Mapping the Interactions of Dengue Virus NS1 Protein with Human Liver Proteins Using a Yeast Two-Hybrid System: Identification of C1q as an Interacting Partner

Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection.     

ESCRT-0 Is Not Required for Ectopic Notch Activation and Tumor Suppression in Drosophila

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.     

Rapid Clinical Assessment to Facilitate the Triage of Adults with Falciparum Malaria,a Retrospective Analysis

Background

Most adults dying from falciparum malaria will die within 48 hours of their hospitalisation. An essential component of early supportive care is the rapid identification of patients at greatest risk. In resource-poor settings, where most patients with falciparum malaria are managed, decisions regarding patient care must frequently be made using clinical evaluation alone.

Methods

We retrospectively analysed 4 studies of 1801 adults with severe falciparum malaria to determine whether the presence of simple clinical findings might assist patient triage.

Results

If present on admission, shock, oligo-anuria, hypo- or hyperglycaemia, an increased respiratory rate, a decreased Glasgow Coma Score and an absence of fever were independently predictive of death. The variables were used to construct a simple clinical algorithm. When applied to the 1801 patients, this algorithm’s positive predictive value for survival to 48 hours was 99.4 (95% confidence interval (CI) 97.8–99.9) and for survival to discharge 96.9% (95% CI 94.3–98.5). In the 712 patients receiving artesunate, the algorithm’s positive predictive value for survival to 48 hours was 100% (95% CI 97.3–100) and to discharge was 98.5% (95% CI 94.8–99.8).

Conclusions

Simple clinical findings are closely linked to the pathophysiology of severe falciparum malaria in adults. A basic algorithm employing these indices can facilitate the triage of patients in settings where intensive care services are limited. Patients classified as low-risk by this algorithm can be safely managed initially on a general ward whilst awaiting senior clinical review and laboratory data.     

Role of mitogen-activated protein kinases in the iNOS production and cytokine secretion by Salmonella enterica serovar Typhimurium porins

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both physiological and pathological functions. The present study examined the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and proinflammatory cytokine production in RAW 264.7 cells in response to Salmonella enterica serovar Typhimurium porins. By use of Western blotting for iNOS detection and enzyme-linked immunosorbent assay (ELISA) for quantization of cytokine secretion, selective pharmacological inhibitors of MAPK pathways were tested for dissecting the molecular mechanisms underlying the mediation of these signaling in porins-stimulated murine macrophages. S. enterica serovar Typhimurium porins activated iNOS expression, NO production and interleukin (IL)-6, IL-8 and tumor necrosis factor-α (TNF-α) release. Treatment of cells with SB203580 and SP600125 (inhibitors of p38 and JNK, respectively) significantly affected porin-stimulated iNOS and NO production. Concomitant decrease in the proinflammatory cytokine secretion was detected. These data confirm the importance of the MAPKs cascade in macrophage activation by bacterial product opening up new strategies for therapy of septic shock.     

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N₂-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N₂ might explain why they are so abundant in the mucus of different coral species.     

Evaluation of recovery methods to detect faecal streptococci in polluted waters

AIMS: This paper compares the faecal streptococci count on 25 samples of polluted waters obtained with three techniques: most probable number (MPN), membrane filtration (MF) and pour plate (PP) methods. Although the PP method is a simple technique, familiar to water bacteriologists, it is not recommended in the international methods. METHODS AND RESULTS: For the MPN method, azide dextrose broth and ethyl violet azide broth were employed. For the MF technique, Millipore filters were placed onto azide maltose agar (KF agar), while for the PP method, 1 ml of a decimal water dilution was added to (Kennel Faecal) KF medium. Regression analysis and Friedman's ANOVA were performed to determine the relationship between faecal streptococci counts obtained with the three techniques. Statistical analysis of the results showed that the MPN, MF and PP techniques were equally valid with respect to faecal streptococci enumeration in polluted waters. CONCLUSION: Since the PP method was found to be as good as the other techniques, it may be preferred in polluted waters. It is more economical in terms of both time and materials than the MPN count, and it is as accurate as the MF count. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that the PP method, although not recommended internationally, is a reliable alternative to MF and MPN.     

Oligodendrocyte ablation impairs cerebellum development

Oligodendrocytes (OLs) are the glial cells of the central nervous system and are classically known to form myelin sheaths around most axons of higher vertebrates. Whether these cells might have other roles, in particular during development, has not been studied. Taking advantage of a transgenic mouse model in which OLs can be selectively killed in a desired time-frame, we have investigated the impact of OL ablation on cerebellar development. OL ablation was induced during the first 3 postnatal weeks, a time at which cerebellum development is ongoing. Strikingly, OL ablation triggers a profound perturbation of the known cerebellum developmental program, characterized by the disorganization of the cortical layers, abnormal foliation and a complete alteration of Purkinje cell dendritic arborization and axonal fasciculation. This phenotype is accompanied by decreased granule cell density, a disorganized Bergmann glia network and impaired migration of interneurons in the molecular layer. These results demonstrate a previously ignored role of OLs in the formation of the cerebellar cytoarchitecture.     

Without DJ-1, the D2 receptor doesn't play

Dopamine is an essential neuromodulator of the central nervous system whose activity is mediated through interaction with membrane receptors. Among these, the D2 receptor plays a pivotal role in regulating postsynaptic functions as well as dopamine synthesis and release from dopaminergic neurons. Parkinson's disease and parkinsonism are neurodegenerative diseases of dopaminergic neurons affecting the human brain. These diseases are clinically defined by a decrease in dopamine levels that manifest as motor impairements. In this issue of Neuron, Goldberg et al. report of an exciting link between the loss of function of DJ-1, a protein associated with familial parkinsonism, and D2 receptor activity.