Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1alpha, and MIP-1beta, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-kappaB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.     

TYRA-2 (F01E11.5): a Caenorhabditis elegans tyramine receptor expressed in the MC and NSM pharyngeal neurons

Tyramine appears to regulate key processes in nematodes, such as pharyngeal pumping, and more complex behaviors, such as foraging. Recently, a Caenorhabditis elegans tyramine receptor, SER-2, was identified that is involved in the TA-dependent regulation of these processes. In the present study, we have identified a second C. elegans gene, tyra-2 (F01E11.5) that encodes a tyramine receptor. This is the first identification of multiple tyramine receptor genes in any invertebrate. Membranes from COS-7 cells expressing TYRA-2 bind [(3)H]tyramine with high affinity with a K(d) of 20 +/- 5 nM. Other physiologically relevant biogenic amines, such as octopamine and dopamine, inhibit [(3)H]tyramine binding with much lower affinity (K(i)s of 1.55 +/- 0.5 and 1.78 +/- 0.6 microM, respectively), supporting the identification of TYRA-2 as a tyramine receptor. Indeed, tyramine also dramatically increases GTPgammaS binding to membranes from cells expressing TYRA-2 (EC(50) of 50 +/- 13 nM) and the TA-dependent GTPgammaS binding is PTX-sensitive suggesting that TYRA-2 may couple to Galpha(i/o). Based on fluorescence from tyra::gfp fusion constructs, TYRA-2 expression appears to be exclusively neuronal in the MC and NSM pharyngeal neurons, the AS family of amphid neurons and neurons in the nerve ring, body and tail. Taken together, these results suggest that TYRA-2 encodes a second Galpha(i/o)-coupled tyramine receptor and suggests that TA-dependent neuromodulation may be mediated by multiple receptors and more complex than previously appreciated.     

The Effects of Sub-Regional Climate Velocity on the Distribution and Spatial Extent of Marine Species Assemblages

Many studies illustrate variable patterns in individual species distribution shifts in response to changing temperature. However, an assemblage, a group of species that shares a common environmental niche, will likely exhibit similar responses to climate changes, and these community-level responses may have significant implications for ecosystem function. Therefore, we examine the relationship between observed shifts of species in assemblages and regional climate velocity (i.e., the rate and direction of change of temperature isotherms). The assemblages are defined in two sub-regions of the U.S. Northeast Shelf that have heterogeneous oceanography and bathymetry using four decades of bottom trawl survey data and we explore temporal changes in distribution, spatial range extent, thermal habitat area, and biomass, within assemblages. These sub-regional analyses allow the dissection of the relative roles of regional climate velocity and local physiography in shaping observed distribution shifts. We find that assemblages of species associated with shallower, warmer waters tend to shift west-southwest and to shallower waters over time, possibly towards cooler temperatures in the semi-enclosed Gulf of Maine, while species assemblages associated with relatively cooler and deeper waters shift deeper, but with little latitudinal change. Conversely, species assemblages associated with warmer and shallower water on the broad, shallow continental shelf from the Mid-Atlantic Bight to Georges Bank shift strongly northeast along latitudinal gradients with little change in depth. Shifts in depth among the southern species associated with deeper and cooler waters are more variable, although predominantly shifts are toward deeper waters. In addition, spatial expansion and contraction of species assemblages in each region corresponds to the area of suitable thermal habitat, but is inversely related to assemblage biomass. This suggests that assemblage distribution shifts in conjunction with expansion or contraction of thermal habitat acts to compress or stretch marine species assemblages, which may respectively amplify or dilute species interactions to an extent that is rarely considered. Overall, regional differences in climate change effects on the movement and extent of species assemblages hold important implications for management, mitigation, and adaptation on the U.S. Northeast Shelf.     

Endovascular Treatment with Stent-Retriever Devices for Acute Ischemic Stroke: A Meta-Analysis of Randomized Controlled Trials

Importance

Acute ischemic stroke is a leading cause of death and disability worldwide. Several recent clinical trials have shown that endovascular treatment improves clinical outcomes among patients with acute ischemic stroke.

