Carriage of stx2a Differentiates Clinical and Bovine-Biased Strains of Escherichia coli O157

Background

Shiga toxin (Stx) are cardinal virulence factors of enterohemorrhagic E. coli O157:H7 (EHEC O157). The gene content and genomic insertion sites of Stx-associated bacteriophages differentiate clinical genotypes of EHEC O157 (CG, typical of clinical isolates) from bovine-biased genotypes (BBG, rarely identified among clinical isolates). This project was designed to identify bacteriophage-mediated differences that may affect the virulence of CG and BBG.

Methods

Stx-associated bacteriophage differences were identified by whole genome optical scans and characterized among >400 EHEC O157 clinical and cattle isolates by PCR.

Results

Optical restriction maps of BBG strains consistently differed from those of CG strains only in the chromosomal insertion sites of Stx2-associated bacteriophages. Multiplex PCRs (stx1, stx2a, and stx2c as well as Stx-associated bacteriophage - chromosomal insertion site junctions) revealed four CG and three BBG that accounted for >90% of isolates. All BBG contained stx2c and Stx2c-associated bacteriophage – sbcB junctions. All CG contained stx2a and Stx2a-associated bacteriophage junctions in wrbA or argW.

Conclusions

Presence or absence of stx2a (or another product encoded by the Stx2a-associated bacteriophage) is a parsimonious explanation for differential virulence of BBG and CG, as reflected in the distributions of these genotypes in humans and in the cattle reservoir.     

Oral administration of GW788388, an inhibitor of transforming growth factor beta signaling, prevents heart fibrosis in Chagas disease

Background

Chagas disease induced by Trypanosoma cruzi (T. cruzi) infection is a major cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are largely inadequate. Transforming Growth Factor beta (TGFß) has been involved in several regulatory steps of T. cruzi invasion and in host tissue fibrosis. {"type":"entrez-nucleotide","attrs":{"text":"GW788388","term_id":"293585730","term_text":"GW788388"}}GW788388 is a new TGFß type I and type II receptor kinase inhibitor that can be orally administered. In the present work, we studied its effects in vivo during the acute phase of experimental Chagas disease.

Methodology/Principal Findings

Male Swiss mice were infected intraperitoneally with 10⁴ trypomastigotes of T. cruzi (Y strain) and evaluated clinically. We found that this compound given once 3 days post infection (dpi) significantly decreased parasitemia, increased survival, improved cardiac electrical conduction as measured by PR interval in electrocardiography, and restored connexin43 expression. We could further show that cardiac fibrosis development, evaluated by collagen type I and fibronectin expression, could be inhibited by this compound. Interestingly, we further demonstrated that administration of {"type":"entrez-nucleotide","attrs":{"text":"GW788388","term_id":"293585730","term_text":"GW788388"}}GW788388 at the end of the acute phase (20 dpi) still significantly increased survival and decreased cardiac fibrosis (evaluated by Masson''s trichrome staining and collagen type I expression), in a stage when parasite growth is no more central to this event.

Conclusion/Significance

This work confirms that inhibition of TGFß signaling pathway can be considered as a potential alternative strategy for the treatment of the symptomatic cardiomyopathy found in the acute and chronic phases of Chagas disease.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

Sensitive and Specific Immunohistochemical Diagnosis of Human Alveolar Echinococcosis with the Monoclonal Antibody Em2G11

Background

Alveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis. Differential diagnosis with cystic echinococcosis (CE) caused by E. granulosus and AE is challenging. We aimed at improving diagnosis of AE on paraffin sections of infected human tissue by immunohistochemical testing of a specific antibody.

Methodology/Principal Findings

We have analysed 96 paraffin archived specimens, including 6 cutting needle biopsies and 3 fine needle aspirates, from patients with suspected AE or CE with the monoclonal antibody (mAb) Em2G11 specific for the Em2 antigen of E. multilocularis metacestodes. In human tissue, staining with mAb Em2G11 is highly specific for E. multilocularis metacestodes while no staining is detected in CE lesions. In addition, the antibody detects small particles of E. multilocularis (spems) of less than 1 µm outside the main lesion in necrotic tissue, liver sinusoids and lymphatic tissue most probably caused by shedding of parasitic material. The conventional histological diagnosis based on haematoxylin and eosin and PAS stainings were in accordance with the immunohistological diagnosis using mAb Em2G11 in 90 of 96 samples. In 6 samples conventional subtype diagnosis of echinococcosis had to be adjusted when revised by immunohistology with mAb Em2G11.

Conclusions/Significance

Immunohistochemistry with the mAb Em2G11 is a new, highly specific and sensitive diagnostic tool for AE. The staining of small particles of E. multilocularis (spems) outside the main lesion including immunocompetent tissue, such as lymph nodes, suggests a systemic effect on the host.     

Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species

Background

The carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO₂ fixation via the Calvin-Benson-Bassham cycle.

Methodology/Principal Findings

To analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into α-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition.

Conclusions/Significance

These results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.     

Social Isolation Impairs Oral Palatal Wound Healing in Sprague-Dawley Rats: A Role for miR-29 and miR-203 via VEGF Suppression

Objective

To investigate the effects of social isolation on oral mucosal healing in rats, and to determine if wound-associated genes and microRNAs (miRNAs) may contribute to this response.

Methods

Rats were group housed or socially isolated for 4 weeks before a 3.5 mm wound was placed on the hard oral palate. Wound closure was assessed daily and tissues were collected for determination of gene expression levels and miRNAs (i.e., miR-29a,b,c and miR-203). The predicted target of these microRNAs (i.e., vascular endothelial growth factor A, VEGFA) was functionally validated.

Results

Social isolation stress delayed the healing process of oral palatal mucosal wounds in rats. Lower mRNA levels of interleukin-1β (IL1β), macrophage inflammatory p r o t e i n-1α (MIP1α), fibroblast growth factor 7 (FGF7), and VEGFA were found in the biopsied tissues of isolated animals on days 1 and/or 3 post-wounding. Intriguingly, the isolated rats persistently exhibited higher levels of miR-29 family members and miR-203. Our results confirmed that VEGFA is a direct target of these miRNAs, as both miR-29a,c and miR-203 strongly and specifically suppressed endogenous VEGFA expression in vitro.

Conclusions

This study in rats demonstrates for the first time that social isolation delays oral mucosal healing, and suggests a potential role for healing-associated gene and miRNA interactions during this process via modulation of VEGF expression.     

Schistosoma mansoni venom allergen like proteins present differential allergic responses in a murine model of airway inflammation

Background

The Schistosoma mansoni Venom-Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in the host-pathogen interaction. Some of these molecules were suggested by us and others as potential immunomodulators and vaccine candidates, due to their functional classification, expression profile and predicted localization. From a vaccine perspective, one of the concerns is the potential allergic effect of these molecules.

Methodology/Principal Findings

Herein, we characterized the putative secreted proteins SmVAL4 and SmVAL26 and explored the mouse model of airway inflammation to investigate their potential allergenic properties. The respective recombinant proteins were obtained in the Pichia pastoris system and the purified proteins used to produce specific antibodies. SmVAL4 protein was revealed to be present only in the cercarial stage, increasing from 0–6 h in the secretions of newly transformed schistosomulum. SmVAL26 was identified only in the egg stage, mainly in the hatched eggs'' fluid and also in the secretions of cultured eggs. Concerning the investigation of the allergic properties of these proteins in the mouse model of airway inflammation, SmVAL4 induced a significant increase in total cells in the bronchoalveolar lavage fluid, mostly due to an increase in eosinophils and macrophages, which correlated with increases in IgG1, IgE and IL-5, characterizing a typical allergic airway inflammation response. High titers of anaphylactic IgG1 were revealed by the Passive Cutaneous Anaphylactic (PCA) hypersensitivity assay. Additionally, in a more conventional protocol of immunization for vaccine trials, rSmVAL4 still induced high levels of IgG1 and IgE.

Conclusions

Our results suggest that members of the SmVAL family do present allergic properties; however, this varies significantly and therefore should be considered in the design of a schistosomiasis vaccine. Additionally, the murine model of airway inflammation proved to be useful in the investigation of allergic properties of potential vaccine candidates.     

Wolfram Syndrome in the Japanese Population; Molecular Analysis of WFS1 Gene and Characterization of Clinical Features

Background

Wolfram syndrome (WFS) is a recessive neurologic and endocrinologic degenerative disorder, and is also known as DIDMOAD (Diabetes Insipidus, early-onset Diabetes Mellitus, progressive Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene (WFS1). However, the phenotypic pleiomorphism, rarity and molecular complexity of this disease complicate our efforts to understand WFS. To address this limitation, we aimed to describe complications and to elucidate the contributions of WFS1 mutations to clinical manifestations in Japanese patients with WFS.

Methodology

The minimal ascertainment criterion for diagnosing WFS was having both early onset diabetes mellitus and bilateral optic atrophy. Genetic analysis for WFS1 was performed by direct sequencing.

