Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus

We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG), but not undifferentiated neuronal progenitor cells (NPCs) from ventral subventricular zone (SVZ), results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2). NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control). By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+), whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+). At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative). Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78%) expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG.     

Microbial Communities Associated with the Larval Gut and Eggs of the Western Corn Rootworm

Background

The western corn rootworm (WCR) is one of the economically most important pests of maize. A better understanding of microbial communities associated with guts and eggs of the WCR is required in order to develop new pest control strategies, and to assess the potential role of the WCR in the dissemination of microorganisms, e.g., mycotoxin-producing fungi.

Methodology/Principal Findings

Total community (TC) DNA was extracted from maize rhizosphere, WCR eggs, and guts of larvae feeding on maize roots grown in three different soil types. Denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene and ITS fragments, PCR-amplified from TC DNA, were used to investigate the fungal and bacterial communities, respectively. Microorganisms in the WCR gut were not influenced by the soil type. Dominant fungal populations in the gut were affiliated to Fusarium spp., while Wolbachia was the most abundant bacterial genus. Identical ribosomal sequences from gut and egg samples confirmed a transovarial transmission of Wolbachia sp. Betaproteobacterial DGGE indicated a stable association of Herbaspirillum sp. with the WCR gut. Dominant egg-associated microorganisms were the bacterium Wolbachia sp. and the fungus Mortierella gamsii.

Conclusion/Significance

The soil type-independent composition of the microbial communities in the WCR gut and the dominance of only a few microbial populations suggested either a highly selective environment in the gut lumen or a high abundance of intracellular microorganisms in the gut epithelium. The dominance of Fusarium species in the guts indicated WCR larvae as vectors of mycotoxin-producing fungi. The stable association of Herbaspirillum sp. with WCR gut systems and the absence of corresponding sequences in WCR eggs suggested that this bacterium was postnatally acquired from the environment. The present study provided new insights into the microbial communities associated with larval guts and eggs of the WCR. However, their biological role remains to be explored.     

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with alpha-synuclein at the single molecule level

ABSTRACT: BACKGROUND: Fibrillar amyloid-like deposits and co-deposits of tau and alpha-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and alpha-synuclein are not well understood. RESULTS: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with alpha-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3beta exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of alpha-synuclein in pre-formed tau oligomers. CONCLUSIONS: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.     

Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

Background

Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process.

Methodology/Principal Findings

We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion.

Conclusions/Significance

Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.     

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.     

Identification of novel small-molecule histone deacetylase inhibitors by medium-throughput screening using a fluorigenic assay

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure-activity relationships in the bacterial membrane disruptor betapep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of betapep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel beta-sheet structure is the bioactive conformation of betapep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, betapep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.     

Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes

Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after θ-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor–dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.     

Carotid geometry effects on blood flow and on risk for vascular disease

It has been widely observed that atherosclerotic diseases occur at sites with complex hemodynamics, such as artery bifurcations, junctions, and regions of high curvature. These regions usually have very low or highly oscillatory wall shear stress (WSS). In the present work, 3D pulsatile blood flow through a model of the carotid artery bifurcation was simulated using a finite volume numerical method. The goal was to quantify the risk of atherogenesis associated with different carotid artery geometries. A risk scale based on the average WSS on the sinus wall of the internal carotid artery was proposed-a scale that can be used to quantify the effect of the carotid geometry on the relative risk for developing vascular disease. It was found that the bifurcation angle and the out-of-plane angle of the internal carotid artery affect the formation of low stress regions on the carotid walls. The main conclusions are: (a) larger internal carotid artery angles (theta(IC)) generally increase the frequency and the area of blood recirculation and lower the WSS on the sinus wall, hence increasing the risk of plaque build-up; (b) off-plane angles were found to lower the WSS on the sinus for geometries with theta(IC)25 degrees . Larger off-plane angles generally increase the danger of plague build-up; (c) for theta(IC) < 25 degrees , the off-plane angle does not have an obvious effect on the hemodynamic WSS; (d) symmetric bifurcations were found to increase the WSS on the sinus wall and ease the risk of vascular disease.