Objective

To provide an overall and precise estimate of the efficacy of endovascular treatment predominantly using second-generation mechanical thrombectomy devices (stent-retriever devices) compared to medical management on clinical and functional outcomes among patients with acute ischemic stroke.

Data Sources

MEDLINE, EMBASE, Cochrane Collaboration Central Register of Controlled Clinical Trials, Web of Science, and NIH ClinicalTrials.gov were searched through November 2015.

Study Selection

Searches returned 3,045 articles. After removal of duplicates, two authors independently screened titles and abstracts to assess eligibility of 2,495 potentially relevant publications. From these, 38 full-text publications were more closely assessed. Finally, 5 randomized controlled trials of endovascular treatment with predominant use of retrievable stents were selected.

Data Extraction and Synthesis

Three authors independently extracted information on participant and trial characteristics and clinical events using a standardized protocol. Random effects models were used to pool endovascular treatment effects across outcomes.

Main Outcomes and Measures

The primary outcome was better functional outcome as measured on the modified Rankin Scale at 90 days of follow-up. Secondary outcomes included all-cause mortality and symptomatic intra-cerebral hemorrhage.

Results

Five trials representing 1,287 patients were included. Overall, patients randomized to endovascular therapy experienced 2.22 times greater odds of better functional outcome compared to those randomized to medical management (95% CI, 1.66 to 2.98; P < 0.0001). Endovascular therapy was not associated with mortality [OR (95% CI), 0.78 (0.54, 1.12); P = 0.1056] or symptomatic intracerebral hemorrhage [OR (95% CI), 1.19 (0.69, 2.05); P = 0.5348]. Meta-regression analysis suggested that shorter times from stroke onset to groin puncture and from stroke onset to reperfusion result in better functional outcomes in ischemic stroke patients (P = 0.0077 and P = 0.0089). There were no significant differences in the beneficial effects of endovascular treatment on functional outcomes across categories of gender, age, stroke severity, ischemic changes on computed tomography, or intravenous tissue plasminogen activator administration.

Conclusions and Relevance

This meta-analysis demonstrated superior functional outcomes in subjects receiving endovascular treatment compared to medical management. Further, this analysis showed that acute ischemic stroke patients may receive enhanced functional benefit from earlier endovascular treatment.     

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.     

Hepatocyte Growth Factor-induced Asef-IQGAP1 Complex Controls Cytoskeletal Remodeling and Endothelial Barrier

Hepatocyte growth factor (HGF) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via Rac-dependent enhancement of the peripheral actin cytoskeleton. However, the precise mechanisms of HGF effects on the peripheral cytoskeleton are not well understood. This study evaluated a role for Rac/Cdc42-specific guanine nucleotide exchange factor Asef and the multifunctional Rac effector, IQGAP1, in the mechanism of HGF-induced EC barrier enhancement. HGF induced Asef and IQGAP1 co-localization at the cell cortical area and stimulated formation of an Asef-IQGAP1 functional protein complex. siRNA-induced knockdown of Asef or IQGAP1 attenuated HGF-induced EC barrier enhancement. Asef knockdown attenuated HGF-induced Rac activation and Rac association with IQGAP1, and it abolished both IQGAP1 accumulation at the cell cortical layer and IQGAP1 interaction with actin cytoskeletal regulators cortactin and Arp3. Asef activation state was essential for Asef interaction with IQGAP1 and protein complex accumulation at the cell periphery. In addition to the previously reported role of the IQGAP1 RasGAP-related domain in the Rac-dependent IQGAP1 activation and interaction with its targets, we show that the IQGAP1 C-terminal domain is essential for HGF-induced IQGAP1/Asef interaction and Asef-Rac-dependent activation leading to IQGAP1 interaction with Arp3 and cortactin as a positive feedback mechanism of IQGAP1 activation. These results demonstrate a novel feedback mechanism of HGF-induced endothelial barrier enhancement via Asef/IQGAP1 interactions, which regulate the level of HGF-induced Rac activation and promote cortical cytoskeletal remodeling via IQGAP1-Arp3/cortactin interactions.