Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

BACKGROUND: Over 6 million esophagogastroduodenoscopy (EGD) procedures are performed in the United States each year. Patients having anesthesia for advanced EGD procedures, such as interventional procedures, are at high risk for hypoxemia. METHODS: Our primary study aim was to evaluate whether high-flow nasal cannula (HFNC) oxygen reduces the incidence of hypoxemia during anesthesia for advanced EGD. Secondarily, we studied whether HFNC oxygen reduces hypercarbia or hypotension. After obtaining written informed consent, adults having anesthesia for advanced EGD, expected to last longer than 15 minutes, were randomly assigned to receive HFNC oxygen or standard nasal cannula (SNC) oxygen. The primary outcome was occurrence of one or more hypoxemia events during anesthesia, defined by arterial oxygen saturation <92% for at least 15 consecutive seconds. Secondary outcomes were occurrence of one or more hypercarbia or hypotension events. A hypercarbia event was defined by a transcutaneous CO2 measurement 20 mm Hg or more above baseline, and a hypotension event was defined by a mean arterial blood pressure measurement 25% or more below baseline. RESULTS: Two hundred seventy-one adult patients were enrolled and randomized, and 262 patients completed study procedures. Eight randomized patients did not complete study procedures due to changes in their anesthesia or endoscopy plan. One patient was excluded from analysis because their procedure was aborted after 1 minute. Patients who received HFNC oxygen (N = 132) had a significantly lower incidence of hypoxemia than those who received SNC oxygen (N = 130; 21.2% vs 33.1%; hazard ratio [HR] = 0.59 [95% confidence interval {CI}, 0.36–0.95]; P = .03). There was no difference in the incidence of hypercarbia or hypotension between the groups. The HR for hypercarbia with HFNC oxygen was 1.29 (95% CI, 0.89–1.88; P = .17), and the HR for hypotension was 1.25 (95% CI, 0.86–1.82; P = .25). CONCLUSIONS: HFNC oxygen reduces the incidence of hypoxemia during anesthesia for advanced EGD and may offer an opportunity to enhance patient safety during these procedures.

The gamma aminobutyric acid (GABA) neurotransmission system has been implicated in autism spectrum disorder (ASD). Molecular neuroimaging studies incorporating simultaneous acquisitions of GABA concentrations and GABA A receptor densities can identify objective molecular markers in ASD. We measured both total GABA A receptor densities by using [ 18 F]flumazenil positron emission tomography ([ 18 F]FMZ-PET) and GABA concentrations by using proton magnetic resonance spectroscopy ( 1 H-MRS) in 28 adults with ASD and in 29 age-matched typically developing (TD) individuals. Focusing on the bilateral thalami and the left dorsolateral prefrontal cortex (DLPFC) as our regions of interest, we found no differences in GABA A receptor densities between ASD and TD groups. However, 1 H-MRS measurements revealed significantly higher GABA/Water (GABA normalized by water signal) in the left DLPFC of individuals with ASD than that of TD controls. Furthermore, a significant gender effect was observed in the thalami, with higher GABA/Water in males than in females. Hypothesizing that thalamic GABA correlates with ASD symptom severity in gender-specific ways, we stratified by diagnosis and investigated the interaction between gender and thalamic GABA/Water in predicting Autism Quotient (AQ) and Ritvo Autism Asperger’s Diagnostic Scale – Revised (RAADS-R) total scores. We found that gender is a significant effect modifier of thalamic GABA/Water’s relationship with AQ and RAADS-R scores for individuals with ASD, but not for TD controls. When we separated the ASD participants by gender, a negative correlation between thalamic GABA/Water and AQ was observed in male ASD participants. Remarkably, in female ASD participants, a positive correlation between thalamic GABA/Water and AQ was found.

The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.

Soluble amyloid-␤ (A␤) oligomers, also known as A␤-derived diffusible ligands (ADDLs), are thought to be the key pathogenic factor in Alzheimer's disease (AD), but there is still no effective treatment for preventing or reversing the progression of the disease. Targeting NMDA receptor trafficking and regulation is a new strategy for early treatment of AD. A␤ oligomers have been found to bind to the fibronectin (FN) type III repeat domain of EphB2 to trigger EphB2 degradation, thereby impairing the normal functioning of NMDA receptors and resulting in cognitive deficits. Here, we identified for the first time the interaction sites of the EphB2 FN domain with ADDLs by applying the peptide array method to design and synthesize four candidate peptides (Pep21, Pep25, Pep32, and Pep63) that might be able to block the EphB2-ADDL interaction. Among them, Pep63 was found to be the most effective at inhibiting the binding between EphB2 and ADDLs. We found that Pep63 not only rescued the ADDL-induced depletion of EphB2-and GluN2B-containing NMDA receptors from the neuronal surface in cultured hippocampal neurons, but also improved impaired memory deficits in APPswe/PS1dE9 (APP/PS1) transgenic mice and the phosphorylation and surface expression of GluN2B-containing NMDA receptors in cultures. Together, these results suggest that blocking the EphB2-ADDL interaction by small interfering peptides may be a promising strategy for AD treatment.

