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This study was the first to compare the neuroprotective activity of Cerebrolysin ® , Actovegin ® and Cortexin ® in rodent models of acute and chronic brain ischemia. The neuroprotective action was evaluated in animals with acute (middle cerebral artery occlusion) or chronic (common carotid artery stenosis) brain ischemia models in male rats. Cortexin ® (1 or 3 mg/kg/day), Cerebrolysin ® (538 or 1614 mg/kg/day) and Actovegin ® (200 mg/kg/day) were administered for 10 days. To assess the neurological and motor impairments, open field test, adhesive removal test, rotarod performance test and Morris water maze test were performed. Brain damage was assessed macro- and microscopically, and antioxidant system activity was measured in brain homogenates. In separate experiments in vitro binding of Cortexin ® to a wide panel of receptors was assessed, and blood-brain barrier permeability of Cortexin ® was assessed in mice in vivo . Cortexin ® or Cerebrolysin ® and, to a lesser extent, Actovegin ® improved the recovery of neurological functions, reduced the severity of sensorimotor and cognitive impairments in rats. Cortexin ® reduced the size of necrosis of brain tissue in acute ischemia, improved functioning of the antioxidant system and prevented the development of severe neurodegenerative changes in chronic ischemia model. Radioactively labeled Cortexin ® crossed the blood-brain barrier in mice in vivo with concentrations equal to 6–8% of concentrations found in whole blood. During in vitro binding assay Cortexin ® (10 μg/ml) demonstrated high or moderate binding to AMPA-receptors (80.1%), kainate receptors (73.5%), mGluR1 (49.0%), GABAA1 (44.0%) and mGluR5 (39.7%), which means that effects observed in vivo could be related on the glutamatergic and GABAergic actions of Cortexin ® . Thus, Cortexin, 1 or 3 mg/kg, or Cerebrolysin ® , 538 or 1614 mg/kg, were effective in models acute and chronic brain ischemia in rats. Cortexin ® contains compounds acting on AMPA, kainate, mGluR1, GABAA1 and mGluR5 receptors in vitro , and readily crosses the blood-brain barrier in mice.

Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for tomato bushy stunt virus (TBSV). The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.

Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.

Positive-strand RNA viruses assemble numerous membrane-bound viral replicase complexes within large replication compartments to support their replication in infected cells. Yet the detailed mechanism of how given subcellular compartments are subverted by viruses is incompletely understood. Although, Tomato bushy stunt virus (TBSV) uses peroxisomal membranes for replication, in this paper, we show evidence that the ER-resident SNARE (soluble NSF attachment protein receptor) proteins play critical roles in the formation of active replicase complexes in yeast model host and in plants. Depletion of the syntaxin 18-like Ufe1 and Use1, which are components of the ER SNARE complex in the ERAS (ER arrival site) subdomain, in yeast resulted in greatly reduced tombusvirus accumulation. Over-expression of a dominant-negative mutant of either the yeast Ufe1 or the orthologous plant Syp81 syntaxin greatly interferes with tombusvirus replication in yeast and plants, thus further supporting the role of this host protein in tombusvirus replication. Moreover, tombusvirus RNA replication was low in cell-free extracts from yeast with repressed Ufe1 or Use1 expression. We also present evidence for the mislocalization of the tombusviral p33 replication protein to the ER membrane in Ufe1p-depleted yeast cells. The viral p33 replication protein interacts with both Ufe1p and Use1p and co-opts them into the TBSV replication compartment in yeast and plant cells. The co-opted Ufe1 affects the virus-driven membrane contact site formation, sterol-enrichment at replication sites, recruitment of several pro-viral host factors and subversion of the Rab5-positive PE-rich endosomes needed for robust TBSV replication. In summary, we demonstrate a critical role for Ufe1 and Use1 SNARE proteins in TBSV replication and propose that the pro-viral functions of Ufe1 and Use1 are to serve as assembly hubs for the formation of the extensive TBSV replication compartments in cells. Altogether, these findings point clearly at the ERAS subdomain of ER as a critical site for the biogenesis of the TBSV replication compartment.

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC), template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3' terminal promoter region in the viral minus-strand (-)RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5' proximal RIII(-) replication enhancer (REN) element in the TBSV (-)RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (-)RNA could unwind the dsRNA structure within the RIII(-) REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(-) REN in stimulation of plus-strand (+)RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(-) REN that promotes bringing the 5' and 3' terminal (-)RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication.

Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (endosomal sorting complexes required for transport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for in vitro assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in pex3Δ yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3'-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3'-end of the TBSV (-)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (-)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells.

Typically, a search operation on Big Data is complicated task due to different formats and different nature of the data. To simplify it the user may implement special algorithms to process different data for uncertain criteria. The “self-learning query” algorithm presented in this work allows to search in Big Data either for certain or uncertain search criteria’s with minimum attention from the data programmer. It uses accumulated search statistics as the basis to make the result set more precisely according to the search criteria, so as long the user work with the system as more precise will be the results. The algorithm presented in this work allows data scientists to search for uncertain data and potentially discover results faster by offloading the burdens of data management and provenance to the expert system.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.

Directivity patters of some antenna systems depend on duration of radiated microwave pulses. It is shown that for large enough twodimensional systems the shortening of pulses leads to suppression of side lobe radiation. We propose to use a novel for two-dimensional antenna systems parameter - a correlation length which is useful for estimation of pulse shortening influence on antenna pattern. Correlation length depends both on the pulse duration and direction of radiation. The effect of side lobes suppression can be used for estimation of pulse duration.