Lack of a 5.9 kDa Peptide C-Terminal Fragment of Fibrinogen α Chain Precedes Fibrosis Progression in Patients with Liver Disease

Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT). We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease.     

Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells: A POTENTIAL ROLE FOR REDUCING LIPID ACCUMULATION*?

Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has beneficial effects in the prevention of cardiovascular disease. Autophagy is a cellular process that protects cells from stressful conditions. To determine whether the beneficial effect of EGCG is mediated by a mechanism involving autophagy, the roles of the EGCG-stimulated autophagy in the context of ectopic lipid accumulation were investigated. Treatment with EGCG increased formation of LC3-II and autophagosomes in primary bovine aortic endothelial cells (BAEC). Activation of calmodulin-dependent protein kinase kinase β was required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation was significantly impaired by knockdown of calmodulin-dependent protein kinase kinase β. This effect is most likely due to cytosolic Ca²⁺ load. To determine whether EGCG affects palmitate-induced lipid accumulation, the effects of EGCG on autophagic flux and co-localization of lipid droplets and autophagolysosomes were examined. EGCG normalized the palmitate-induced impairment of autophagic flux. Accumulation of lipid droplets by palmitate was markedly reduced by EGCG. Blocking autophagosomal degradation opposed the effect of EGCG in ectopic lipid accumulation, suggesting the action of EGCG is through autophagosomal degradation. The mechanism for this could be due to the increased co-localization of lipid droplets and autophagolysosomes. Co-localization of lipid droplets with LC3 and lysosome was dramatically increased when the cells were treated with EGCG and palmitate compared with the cells treated with palmitate alone. Collectively, these findings suggest that EGCG regulates ectopic lipid accumulation through a facilitated autophagic flux and further imply that EGCG may be a potential therapeutic reagent to prevent cardiovascular complications.     

(R)-sila-venlafaxine: a selective noradrenaline reuptake inhibitor for the treatment of emesis

Sila-substitution of drugs (the carbon/silicon switch) is a concept that is being successfully used for the development of new chemical entities. The (R)-sila-analogue of the antidepressant venlafaxine is devoid of the serotonin reuptake inhibition observed with the marketed drug, leading to a selective noradrenaline reuptake inhibitor displaying anti-emetic properties.     

Smchd1-dependent and -independent pathways determine developmental dynamics of CpG island methylation on the inactive x chromosome

X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 was recently shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation?pathway that is required for long-term maintenance of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1-dependent or -independent pathways. Smchd1-dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1-independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ with respect to their sequence characteristics and immediate chromosomal environment. We discuss the implications of these results for understanding CGI methylation during development.     

EGFR signalling is required for Paracentrotus lividus endomesoderm specification

The EGFR pathway is critical for cell fate specification throughout the development of several organisms. Here we identified in sea urchin an EGFR-related antigen maternally expressed and showing a dynamic pattern of localization during development. To investigate the role played by the EGFR in Paracentrotus lividus development we blocked its activity by using the EGFR kinase inhibitor AG1478. This treatment produces decrease of EGFR phosphorylation, and embryos with various defects especially in the endomesoderm territory until to obtain an animalized phenotype. These effects are rescued by the addition of TGF-α, an EGFR ligand. The role played by EGFR-like along the animal/vegetal axis was also detected, after AG1478 treatment, by the extended distribution of HE and decreased nuclearization of β-catenin in vegetal cells. Moreover, inhibition of EGFR-like reduced ERK phosphorylation, necessary for cell fate specification in the micromeres and their derivates. Taken together these results indicate that EGFR-like activity is required both for A/V axis formation and endomesoderm differentiation.     