Principal Findings

Sixty-seven patients were identified nationally for a prevalence of one per 710,000, with 33 patients (49%) having all 4 components of DIDMOAD. In 40 subjects who agreed to participate in this investigation from 30 unrelated families, the earliest manifestation was DM at a median age of 8.7 years, followed by OA at a median age of 15.8 years. However, either OA or DI was the first diagnosed feature in 6 subjects. In 10, features other than DM predated OA. Twenty-seven patients (67.5%) had a broad spectrum of recessive mutations in WFS1. Two patients had mutations in only one allele. Eleven patients (27.5%) had intact WFS1 alleles. Ages at onset of both DM and OA in patients with recessive WFS1 mutations were indistinguishable from those in patients without WFS1 mutations. In the patients with predicted complete loss-of-function mutations, ages at the onsets of both DM and OA were significantly earlier than those in patients with predicted partial-loss-of function mutations.

Conclusion/Significance

This study emphasizes the clinical and genetic heterogeneity in patients with WFS. Genotype-phenotype correlations may exist in patients with WFS1 mutations, as demonstrated by the disease onset.     

Fusogenic pairings of vesicle-associated membrane proteins (VAMPs) and plasma membrane t-SNAREs--VAMP5 as the exception

Background

Intracellular vesicle fusion is mediated by the interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs). The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments. To fully understand the specific role of each VAMP in vesicle trafficking, it is important to determine if VAMPs have differential membrane fusion activities.

Methodology/Principal Findings

In this study, we developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase, and examined fusogenic pairings between the seven VAMPs, i.e., VAMPs 1, 2, 3, 4, 5, 7 and 8, and two plasma membrane t-SNARE complexes, syntaxin1/SNAP-25 and syntaxin4/SNAP-25. VAMPs 1, 2, 3, 4, 7 and 8 drove fusion efficiently, whereas VAMP5 was unable to mediate fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compared their membrane fusion activities. VAMPs 1 and 3 had comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 exhibited 30–50% lower fusion activities. Moreover, we determined the dependence of cell fusion activity on VAMP1 expression level. Analysis of the dependence data suggested that there was no cooperativity of VAMP proteins in the cell fusion reaction.

Conclusions/Significance

These data indicate that VAMPs have differential membrane fusion capacities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating fusion with plasma membrane t-SNAREs.     

Treatment of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> with 2-bromopalmitate alters morphology,endocytosis, differentiation and infectivity

Background

The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease.

Results

The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC₅₀/48 h value for culture epimastigotes was estimated as 130 μM. The IC₅₀/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC₅₀/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity.

Conclusion

Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.

     

The MASP Family of Trypanosoma cruzi: Changes in Gene Expression and Antigenic Profile during the Acute Phase of Experimental Infection

Background

Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host–parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.

Methods

By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.

Findings and Conclusions

We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.     

Evaluation of pulsed-FRAP and conventional-FRAP for determination of protein mobility in prokaryotic cells

Background

Macromolecule mobility is often quantified with Fluorescence Recovery After Photobleaching (FRAP). Throughout literature a wide range of diffusion coefficients for GFP in the cytoplasm of Escherichia coli (3 to 14 µm²/s) is reported using FRAP-based approaches. In this study, we have evaluated two of these methods: pulsed-FRAP and “conventional”-FRAP.

Principal Findings

To address the question whether the apparent discrepancy in the diffusion data stems from methodological differences or biological variation, we have implemented and compared the two techniques on bacteria grown and handled in the same way. The GFP diffusion coefficients obtained under normal osmotic conditions and upon osmotic upshift were very similar for the different techniques.

Conclusions

Our analyses indicate that the wide range of values reported for the diffusion coefficient of GFP in live cells are due to experimental conditions and/or biological variation rather than methodological differences.     

Schistosoma mansoni Stomatin Like Protein-2 Is Located in the Tegument and Induces Partial Protection against Challenge Infection

Background

Schistosomiasis affects more than 200 million individuals worldwide, with a further 650 million living at risk of infection, constituting a severe health problem in developing countries. Even though an effective treatment exists, it does not prevent re-infection, and the development of an effective vaccine still remains the most desirable means of control for this disease.

Methodology/Principal Findings

Herein, we report the cloning and characterization of a S. mansoni Stomatin-like protein 2 (SmStoLP-2). In silico analysis predicts three putative sites for palmitoylation (Cys11, Cys61 and Cys330), which could contribute to protein membrane association; and a putative mitochondrial targeting sequence, similar to that described for human Stomatin-like protein 2 (HuSLP-2). The protein was detected by Western blot with comparable levels in all stages across the parasite life cycle. Fractionation by differential centrifugation of schistosome tegument suggested that SmStoLP-2 displays a dual targeting to the tegument membranes and mitochondria; additionally, immunolocalization experiments confirm its localization in the tegument of the adult worms and, more importantly, in 7-day-old schistosomula. Analysis of the antibody isotype profile to rSmStoLP-2 in the sera of patients living in endemic areas for schistosomiasis revealed that IgG1, IgG2, IgG3 and IgA antibodies were predominant in sera of individuals resistant to reinfection as compared to those susceptible. Next, immunization of mice with rSmStoLP-2 engendered a 30%–32% reduction in adult worm burden. Protective immunity in mice was associated with specific anti-rSmStoLP-2 IgG1 and IgG2a antibodies and elevated production of IFN-γ and TNF-α, while no IL-4 production was detected, suggesting a Th1-predominant immune response.

