Acute exposure to long-chain fatty acids impairs {alpha}2-adrenergic receptor-mediated antilipolysis in human adipose tissue

The acute in vitro and in vivo effects of long-chain fatty acids (LCFAs) on the regulation of adrenergic lipolysis were investigated in human adipose tissue. The effect of a 2 h incubation, without or with LCFA (200 mumol/l), on basal and hormonally induced lipolysis was tested in vitro on isolated fat cells. The lipolytic response to epinephrine was enhanced by suppression of the antilipolytic alpha(2)-adrenergic effect. Then, healthy lean and obese male subjects performed a 45 min exercise bout at 50% of their heart rate reserve either after an overnight fast or 3 h after a high-fat meal (HFM: 95% fat, 5% carbohydrates). Subcutaneous adipose tissue lipolysis was measured by microdialysis in the presence or absence of an alpha-antagonist (phentolamine). In vivo, a HFM increased plasma levels of nonesterified fatty acids in lean and obese subjects. In both groups, the HFM did not alter hormonal responses to exercise. Under fasting conditions, the alpha(2)-adrenergic antilipolytic effect was more pronounced in obese than in lean subjects. The HFM totally suppressed the alpha(2)-adrenergic antilipolytic effect in lean and obese subjects during exercise. LCFAs per se, in vitro as well as in vivo, suppress alpha(2)-adrenergic-mediated antilipolysis in adipose tissue. LCFA-mediated suppression of antilipolytic pathways represents another mechanism whereby a high fat content in the diet might increase adipose tissue lipolysis.     

The lipid droplet binding domain of hepatitis C virus core protein is a major determinant for efficient virus assembly

Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.     

Electrogenerated indium tin oxide-coated glass surface with photosensitive interfaces: surface analysis

We present herein a photo-immobilization technique for the localized and specific conjugation of biochip platforms with different proteinaceous bioreceptors, such as antigen or antibodies. This methodology based on a photoactivable electrogenerated polymer film, pyrrole-benzophenone, allows the covalent immobilization of biomolecules through light mediation. The surface-conductive glass platform electropolymerized with poly(pyrrole-benzophenone) thin film may then be used to affinity-coat the chip with molecular recognition probes. This glass chip electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide (ITO). Thereafter, pyrrole-benzophenone monomers are electropolymerized onto the conductive metal oxide surface and then exposed to an antigen Staphylococcal Enterotoxin B (SEB)) solution and illuminated with UV light (wavelength approximately 345 nm) through a mask. As a result of the photochemical reaction, a pattern thin layer of the antigen was covalently bound to the benzophenone-modified surface. Then the sample to be analyzed, along with its specific target antibody (anti-SEB antibodies), is introduced onto the glass surface and left to react with the previously photo-immobilized antigen. When the immuno-reaction is completed, the specifically attached immunoglobulin analytes are detected by using secondary antibodies conjugated with Fluorescein isothiocyanate (FITC). The fluorescence signal emanating from the biochip surface is then quantified by two methods, using a filtered intensified charge-coupled device (CCD) camera and a grating spectrometer.     

Inactivation of a subpopulation of human neutrophils by exposure to ultrahigh-molecular-weight polyethylene wear debris

Polymorphonuclear neutrophils, a first line of defence against invading microbial pathogens, may be attracted by inflammatory mediators triggered by ultrahigh-molecular-weight polyethylene (UHMWPE) wear particles released from orthopaedic prostheses. Phagocytosis of UHMWPE particles by neutrophils may indirectly compromise their phagocytic-bactericidal mechanisms, thus enhancing host susceptibility to microbial infections. In an in vitro assay, pre-exposure of purified human neutrophils to UHMWPE micrometre- and submicrometre-sized wear particles interfered with subsequent Staphylococcos aureus uptake in a heterogeneous way, as assessed by a dual label fluorescence microscopic assay that discriminated intracellular rhodamine-labelled UHMWPE particles from fluorescein isothiocyanate-labelled S. aureus. Indeed, a higher percentage (44%) of neutrophils having engulfed UHMWPE particles lost the ability to phagocytize S. aureus, compared with UHMWPE-free neutrophils (<3%). Pre-exposure of neutrophils to UHMWPE wear particles did not impair but rather stimulated their oxidative burst response in a chemoluminescence assay. The presence of UHMWPE wear particles did not lead to significant overall consumption of complement-mediated opsonic factors nor decreased surface membrane display of neutrophil complement receptors. In conclusion, engulfment of UHMWPE wear particles led to inactivation of S. aureus uptake in nearly half of the neutrophil population, which may potentially impair host clearance mechanisms against pyogenic infections.     

