Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets

Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.     

Clinical Features and Risk Factors for Atazanavir (ATV)-Associated Urolithiasis: A Case-Control Study

Objectives

Clinical features and risk factors for atazanavir (ATV)-associated urolithiasis have not been fully investigated.

Methods

We reviewed all cases of ATV-containing urolithiasis identified by infrared spectrophotometry among HIV-infected patients over a 5-year period to describe their clinical features and outcome. A case-control study was performed to identify risk factors associated with ATV-associated urolithiasis using univariate and multivariate logistic regression analyses.

Results

30 cases of ATV-associated urolithiasis were analyzed. Patients were mostly men (87%), median age: 45.5 years, median CD4 cell count: 443 cells/µL and 97% had plasma HIV RNA level <50 cp/mL. Median time between the initiation of ATV-containing regimen and the diagnosis of urolithiasis was 3.1 years. Patients presented with flank pain in 90% and macroscopic hematuria in 82.6%, 34% had renal dysfunction and 44.8% needed ureteroscopic treatment. In univariate analysis, chronic hepatitis C, a history of urolithiasis, prior use of indinavir, ATV duration, undetectable plasma HIV RNA, use of ritonavir as a booster and serum free bilirubin level were associated with ATV-urolithiasis. Multivariate models retained serum free bilirubin level (OR: 2.31, p<0.02) and either ATV duration (OR:  = 1.42, p = <0.03) or a history of urolithiasis (OR = 4.79, p<0.02) when adjusting on serum free bilirubin level as risk factors associated with urolithiasis.

Conclusions

ATV-containing urolithiasis are associated with frank clinical symptoms and may require surgical intervention. A high serum bilirubin level, a long exposure to ATV and a history of urolithiasis are risk factors for this rare adverse event.     

Longitudinal Strain Is a Marker of Microvascular Obstruction and Infarct Size in Patients with Acute ST-Segment Elevation Myocardial Infarction

Objectives

We assessed the value of speckle tracking imaging performed early after a first ST-segment elevation myocardial infarction (STEMI) in order to predict infarct size and functional recovery at 3-month follow-up.

Methods

44 patients with STEMI who underwent revascularization within 12 h of symptom onset were prospectively enrolled. Echocardiography was performed 3.9±1.2 days after myocardial reperfusion, assessing circumferential (CGS), radial (RGS), and longitudinal global (GLS) strains. Late gadolinium-enhanced cardiac magnetic imaging (CMR), for assessing cardiac function, infarct size, and microvascular obstruction (MVO), was conducted 5.6±2.5 days and 99.4±4.6 days after myocardial reperfusion.

Results

GLS was evaluable in 97% of the patients, while CGS and RGS could be assessed in 85%. Infarct size significantly correlated with GLS (R = 0.601, p<0.001), RGS (R = −0.405, p = 0.010), CGS (R = 0.526, p = 0.001), ejection fraction (R = −0.699, p<0.001), wall motion score index (WMSI) (R = 0.539, p = 0.001), and left atrial volume (R = 0.510, p<0.001). Baseline ejection fraction and GLS were independent predictors of 3-month infarct size. MVO mass significantly correlated with GLS (R = 0.376, p = 0.010), WMSI (R = 0.387, p = 0.011), and ejection fraction (R = −0.389, p = 0.011). In multivariate analysis, GLS was the only independent predictor of MVO mass (p = 0.015). Longitudinal strain >−6.0% within the infarcted area exhibited 96% specificity and 61% sensitivity for predicting the persistence of akinesia (≥3 segments) at 3-month follow-up.

Conclusions

Speckle-tracking strain imaging performed early after a STEMI is easy-to-use as a marker for persistent akinetic territories at 3 months. In addition, GLS correlated significantly with MVO and final infarct size, both parameters being relevant post-MI prognostic factors, usually obtained via CMR.     

IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn''s disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin⁺ fibroblast-like synoviocytes and CD68⁺ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t⁺ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R⁻ or CCR6⁻ CD161⁻) CD4⁺ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.     

GW501516-activated PPARβ/δ promotes liver fibrosis via p38-JNK MAPK-induced hepatic stellate cell proliferation

Background

After liver injury, the repair process comprises activation and proliferation of hepatic stellate cells (HSCs), which produce extracellular matrix (ECM) proteins. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in these cells, but its function in liver repair remains incompletely understood. This study investigated whether activation of PPARβ/δ with the ligand GW501516 influenced the fibrotic response to injury from chronic carbon tetrachloride (CCl₄) treatment in mice. Wild type and PPARβ/δ-null mice were treated with CCl₄ alone or CCl₄ co-administered with GW501516. To unveil mechanisms underlying the PPARβ/δ-dependent effects, we analyzed the proliferative response of human LX-2 HSCs to GW501516 in the presence or absence of PPARβ/δ.

Results

We found that GW501516 treatment enhanced the fibrotic response. Compared to the other experimental groups, CCl₄/GW501516-treated wild type mice exhibited increased expression of various profibrotic and pro-inflammatory genes, such as those involved in extracellular matrix deposition and macrophage recruitment. Importantly, compared to healthy liver, hepatic fibrotic tissues from alcoholic patients showed increased expression of several PPAR target genes, including phosphoinositide-dependent kinase-1, transforming growth factor beta-1, and monocyte chemoattractant protein-1. GW501516 stimulated HSC proliferation that caused enhanced fibrotic and inflammatory responses, by increasing the phosphorylation of p38 and c-Jun N-terminal kinases through the phosphoinositide-3 kinase/protein kinase-C alpha/beta mixed lineage kinase-3 pathway.

