Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology

Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure

<p>Abstract</p> <p>Background</p> <p>Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation.</p> <p>Methods</p> <p>Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment.</p> <p>Results</p> <p>The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure.</p> <p>Conclusion</p> <p>Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus <it>in vivo</it>.</p

Maternal postnatal depression and child growth: a European cohort study

<p>Abstract</p> <p>Background</p> <p>Previous studies have reported postpartum depression to be associated with both positive and negative effects on early infant growth. This study examined the hypothesis that maternal postnatal depression may be a risk factor for later child growth faltering or overweight.</p> <p>Methods</p> <p>A total of 929 women and their children participating in a European multicenter study were included at a median age of 14 days. Mothers completed the Edinburgh postnatal depression scale (EPDS) at 2, 3 and 6 months after delivery. EPDS scores of 13 and above at any time were defined as maternal depression. Weight, length, triceps and subscapular skinfold thicknesses were measured, and body mass index (BMI) were calculated when the children were two years old and converted to standard deviation scores based on the WHO Multicentre Growth Reference Study (MGRS).</p> <p>Results</p> <p>Z-scores for weight-for-length at inclusion of infants of mothers with high EPDS scores (-0.55, SD 0.74) were lower than of those with normal scores (-0.36, SD 0.74; p = 0.013). BMI at age 24 months did not differ in the high (16.3 kg/m2, SD 1.3) and in the normal EPDS groups (16.2 kg/m2, SD 1.3; p = 0.48). All other anthropometric indices also did not differ between groups, with no change by multivariate adjustment.</p> <p>Conclusions</p> <p>We conclude that a high maternal postnatal depression score does not have any major effects on offspring growth in high income countries.</p

Assessment of Blood Hemodynamics by USPIO-Induced R1 Changes in MRI of Murine Colon Carcinoma

The objective of this study is to assess whether ultrasmall superparamagnetic iron oxide (USPIO)-induced changes of the water proton longitudinal relaxation rate (R1) provide a means to assess blood hemodynamics of tumors. Two types of murine colon tumors (C26a and C38) were investigated prior to and following administration of USPIO blood-pool contrast agent with fast R1 measurements. In a subpopulation of mice, R1 was measured following administration of hydralazine, a well-known blood hemodynamic modifier. USPIO-induced R1 increase in C38 tumors (ΔR1 = 0.072 ± 0.0081 s−1) was significantly larger than in C26a tumors (ΔR1 = 0.032 ± 0.0018 s−1, N = 9, t test, P < 0.001). This was in agreement with the immunohistochemical data that showed higher values of relative vascular area (RVA) in C38 tumors than in C26a tumors (RVA = 0.059 ± 0.015 vs. 0.020 ± 0.011; P < 0.05). Following administration of hydralazine, a decrease in R1 value was observed. This was consistent with the vasoconstriction induced by the steal effect mechanism. In conclusion, R1 changes induced by USPIO are sensitive to tumor vascular morphology and to blood hemodynamics. Thus, R1 measurements following USPIO administration can give novel insight into the effects of blood hemodynamic modifiers, non-invasively and with a high temporal resolution

Histone deacetylases suppress cgg repeat-induced neurodegeneration via transcriptional silencing in models of Fragile X Tremor Ataxia Syndrome

Fragile X Tremor Ataxia Syndrome (FXTAS) is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 59UTR of the fragile X syndrome (FXS) gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11) suppresses CGG repeat-induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat-containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT) inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat-expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.open293

CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp

The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound ∼40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection

Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22–54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ–related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyt

α5β1 Integrin-Mediated Adhesion to Fibronectin Is Required for Axis Elongation and Somitogenesis in Mice

