Next-Generation Sequencing of Connective Tissue Genes in Patients with Classical Ehlers-Danlos Syndrome

Background: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been described. Material and methods: The study group included 59 patients of Polish origin, diagnosed with cEDS. The analysis was performed on genomic DNA (gDNA) with NGS technology, using an Illumina sequencer. Thirty-five genes related to connective tissue were investigated. The pathogenicity of the detected variants was assessed by VarSome. Results: The NGS of 35 genes revealed variants within the COL5A1, COL5A2, COL1A1, and COL1A2 genes for 30 of the 59 patients investigated. Our panel detected no sequence variations for the remaining 29 patients. Discussion: Next-generation sequencing, with an appropriate multigene panel, showed great potential to assist in the diagnosis of EDS and other connective tissue disorders. Our data also show that not all causative genes giving rise to cEDS have been elucidated yet.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis

The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan.     

Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

Background

Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.

Methodology/Findings

We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.

Conclusion/Significance

Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.     

Comparative analysis of phospholipid composition in blood erythrocytes of various species of mouse-like rodents

Comparative analysis of quantitative phospholipid composition of blood erythrocytes was performed on various species of mouse-like rodents. In erythrocyte phospholipids of common voles there was revealed a relatively low sphingomyelin content, not characteristic of mammals. A hypothesis is put forwards that the unique lipid composition in common vole erythrocytes might be one of adaptation mechanisms for survival of the animals under conditions of pathogenic stress.     

Early myocardial deformation changes associated to isolated obesity: a study based on 3D-wall motion tracking analysis

Obesity is considered as a strong risk factor for cardiovascular morbidity and mortality. 3D-wall motion tracking echocardiography (3D-WMT) provides information regarding different parameters of left ventricular (LV) myocardial deformation. Our aim was to assess the presence of early myocardial deformation abnormalities in nonselected obese children free from other cardiovascular risk factors. Thirty consecutive nonselected obese children and 42 healthy volunteer children were enrolled. None of them had any cardiovascular risk factor. Every subject underwent a 2D-echo examination and a 3D-WMT study. Mean age was 13.9 ± 2.56 and 13.25 ± 2.68 years in the nonobese and obese groups, respectively (59.7% and 40.3% male). Statistically significant differences were found for: interventricular septum thickness, LV posterior wall thickness, LV end-diastolic volume, LV end-systolic volume, left atrium volume, LV mass, and lateral annulus peak velocity. Regarding the results obtained by 3D-WMT assessment, all the evaluated parameters were statistically significantly different between the two groups. When the influence of obesity on the different echocardiographic variables was evaluated by means of multivariate logistic regression analysis, the strongest relationship with obesity was found for LV average circumferential strain (β-coefficient: 0.74; r(2): 0.55; P: 0.003). Thus, obesity cardiomyopathy is associated not only with structural cardiac changes, but also with myocardial deformation changes. Furthermore, this association occurs as early as in the childhood and it is independent from any other cardiovascular risk factor. The most related parameter to obesity is LV circumferential strain.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.