Conclusions/Significance

Data presented here demonstrate that SmStoLP-2 is a novel tegument protein located in the host-parasite interface. It is recognized by different subclasses of antibodies in patients resistant and susceptible to reinfection and, based on the data from murine studies, shows protective potential against schistosomiasis. These results indicate that SmStoLP-2 could be useful in a combination vaccine.     

Up-regulation of intestinal vascular endothelial growth factor by Afa/Dr diffusely adhering Escherichia coli

Background

Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn''s disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC).

Methodology

VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors.

Principal Findings

C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways.

Conclusions

Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain and angiogenesis which appeared recently as a novel component of IBD pathogenesis.     

Attomolar detection of botulinum toxin type A in complex biological matrices

Background

A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs.

Methodology/Principal Findings

Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the “gold standard” mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity.

Conclusions/Significance

The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.     

Right Bundle Branch Block: A Predictor of Mortality in Early Systemic Sclerosis

Objective

To evaluate the prognostic significance of baseline electrocardiogram (ECG) abnormalities in a multiethnic cohort of patients with early systemic sclerosis (SSc) and to determine the serological, clinical, and echocardiogram correlates of ECG findings.

Methods

SSc patients with disease duration of≤5 years were enrolled in the GENISOS (Genetics versus Environment in Scleroderma Outcome Study) cohort. At the first visit, a standard 12 lead ECG was obtained along with demographic information, clinical data, and autoantibodies. The results of echocardiograms were also recorded. All ECGs were interpreted by a cardiologist unaware of the patients'' clinical data.

Results

Of 265 SSc patients with average disease duration at enrollment of 2.5 years, 140 (52.8%) had abnormal ECG findings. These findings were not associated with SSc disease type or autoantibody profile but were associated with more severe heart and lung involvement. A total of 75 patients (28.3%) died over a follow up time of 9.9 years. Complete right bundle branch block (± left anterior hemiblock) on ECG, present in 7 (2.6%) patients, predicted a higher risk of mortality (HR: 5.3; 95% CI: 2.1 to 13.4; p<0.001). The predictive significance of right bundle branch block was independent of age at enrollment, gender, ethnicity and risk factors for coronary artery disease.

Conclusion

ECG abnormalities are common in patients with early SSc and are associated with the severity of lung and heart involvement. Right bundle branch block is an independent predictor of mortality, and should be considered a marker of disease severity in SSc.     

Early acquisition of neural crest competence during hESCs neuralization

Background

Neural crest stem cells (NCSCs) are a transient multipotent embryonic cell population that represents a defining characteristic of vertebrates. The neural crest (NC) gives rise to many derivatives including the neurons and glia of the sensory and autonomic ganglia of the peripheral nervous system, enteric neurons and glia, melanocytes, and the cartilaginous, bony and connective tissue of the craniofacial skeleton, cephalic neuroendocrine organs, and some heart vessels.

Methodology/Principal Findings

We present evidence that neural crest (NC) competence can be acquired very early when human embryonic stem cells (hESCs) are selectively neuralized towards dorsal neuroepithelium in the absence of feeder cells in fully defined conditions. When hESC-derived neurospheres are plated on fibronectin, some cells emigrate onto the substrate. These early migratory Neural Crest Stem Cells (emNCSCs) uniformly upregulate Sox10 and vimentin, downregulate N-cadherin, and remodel F-actin, consistent with a transition from neuroepithelium to a mesenchymal NC cell. Over 13% of emNCSCs upregulate CD73, a marker of mesenchymal lineage characteristic of cephalic NC and connexin 43, found on early migratory NC cells. We demonstrated that emNCSCs give rise in vitro to all NC lineages, are multipotent on clonal level, and appropriately respond to developmental factors. We suggest that human emNCSC resemble cephalic NC described in model organisms. Ex vivo emNCSCs can differentiate into neurons in Ret.k^- mouse embryonic gut tissue cultures and transplanted emNCSCs incorporate into NC-derived structures but not CNS tissues in chick embryos.

Conclusions/Significance

These findings will provide a framework for further studying early human NC development including the epithelial to mesenchymal transition during NC delamination.