of transcription factor binding sites lead to a notoriously high number of false discoveries. To alleviate this problem, we consider only binding sites that are conserved in man-mouse genomic sequence comparisons. We employ two alternative methods for predicting binding sites: exact matches to validated binding site sequences and weight matrix scans. We then ask the question whether there is a characteristic association between a transcription factor or set thereof to a particular group of genes. Our approach is tested on genes, which are induced in dendritic cells in response to the cells' exposure to LPS. We chose this example because the underlying signaling pathways are well understood. We demonstrate the benefit of conserved predicted binding sites in interpreting the LPS experiment. Additionally, we find that both methods for the prediction of conserved binding sites complement one another. Finally, our results suggest a distinct role for SRF in the context of LPS-induced gene expression.

CORG: a database for COmparative Regulatory Genomics.

Sequence conservation in non-coding, upstream regions of orthologous genes from man and mouse is likely to reflect common regulatory DNA sites. Motivated by this assumption we have delineated a catalogue of conserved non-coding sequence blocks and provide the CORG-'COmparative Regulatory Genomics'-database. The data were computed based on statistically significant local suboptimal alignments of 15 kb regions upstream of the translation start sites of, currently, 10 793 pairs of orthologous genes. The resulting conserved non-coding blocks were annotated with EST matches for easier detection of non-coding mRNA and with hits to known transcription factor binding sites. CORG data are accessible from the ENSEMBL web site via a DAS service as well as a specially developed web service (http://corg.molgen.mpg.de) for query and interactive visualization of the conserved blocks and their annotation.

In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion.

The individual and synergistic contributions of two transcription factors, EFG1 and CPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia by Candida albicans. For this purpose two in vitro reconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown that C. albicans deleted for both transcription factors CPH1 and EFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted for EFG1 alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted for CPH1 showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multi-layered human tissues reconstructed in vitro the individual contributions of Efg1p and Cph1p to two important virulence factors of C. albicans, namely adhesion and invasion, could be defined.

Cystatin C in a rat model of end-stage renal failure.

BACKGROUND: Cystatin C (MW 13kDa) serum concentration reflects glomerular filtration rate better than creatinine. Like other low-molecular weight proteins it is not eliminated by dialysis. Still, cystatin C serum concentrations do not rise progressively in end-stage renal failure and rarely exceed 10 mg/L (i.e. 8 times the upper limit of normal). OBJECTIVE: To study cystatin C kinetics in a rat model of end-stage renal failure. METHODS: Sequential bilateral nephrectomy was performed seven days apart in 13 male Sprague-Dawley rats as described by Levine and Saltzman. Serum cystatin C (Cystatin C PET-kit, DAKO), creatinine and total protein were measured in daily intervals after the second nephrectomy. Linearity of the anti-human cystatin C assay for rat cystatin C was tested using dilutions of uremic rat serum. Rats were sacrificed for signs of severe uremia on days 10 (n=5), 11 (n=4) and 12 (n = 5). RESULTS: At baseline, mean (+/- SE) cystatin C was 1.59+/-0.041 mg/L, creatinine 19.6+/-1.2 micromol/L. Following bilateral nephrectomy, cystatin C immediately rose to 3.82+/-0.15 mg/L, creatinine to 312+/-20 micromol/L. During the following days, cystatin C concentration stabilized to 4 mg/L approximately whereas creatinine continued to rise to 822+/-185 kmol/L on day 12. Correction for the decrease in serum total protein concentration from 48.9+/-2.3 g/L to 37.4+/-3.6 g/L did not alter these results. CONCLUSION: The kinetics of cystatin C and creatinine in this rat model of end-stage renal failure are in accordance with human data suggesting a change in cystatin C production or extra-renal elimination in severe chronic uremia.

Fetal serum concentrations of cystatin C and beta2-microglobulin as predictors of postnatal kidney function.