Conclusions

This study clarified the mechanism underlying GW501516-dependent promotion of hepatic repair by stimulating proliferation of HSCs via the p38 and JNK MAPK pathways.     

Isotopic tracing reveals single-cell assimilation of a macroalgal polysaccharide by a few marine Flavobacteria and Gammaproteobacteria

Algal polysaccharides constitute a diverse and abundant reservoir of organic matter for marine heterotrophic bacteria, central to the oceanic carbon cycle. We investigated the uptake of alginate, a major brown macroalgal polysaccharide, by microbial communities from kelp-dominated coastal habitats. Congruent with cell growth and rapid substrate utilization, alginate amendments induced a decrease in bacterial diversity and a marked compositional shift towards copiotrophic bacteria. We traced ¹³C derived from alginate into specific bacterial incorporators and quantified the uptake activity at the single-cell level, using halogen in situ hybridization coupled to nanoscale secondary ion mass spectrometry (HISH-SIMS) and DNA stable isotope probing (DNA-SIP). Cell-specific alginate uptake was observed for Gammaproteobacteria and Flavobacteriales, with carbon assimilation rates ranging from 0.14 to 27.50 fg C µm⁻³ h⁻¹. DNA-SIP revealed that only a few initially rare Flavobacteriaceae and Alteromonadales taxa incorporated ¹³C from alginate into their biomass, accounting for most of the carbon assimilation based on bulk isotopic measurements. Functional screening of metagenomic libraries gave insights into the genes of alginolytic Alteromonadales active in situ. These results highlight the high degree of niche specialization in heterotrophic communities and help constraining the quantitative role of polysaccharide-degrading bacteria in coastal ecosystems.Subject terms: Water microbiology, Microbial ecology, Marine microbiology, Biogeochemistry, Microbial ecology     

Novel uncultured Epsilonproteobacteria dominate a filamentous sulphur mat from the 13 degrees N hydrothermal vent field, East Pacific Rise

Rapid growth of microbial sulphur mats have repeatedly been observed during oceanographic cruises to various deep-sea hydrothermal vent sites. The microorganisms involved in the mat formation have not been phylogenetically characterized, although the production of morphologically similar sulphur filaments by a Arcobacter strain coastal marine has been documented. An in situ collector deployed for 5 days at the 13 degrees N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) was rapidly colonized by a filamentous microbial mat. Microscopic and chemical analyses revealed that the mat consisted of a network of microorganisms embedded in a mucous sulphur-rich matrix. Molecular surveys based on 16S rRNA gene and aclB genes placed all the environmental clone sequences within the Epsilonproteobacteria. Although few 16S rRNA gene sequences were affiliated with that of cultured organisms, the majority was related to uncultured representatives of the Arcobacter group (< or = 95% sequence similarity). A probe designed to target all of the identified lineages hybridized with more than 95% of the mat community. Simultaneous hybridizations with the latter probe and a probe specific to Arcobacter spp. confirmed the numerical dominance of Arcobacter-like bacteria. This study provides the first example of the prevalence and ecological significance of free-living Arcobacter at deep-sea hydrothermal vents.     

Exclusion of a proton ATPase from the apical membrane is associated with cell polarity and tip growth in Nicotiana tabacum pollen tubes

Polarized growth in pollen tubes results from exocytosis at the tip and is associated with conspicuous polarization of Ca(2+), H(+), K(+), and Cl(-) -fluxes. Here, we show that cell polarity in Nicotiana tabacum pollen is associated with the exclusion of a novel pollen-specific H(+)-ATPase, Nt AHA, from the growing apex. Nt AHA colocalizes with extracellular H(+) effluxes, which revert to influxes where Nt AHA is absent. Fluorescence recovery after photobleaching analysis showed that Nt AHA moves toward the apex of growing pollen tubes, suggesting that the major mechanism of insertion is not through apical exocytosis. Nt AHA mRNA is also excluded from the tip, suggesting a mechanism of polarization acting at the level of translation. Localized applications of the cation ionophore gramicidin A had no effect where Nt AHA was present but acidified the cytosol and induced reorientation of the pollen tube where Nt AHA was absent. Transgenic pollen overexpressing Nt AHA-GFP developed abnormal callose plugs accompanied by abnormal H(+) flux profiles. Furthermore, there is no net flux of H(+) in defined patches of membrane where callose plugs are to be formed. Taken together, our results suggest that proton dynamics may underlie basic mechanisms of polarity and spatial regulation in growing pollen tubes.     

Oligocarrageenans and tissue-dependant oxidative burst in Solieria chordalis (Rhodophyceae, Gigartinales)

The release of hydrogen peroxide by thallus fragments of the rhodophycean Solieria chordalis (C. Agardh) J. Agardh has been documented both in the presence and in the absence of oligosaccharides. Within 1 h, ramuli were able to release large amounts of peroxide in the absence of any chemical stress. Among potential elicitors tested, only degree of polymerization 1 (DP1) and DP7‐8 oligo‐iota‐carrageenans stimulated defense mechanisms in both axes and ramuli as shown by the occurrence of an oxidative burst. Chopping of the tissues had no effect on the intensity of the burst, therefore suggesting that mainly cortical cell layers were involved in the process. After 5 min incubation, a dose of 125 μg mL^?1 of an oligomeric mixture containing a large proportion of DP1 units proved to be sufficient to obtain a maximal response. The intensity of the burst was significantly higher with isolated ramuli than with pieces of the axis, with outer peroxide accumulations reaching 200 nmol g^?1 fresh weight of treated tissue. Altogether, our results show that S. chordalis is able to react to a simulated pathogen attack by an oxidative burst and that the capacity to carry out an oxidative burst is stronger in ramuli than in axes.