Association of TNF promoter polymorphisms with type 1 diabetes in the South Croatian population

Type 1 diabetes mellitus (TIDM) is an autoimmune disease characterized by the destruction of pancreatic p cells. Tumor necrosis factor (TNF) is a pleotropic cytokine with potent immunomodulatory and inflammatory activity. Association studies of TNF polymorphisms and type 1 diabetes (TIDM) frequently demonstrated TNF involvement with TIDM. Although TNF may play an important role in the pathogenesis of TIDM, the genetic association of TNF región with the disease has not been conclusive because of the strong linkage disequilibrium with HLA genes. In this study, we examined two TNF promoter variants (rs 1800629 at position -308, and rs361525 at position -238) for TIDM association in 233 patients and 144 controls from the population of South Croatia. A higher frequency of TNF -308 A alíele and also, a more frequent specific -308A -238G haplotype in TIDM patients were observed with a limited significance. However, we did not find strong evidence of association of TNF promoter polymorphisms with TIDM. In order to elucidate the trae contribution of TNF to TIDM susceptibility in our population, more comprehensive studies with HLA adjustment in a larger sample are required.     

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.     

Genetic structure and outcrossing rates in Flourensia cernua (Asteraceae) growing at different densities in the South-western Chihuahuan Desert

BACKGROUNDS AND AIMS: Flourensia cernua is a partially self-incompatible, wind-pollinated shrub that grows in two scrub types of contrasting densities. It was anticipated that differences in plant density would affect the amount of genotype availability, and thus higher outcrossing rates and less genetic differentiation would be found at high-density sites. METHODS: At five high-density sites and at five low-density sites, 11 allozyme loci were analysed in adults. Outcrossing rates were estimated using five allozyme loci sampled from eight families from each scrub type. KEY RESULTS: High levels of genetic variation were found at all sites (ranging from P = 82-100 %, He = 0.33-0.45, and Ho = 0.4-0.59). Heterozygotes were found in excess (FIS = -0.15 +/- 0.06 s.d.), suggesting that natural selection favours heterozygosity, and there was little differentiation between sites (FST = 0.08 +/- 0.02 s.d.). Life history attributes, such as long-lived habit and wide geographic distribution, as well as the presence of a self-incompatibility system may explain these results. Outcrossing rates did not differ from 1.0 in both scrub types, and there was no genetic differentiation between scrub types (FST = -0.01 +/- 0.004 s.d.). CONCLUSIONS: The high rate of outcrossing favoured by partial incompatibility may generate unrestricted gene flow between scrub types and thus may explain the lack of differentiation between them. High heterozygosity could be expected in long-lived plants of arid zones as they confront a variable and stressing environment.     

Maternal Paracentrotus lividus RNAs are differentially localized during the first cell division

In Paracentrotus lividus eggs, there are RNAs localized at the animal and vegetal poles. During the first cell division, some of these RNAs are associated with the mitotic spindle, whereas others are free in the cytoplasm. Among the RNAs bound to mitotic apparatus (MA), we have found the mitochondrial 16S rRNA. By immunohistochemistry we have also detected hsp60, a mitochondrial membrane protein, localized around the MA, suggesting that the entire mitochondria are associated with it. Western blotting of proteins prepared by cellular fractionation after detergent treatment of P. lividus eggs revealed that both hsp60 and cytochrome c are not associated with cytoskeletal elements. All the above data have been confirmed by immunoblot analyses of preparations of microtubules and MA in which the presence of hsp60 and cytochrome c were detected only in the MA fraction. Moreover, mitochondrial succinate dehydrogenase activity was determined in MA and cytoplasm fractions during the first cell division, and the localization and vitality of the organelles were also confirmed by in vivo staining with Mito red. A possible role for mitochondria in the asymmetric distribution of RNAs and in cell division is discussed.     

Localization and association to cytoskeleton of COLL1alpha mRNA in Paracentrotus lividus egg requires cis- and trans-acting factors

COLL1alpha mRNA is asymmetrically distributed in the Paracentrotus lividus egg. Here we examine the involvement of the cytoskeleton in the localization process of collagen mRNA. The use of drugs such as colchicine and cytochalasin B reveals a perturbation of localization collagen mRNA. Moreover, the presence of specific cis-and trans-acting factors involved in cytoskeleton binding and the localization process was investigated. By Northwestern experiment we found that the 3'UTR of COLL1alpha mRNA is also able to bind two proteins of 54 and 40 kDa in a cellular fraction containing the cytoskeleton. Finally, we found that the protein of 54 kDa is LP54, a protein that binds the 3'UTRs of P. lividus maternal bep messengers and is necessary for their localization.