OBJECTIVES: Cystatin C and beta(2)-microglobulin are established serum markers of renal function in children and adults. In contrast to creatinine, diaplacental exchange is minimal. The aim of the study was to establish reference values in fetal serum and to test their efficiency in predicting postnatal kidney function. STUDY DESIGN: This was a prospective noninterventional study measuring cystatin C and beta(2)-microglobulin by particle-enhanced immunoturbidimetry in excess serum from 129 cordocenteses performed in 84 fetuses. Reference intervals (mean +/- 1.96 SD) were calculated in a subgroup of 54 fetuses without evidence of kidney disease, and these reference values were evaluated in 75 sera from 55 fetuses. RESULTS: Mean cystatin C was 1.66 +/- 0.202 mg/L (upper limit 2.06), and mean beta(2)-microglobulin was 4.25 +/- 0.734 mg/L. Unlike cystatin C, beta(2)-microglobulin decreased significantly with gestational age so that the upper reference limit was 7.19-0.052 x gestational age in weeks. beta(2)-Microglobulin had higher sensitivity (90.0% vs 63.6%) and cystatin C a higher specificity (91.8% vs. 85.5%) for the prediction of impaired renal function; diagnostic efficiency was equal (87.6% vs. 86.1%). Fetuses with impaired renal function at birth or who were aborted for renal malformations had higher cystatin C concentrations than those in a control group. beta(2)-Microglobulin was increased only in fetuses who were aborted. CONCLUSION: Fetal serum cystatin C and beta(2)-microglobulin concentrations may be useful predictors of postnatal kidney function.

Homocysteine--a treatable risk factor for allograft vascular disease after heart transplantation?

Growing evidence suggests that elevated total plasma homocysteine (tHCY) levels are associated with cardiac allograft vasculopathy following heart transplantation. To assess the effect of folic acid supplementation on tHCY levels, we performed a prospective study in a cohort of 69 patients (7.0 +/- 3.2 years after heart transplantation; mean age, 55.0 +/- 9.6 years; 61 male) treated with 5 mg folic acid/day (n = 34) vs no medication (n = 35). Therapy with folic acid resulted in significantly decreased tHCY levels, from 22.6 +/- 9.6 micromol/liter to 17.3 +/- 5.5 micromol/liter (p = 0.001) within 3 months, whereas values in the control group remained unchanged. We conclude that folic acid supplementation (5 mg per day) provides a simple and effective measure to lower elevated tHCY levels in heart transplant recipients.

Increased lipoprotein (a) levels as an independent risk factor for venous thromboembolism.

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P <.001, odds ratio [OR] 3.2, 95% confidence interval [CI], 1.9-5.3). The coexistence of FV G1691A and elevated Lp(a) was significantly more prevalent among patients with VTE than in the control group (7% versus 0.8%; P <.001, OR 9.8, 95% CI, 2.4-40.7). No other established prothrombotic risk factor was found to be significantly combined with increased Lp(a). These data suggest that Lp(a) concentrations greater than 30 mg/dL are a frequent and independent risk factor for VTE. Furthermore, elevated Lp(a) levels might contribute to the penetrance of thromboembolic disease in subjects being affected by other prothrombotic defects, such as FV G1691A mutation.

Cystatin C and creatinine after successful kidney transplantation in children.

BACKGROUND: Serum creatinine is commonly used for the monitoring of allograft function following renal transplantation (RTX). Due to lower muscle mass, creatinine production rate is reduced in children, thus decreasing its sensitivity for the detection of allograft dysfunction. In children, the serum concentration of cystatin C, a low molecular weight protein of 13.3 kDa, reflects glomerular filtration rate independent of age, height and body composition. We, therefore, sought to assess the potential of cystatin C as a marker of allograft function in children. METHODS: Cystatin C and creatinine were measured in parallel at least daily in 24 children (14 boys, 10 girls; mean age 10.5+/-5.1 years) during hospitalization after successful RTX. Cystatin was determined immunoturbidimetrically, creatinine enzymatically. RESULTS: Within one hour after RTX, cystatin C (mean+/-SE) almost halved from 6.69+/-0.45 mg/l to 3.69+/-0.38 mg/l while creatinine declined from 862 +/-65.4 to 633+/-62.9 micromol/l. Following a nadir of 1.82+/-0.18 mg/l on day 2, there was a secondary increase in cystatin C concentrations to 2.69+/-0.35 mg/l on day 10. Creatinine concentrations continued to decline until day 9 reaching 80.5+/-13.1 micromol/l. Day-to-day variation at steady-state was comparable. In the course of 9 acute rejection episodes, both parameters rose in parallel, the increase in creatinine concentration being much greater. CONCLUSION: Cystatin C was an early indicator of allograft function following successful RTX in children. It did not prove superior to creatinine for the recognition of acute allograft dysfunction, however.

Urease-based mucosal immunization against Helicobacter heilmannii infection induces corpus atrophy in mice.

Mucosal immunization with Helicobacter heilmannii urease B or Helicobacter pylori urease, given nasally with cholera toxin, protects BALB/c mice against H. heilmannii infection and significantly reduces a preexisting infection. However, immunization aggravates gastric corpus atrophy. Our results underline the necessity of defining immunization regimens that do not enhance mucosal damage.