It is accepted that amyloid β-derived diffusible ligands (ADDLs) have a prominent role in triggering the early cognitive deficits that constitute Alzheimer's disease (AD). However, there is still no effective treatment for preventing or reversing the progression of the disease. Targeting α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor trafficking and its regulation is a new strategy for AD early treatment. Here we investigate the effect and mechanism of L-Stepholidine (L-SPD), which elicits dopamine D1-type receptor agonistic activity, while acting as D2-type receptor antagonist on cognition and synaptic plasticity in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic (APP/PS1) mice, and hippocampal cultures or slices treated with ADDLs. L-SPD could improve the hippocampus-dependent memory, surface expression of glutamate receptor A (GluA1)-containing AMPA receptors and spine density in hippocampus of APP/PS1 transgenic mice. L-SPD not only rescued decreased phosphorylation and surface expression of GluA1 in hippocampal cultures but also protected the long-term potentiation in hippocampal slices induced by ADDLs. Protein kinase A (PKA) agonist Sp-cAMPS or D1-type receptor agonist SKF81297 had similar effects, whereas PKA antagonist Rp-cAMPS or D1-type receptor antagonist SCH23390 abolished the effect of L-SPD on GluA1 trafficking. This was mediated mainly by PKA, which could phosphorylate serine residue at 845 of the GluA1. L-SPD may be explored as a potential therapeutic drug for AD through a mechanism that improves AMPA receptor trafficking and synaptic plasticity via activating D1/PKA signaling pathway.

Positive-stranded RNA virus movement proteins (MPs) generally lack sequence-specific nucleic acid-binding activities and display cross-family movement complementarity with related and unrelated viruses. Negative-stranded RNA plant rhabdoviruses encode MPs with limited structural and functional relatedness with other plant virus counterparts, but the precise mechanisms of intercellular transport are obscure. In this study, we first analyzed the abilities of MPs encoded by five distinct rhabdoviruses to support cell-to-cell movement of two positive-stranded RNA viruses by using trans-complementation assays. Each of the five rhabdovirus MPs complemented the movement of MP-defective mutants of tomato mosaic virus and potato X virus. In contrast, movement of recombinant MP deletion mutants of sonchus yellow net nucleorhabdovirus (SYNV) and tomato yellow mottle-associated cytorhabdovirus (TYMaV) was rescued only by their corresponding MPs, i.e., SYNV sc4 and TYMaV P3. Subcellular fractionation analyses revealed that SYNV sc4 and TYMaV P3 were peripherally associated with cell membranes. A split-ubiquitin membrane yeast two-hybrid assay demonstrated specific interactions of the membraneassociated rhabdovirus MPs only with their cognate nucleoproteins (N) and phosphoproteins (P). More importantly, SYNV sc4-N and sc4-P interactions directed a proportion of the N-P complexes from nuclear sites of replication to punctate loci at the cell periphery that partially colocalized with the plasmodesmata. Our data show that cell-to-cell movement of plant rhabdoviruses is highly specific and suggest that cognate MP-nucleocapsid core protein interactions are required for intra-and intercellular trafficking. IMPORTANCE Local transport of plant rhabdoviruses likely involves the passage of viral nucleocapsids through MP-gated plasmodesmata, but the molecular mechanisms are not fully understood. We have conducted complementation assays with MPs encoded by five distinct rhabdoviruses to assess their movement specificity. Each of the rhabdovirus MPs complemented the movement of MP-defective mutants of two positive-stranded RNA viruses that have different movement strategies. In marked contrast, cell-to-cell movement of two recombinant plant rhabdoviruses was highly specific in requiring their cognate MPs. We have shown that these rhabdovirus MPs are localized to the cell periphery and associate with cellular membranes, and that they interact only with their cognate nucleocapsid core proteins. These interactions are able to redirect viral nucleocapsid core proteins from their sites of replication to the cell periphery. Our study provides a model for the specific interand intracellular trafficking of plant rhabdoviruses that may be applicable to other negative-stranded RNA viruses. Downloaded fromN atural infections of plant viruses are initiated from one or a few primary infected cells, followed by cell-to-cell spread to adjacent cells and then to distal tissues via vascular networks. To accomplish systemic infections, plant viruses must ci...

Nucleorhabdoviruses such as Sonchus yellow net virus (SYNV) replicate in the nuclei and undergo morphogenesis at the inner nuclear membrane (IM) in plant cells. Mature particles are presumed to form by budding of the Matrix (M) protein-nucleocapsid complexes through host IMs to acquire host phospholipids and the surface glycoproteins (G). To address mechanisms underlying nucleorhabdovirus budding, we generated recombinant SYNV G mutants containing a truncated amino-terminal (NT) or carboxyl-terminal (CT) domain. Electron microscopy and sucrose gradient centrifugation analyses showed that the CT domain is essential for virion morphogenesis whereas the NT domain is also required for efficient budding. SYNV infection induces IM invaginations that are thought to provide membrane sites for virus budding. We found that in the context of viral infections, interactions of the M protein with the CT domain of the membrane-anchored G protein mediate M protein translocation and IM invagination. Interestingly, tethering the M protein to endomembranes, either by co-expression with a transmembrane G protein CT domain or by artificial fusion with the G protein membrane targeting sequence, induces IM invagination in uninfected cells. Further evidence to support functions of G-M interactions in virus budding came from dominant negative effects on SYNV-induced IM invagination and viral infections that were elicited by expression of a soluble version of the G protein CT domain. Based on these data, we propose that cooperative G-M interactions promote efficient SYNV budding.