Multi‐causality and spatial non‐stationarity in the determinants of groundwater crustacean diversity in Europe

The recognition of multi‐causality and spatial non‐stationarity in the determinants of large‐scale biodiversity patterns requires to consider the role of multiple mechanisms, their interactions, and how these mechanisms vary in strength relative to each other across geographical space. Here, we challenge the view that historical climate stability primarily drives European patterns of groundwater crustacean diversity by testing also the role of spatial heterogeneity and productive energy. First, we predicted that the three mechanisms would be equally important at continental scale when analyzed separately, but that the importance of historical climate variability would weaken in joint analyses due to co‐variation with the two other mechanisms. Second, we predicted that the three mechanisms would exhibit predictable latitudinal changes in their relative strength. To test these predictions, we selected predictors representing each mechanism and analyzed separately and jointly their effects and interactions using global regression models. We further mapped the independent and overlapping effects of mechanisms across Europe using partial geographically weighted regressions. When analyzed separately, the three mechanisms explained the same amount of variation in species richness, but in the joint analysis, the influence of historical climate stability became hidden in the variation shared with the other mechanisms. Topographic heterogeneity interacted synergistically with actual evapotranspiration and habitat heterogeneity on species richness. Spatial non‐stationarity in the independent and overlapping effects of the three mechanisms was the most plausible explanation for the hump‐shaped latitudinal pattern of crustacean species richness. Productive energy and spatial heterogeneity were important predictors at mid and southern latitudes, whereas historical climate stability overlapped with the two other mechanisms in northern Europe and productive energy in southern Europe. Multi‐causality and spatial non‐stationarity provide a broader perspective of groundwater biodiversity determinants that revives the importance of spatial heterogeneity and the strong dependence of subterranean communities on food supply from the surface.     

RTTN Mutations Cause Primary Microcephaly and Primordial Dwarfism in Humans

Primary microcephaly is a developmental brain anomaly that results from defective proliferation of neuroprogenitors in the germinal periventricular zone. More than a dozen genes are known to be mutated in autosomal-recessive primary microcephaly in isolation or in association with a more generalized growth deficiency (microcephalic primordial dwarfism), but the genetic heterogeneity is probably more extensive. In a research protocol involving autozygome mapping and exome sequencing, we recruited a multiplex consanguineous family who is affected by severe microcephalic primordial dwarfism and tested negative on clinical exome sequencing. Two candidate autozygous intervals were identified, and the second round of exome sequencing revealed a single intronic variant therein (c.2885+8A>G [p.Ser963^∗] in RTTN exon 23). RT-PCR confirmed that this change creates a cryptic splice donor and thus causes retention of the intervening 7 bp of the intron and leads to premature truncation. On the basis of this finding, we reanalyzed the exome file of a second consanguineous family affected by a similar phenotype and identified another homozygous change in RTTN as the likely causal mutation. Combined linkage analysis of the two families confirmed that RTTN maps to the only significant linkage peak. Finally, through international collaboration, a Canadian multiplex family affected by microcephalic primordial dwarfism and biallelic mutation of RTTN was identified. Our results expand the phenotype of RTTN-related disorders, hitherto limited to polymicrogyria, to include microcephalic primordial dwarfism with a complex brain phenotype involving simplified gyration.     

Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h^−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h^−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h^−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.