Presence of multiple "Helicobacter heilmannii" strains in an individual suffering from ulcers and in his two cats.

Circumstantial evidence suggests that "Helicobacter heilmannii" infection is an example of zoonosis. The presence of "H. heilmannii" strains in a human subject with acute gastric erosions, in his two cats, and in two unrelated cats was analyzed, and the genetic relatedness of the human and feline strains was assessed. A 580-bp, PCR-amplified sequence of "H. heilmannii" urease B gene (ureB) obtained from biopsies from the human subject and his two cats was restricted with AluI and cloned for sequencing. Analysis of the restriction fragment length polymorphism of the ureB-amplified product suggested the presence of different individual "H. heilmannii" strains in the cats and of three distinct strains in the human subject. One of the "H. heilmannii" ureB sequences amplified from the human subject's biopsies was identical to that derived from one of his cats. The degree of similarity between the other "H. heilmannii" human and feline nucleotide sequences was higher than 97%. Most of the base substitutions were conservative. We conclude that human and animal "H. heilmannii" strains are closely related and that humans can be infected by more than one "H. heilmannii" strain, as has been observed for Helicobacter pylori.

Detection and prevalence of Helicobacter infection in pet cats.

The presence of spiral bacteria in the feline stomach has been recognized for over a century, but the identities and degrees of prevalence of such organisms in privately owned cats are still poorly documented. The aims of this study were (i) to adapt different diagnostic tools and evaluate their practicality for diagnosing feline gastric Helicobacter colonization, (ii) to determine the prevalence of gastric Helicobacter-like organisms in pet cats, (iii) to identify the feline species, and (iv) to correlate the presence of a Helicobacter infection with gastritis. Biopsy samples were taken gastroscopically from the antra and the corpora of clinically healthy pet cats. Helicobacter-like organisms were detected by Gram staining, Warthin-Starry staining, and rapid urease testing in biopsy specimens and by [13C]urea breath testing in 79, 77, 78, and 85% of cases, respectively. PCR analysis revealed that 78% of the cats (38 of 49) were infected by Helicobacter heilmannii; however, none of them was harboring Helicobacter pylori or Helicobacter felis. Culture was positive for one cat; the organism was identified as Helicobacter pametensis by dot blot DNA hybridization. By a combination of the detection methods, 91% of the pet cats were found to be Helicobacter positive. For 46 cats (79%) diagnostic tests were concordant. All cats showed mild to moderate gastritis in either the antrum or the corpus, regardless of the presence or density of gastric bacteria. In summary, pet cats are frequently colonized by H. heilmannii without a significant correlation between infection and degree of gastritis.

Development of species-specific enzyme-linked immunosorbent assay for diagnosis of Johne's disease in cattle.

The previously described (M. De Kesel, P. Gilot, M.-C. Misonne, M. Coene, and C. Cocito, J. Clin. Microbiol., 31:947-954, 1993) a362 recombinant polypeptide of Mycobacterium paratuberculosis was used as reagent for an enzyme-linked immunosorbent assay (ELISA). This ELISA, which is endowed with species specificity with respect to the other mycobacteria, was applied to the analysis of bovine paratuberculosis (Johne's disease), an endemic mycobacteriosis of cattle caused by M. paratuberculosis. The distribution of anti-a362 antibodies in the cattle population was analyzed by a computer program (mixture population model) to determine a cutoff value for the test. The prevalence of a362 seropositivity in the Belgian bovine population was estimated to be 12%. The sensitivity of the a362 assay was 70%, as determined with reference sera from the U.S. National Repository of Paratuberculosis Specimens. Some 40% of the animals in the herds with paratuberculosis analyzed were found to be positive by the a362 assay. The latter proved to be 95% specific with respect to both healthy and tuberculous cattle.

Occurrence, in Crohn's disease, of antibodies directed against a species-specific recombinant polypeptide of Mycobacterium paratuberculosis.

Sera from patients with Crohn's disease and control were analyzed by an enzyme-linked immunosorbent assay based on the Mycobacterium paratuberculosis-specific recombinant polypeptide a362. Anti-a362 immunoglobulin G (IgG) (P < 0.05) and IgA (P < 0.001) titers were higher in patients with Crohn's disease than in controls. A monomodal Gaussian distribution of anti-a362 IgA levels were found for controls, and a bimodal distribution was found for patients with Crohn's disease. An M. paratuberculosis etiology is suggested for the 36% of patients with Crohn's disease who had an anti-a362 IgA level higher than that of controls.