The arginine-glycine-aspartate (RGD) motif in fibronectin (FN) represents the major binding site for α5β1 and αvβ3 integrins. Mice lacking a functional RGD motif in FN (FNRGE/RGE) or α5 integrin develop identical phenotypes characterized by embryonic lethality and a severely shortened posterior trunk with kinked neural tubes. Here we show that the FNRGE/RGE embryos arrest both segmentation and axis elongation. The arrest is evident at about E9.0, corresponding to a stage when gastrulation ceases and the tail bud-derived presomitic mesoderm (PSM) induces α5 integrin expression and assumes axis elongation. At this stage cells of the posterior part of the PSM in wild type embryos are tightly coordinated, express somitic oscillator and cyclic genes required for segmentation, and form a tapered tail bud that extends caudally. In contrast, the posterior PSM cells in FNRGE/RGE embryos lost their tight associations, formed a blunt tail bud unable to extend the body axis, failed to induce the synchronised expression of Notch1 and cyclic genes and cease the formation of new somites. Mechanistically, the interaction of PSM cells with the RGD motif of FN is required for dynamic formation of lamellipodia allowing motility and cell-cell contact formation, as these processes fail when wild type PSM cells are seeded into a FN matrix derived from FNRGE/RGE fibroblasts. Thus, α5β1-mediated adhesion to FN in the PSM regulates the dynamics of membrane protrusions and cell-to-cell communication essential for elongation and segmentation of the body axis

A prebiotic intervention study in children with autism spectrum disorders (ASDs)

Different dietary approaches, such as gluten and casein free diets, or the use of probiotics and prebiotics have been suggested in autistic spectrum disorders in order to reduce gastrointestinal (GI) disturbances. GI symptoms are of particular interest in this population due to prevalence and correlation with the severity of behavioural traits. Nowadays, there is lack of strong evidence about the effect of dietary interventions on these problems, particularly prebiotics. Therefore, we assessed the impact of exclusion diets and a 6-week Bimuno® galactooligosaccharide (B-GOS®) prebiotic intervention in 30 autistic children. RESULTS: The results showed that children on exclusion diets reported significantly lower scores of abdominal pain and bowel movement, as well as lower abundance of Bifidobacterium spp. and Veillonellaceae family, but higher presence of Faecalibacterium prausnitzii and Bacteroides spp. In addition, significant correlations were found between bacterial populations and faecal amino acids in this group, compared to children following an unrestricted diet. Following B-GOS® intervention, we observed improvements in anti-social behaviour, significant increase of Lachnospiraceae family, and significant changes in faecal and urine metabolites. CONCLUSIONS: To our knowledge, this is the first study where the effect of exclusion diets and prebiotics has been evaluated in autism, showing potential beneficial effects. A combined dietary approach resulted in significant changes in gut microbiota composition and metabolism suggesting that multiple interventions might be more relevant for the improvement of these aspects as well as psychological traits

PrognoScan: a new database for meta-analysis of the prognostic value of genes

<p>Abstract</p> <p>Background</p> <p>In cancer research, the association between a gene and clinical outcome suggests the underlying etiology of the disease and consequently can motivate further studies. The recent availability of published cancer microarray datasets with clinical annotation provides the opportunity for linking gene expression to prognosis. However, the data are not easy to access and analyze without an effective analysis platform.</p> <p>Description</p> <p>To take advantage of public resources in full, a database named "PrognoScan" has been developed. This is 1) a large collection of publicly available cancer microarray datasets with clinical annotation, as well as 2) a tool for assessing the biological relationship between gene expression and prognosis. PrognoScan employs the minimum <it>P</it>-value approach for grouping patients for survival analysis that finds the optimal cutpoint in continuous gene expression measurement without prior biological knowledge or assumption and, as a result, enables systematic meta-analysis of multiple datasets.</p> <p>Conclusion</p> <p>PrognoScan provides a powerful platform for evaluating potential tumor markers and therapeutic targets and would accelerate cancer research. The database is publicly accessible at <url>http://gibk21.bse.kyutech.ac.jp/PrognoScan/index.html</url>.</p