Accelerated aging phenotype in mice with conditional deficiency for mitochondrial superoxide dismutase in the connective tissue

The free radical theory of aging postulates that the production of mitochondrial reactive oxygen species is the major determinant of aging and lifespan. Its role in aging of the connective tissue has not yet been established, even though the incidence of aging-related disorders in connective tissue-rich organs is high, causing major disability in the elderly. We have now addressed this question experimentally by creating mice with conditional deficiency of the mitochondrial manganese superoxide dismutase in fibroblasts and other mesenchyme-derived cells of connective tissues in all organs. Here, we have shown for the first time that the connective tissue-specific lack of superoxide anion detoxification in the mitochondria results in reduced lifespan and premature onset of aging-related phenotypes such as weight loss, skin atrophy, kyphosis (curvature of the spine), osteoporosis and muscle degeneration in mutant mice. Increase in p16(INK4a) , a robust in vivo marker for fibroblast aging, may contribute to the observed phenotype. This novel model is particularly suited to decipher the underlying mechanisms and to develop hopefully novel connective tissue-specific anti-aging strategies.     

Development and characterization of DehaloR^2, a novel anaerobic microbial consortium performing rapid dechlorination of TCE to ethene

A novel anaerobic consortium, named DehaloR^2, that performs rapid and complete reductive dechlorination of trichloroethene (TCE) to ethene is described. DehaloR^2 was developed from estuarine sediment from the Back River of the Chesapeake Bay and has been stably maintained in the laboratory for over 2 years. Initial sediment microcosms showed incomplete reduction of TCE to DCE with a ratio of trans- to cis- isomers of 1.67. However, complete reduction to ethene was achieved within 10 days after transfer of the consortium to sediment-free media and was accompanied by a shift to cis-DCE as the prevailing intermediate metabolite. The microbial community shifted from dominance of the Proteobacterial phylum in the sediment to Firmicutes and Chloroflexi in DehaloR^2, containing the genera Acetobacterium, Clostridium, and the dechlorinators Dehalococcoides. Also present were Spirochaetes, possible acetogens, and Geobacter which encompass previously described dechlorinators. Rates of TCE to ethene reductive dechlorination reached 2.83 mM Cl⁻ d⁻¹ in batch bottles with a Dehalococcoides sp. density of 1.54E+11 gene copies per liter, comparing favorably to other enrichment cultures described in the literature and identifying DehaloR^2 as a promising consortium for use in bioremediation of chlorinated ethene-impacted environments.     

Syndecan contributes to heart cell specification and lumen formation during Drosophila cardiogenesis

The transmembrane proteoglycan Syndecan contributes to cell surface signaling of diverse ligands in mammals, yet in Drosophila, genetic evidence links Syndecan only to the Slit receptor Roundabout and to the receptor tyrosine phosphatase LAR. Here we characterize the requirement for syndecan in the determination and morphogenesis of the Drosophila heart, and reveal two phases of activity, indicating that Syndecan is a co-factor in at least two signaling events in this tissue. There is a stochastic failure to determine heart cell progenitors in a subset of abdominal hemisegments in embryos mutant for syndecan, and subsequent to Syndecan depletion by RNA interference. This phenotype is sensitive to gene dosage in the FGF receptor (Heartless), its ligand, Pyramus, as well as BMP-ligand Decapentaplegic (Dpp) and co-factor Sara. Syndecan is also required for lumen formation during assembly of the heart vessel, a phenotype shared with mutations in the Slit and Integrin signaling pathways. Phenotypic interactions of syndecan with slit and Integrin mutants suggest intersecting function, consistent with Syndecan acting as a co-receptor for Slit in the Drosophila heart.     

Endocytosis and trafficking of human lactoferrin in macrophage-like human THP-1 cells (1)

Different cell types have been reported to internalize lactoferrin (Lf) by specific or nonspecific receptors. Our studies focused on the endocytic pathway of human Lf in macrophage-like THP-1 cells. Lactoferrin was found to be internalized by THP-1 cells differentiated with phorbol myristate acetate. Incubation of cells with chlorpromazine and dansylcadaverine, inhibitors of clathrin-dependent endocytosis, led to a 50% inhibition of Lf internalization compared with untreated cells. Bafilomycin A1 and NH(4)Cl treatment also resulted in 40%-60% inhibition, respectively, suggesting that the internalization of Lf may partly be mediated by acidic endosome-like organelles. Endocytic uptake of Lf was also cholesterol-dependent, as shown by methyl-β-cyclodextrin or nystatin treatment of the cells prior to internalization. Partial colocalization of Lf and EEA-1, a marker specific for early endosomes, could be observed. Colocalization of Lf with a specific endoplasmic reticulum marker was also detected. Our results suggest that Lf is internalized mainly by the clathrin-dependent pathway in THP-1 cells and targets the ER. The physiological consequences of this intracellular trafficking will be the subject of future investigations.     

Artificial neural networks trained to detect viral and phage structural proteins

Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is available but in vitro propagation of the phage may not be practical or possible.     

Candidate proteomic biomarkers for non-alcoholic fatty liver disease (steatosis and non-alcoholic steatohepatitis) discovered with mass-spectrometry: a systematic review

Context: Non-alcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver which is accompanied by a series of metabolic deregulations. There are sustained research efforts focusing upon biomarker discovery for NAFLD diagnosis and its prognosis in order investigate and follow-up patients as minimally invasive as possible.

Objective: The objective of this study is to critically review proteomic studies that used mass spectrometry techniques and summarize relevant proteomic NAFLD candidate biomarkers.

Methods: Medline and Embase databases were searched from inception to December 2014.

Results: A final number of 22 records were included that identified 251 candidate proteomic biomarkers. Thirty-three biomarkers were confirmed – 14 were found in liver samples, 21 in serum samples, and two from both serum and liver samples.

Conclusion: Some of the biomarkers identified have already been extensively studied regarding their diagnostic and prognostic capacity. However, there are also more potential biomarkers that still need to be addressed in future studies.     

The CARD9 Polymorphisms rs4077515, rs10870077 and rs10781499 Are Uncoupled from Susceptibility to and Severity of Pulmonary Tuberculosis

Genetic variants in the CARD9 gene predispose to inflammatory disorders and chronic infectious diseases. Tuberculosis (TB), a chronic infectious disease affecting the lung, is lethal in Card9-deficient mice. We hypothesized that polymorphisms in the CARD9 gene influence TB progression and disease-associated lung damage in humans. We tested genotype distributions of the CARD9 polymorphisms rs4077515, rs10781499 and rs10870077 in TB patients and healthy subjects in a Caucasian cohort. SNPs were in linkage disequilibrium and none of the haplotypes was significantly enriched in the TB group. We determined total and differential leukocyte count, erythrocyte sedimentation rate and plasma abundance of cytokines and chemokines as markers for systemic inflammation and scored chest X-rays to assess lung involvement in TB subjects. Most disease parameters segregated independently of the CARD9 haplotypes. In contrast to multifactorial chronic inflammation, selected genetic variants in the CARD9 gene leave host responses apparently unaffected in TB, at least in the population analyzed here.