Expression analysis of novel striatal-enriched genes in Huntington disease

Selective degeneration of striatal neurons is a pathologic hallmark of Huntington disease (HD). The exact mechanism(s) behind this specific neurodegeneration is still unknown. Expression studies of diseased human post-mortem brain, as well as different mouse models exhibiting striatal degeneration, have demonstrated changes in the expression of many important genes with a large proportion of changes being observed in the striatal-enriched genes. These investigations have raised questions about how enrichment of particular transcripts in the striatum can lead to its selective vulnerability to neurodegeneration. Monitoring the expression changes of striatal-enriched genes during the course of the disease may be informative about their potential involvement in selective degeneration. In this study, we analyzed a Serial Analysis of Gene Expression (SAGE) database (www.mouseatlas.org) and compared the mouse striatum to 18 other brain regions to generate a novel list of striatal-enriched transcripts. These novel striatal-enriched transcripts were subsequently evaluated for expression changes in the YAC128 mouse model of HD, and differentially expressed transcripts were further examined in human post-mortem caudate samples. We identified transcripts with altered expression in YAC128 mice, which also showed consistent expression changes in human post-mortem tissue. The identification of novel striatal-enriched genes with altered expression in HD offers new avenues of study, leading towards a better understanding of specific pathways involved in the selective degeneration of striatal neurons in H

Anticancer Potential of Temozolomide-Loaded Eudragit-Chitosan Coated Selenium Nanoparticles: In Vitro Evaluation of Cytotoxicity, Apoptosis and Gene Regulation

Resistance to temozolomide (TMZ) is the main cause of death in glioblastoma multiforme (GBM). The use of nanocarriers for drug delivery applications is one of the known approaches to overcome drug resistance. This study aimed to investigate the possible effect of selenium-chitosan nanoparticles loaded with TMZ on the efficacy of TMZ on the expression of MGMT, E2F6, and RELA genes and the rate of apoptosis in the C6 cell line. Selenium nanoparticles (SNPs) were loaded with TMZ and then they were coated by Eudragit(R) RS100 (Eud) and chitosan (C-S) to prepare Se@TMZ/Eud-Cs. Physicochemical properties were determined by scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy (EDAX), thermal gravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS) methods. Se@TMZ/Eud-Cs was evaluated for loading and release of TMZ by spectrophotometric method. Subsequently, SNPs loaded with curcumin (as a fluorophore) were analyzed for in vitro uptake by C6 cells. Cytotoxicity and apoptosis assay were measured by MTT assay and Annexin-PI methods. Finally, real-time PCR was utilized to determine the expression of MGMT, E2F6, and RELA genes. Se@TMZ/Eud-Cs was prepared with an average size of 200 nm as confirmed by the DLS and microscopical methods. Se@TMZ/Eud-Cs presented 82.77 +/- 5.30 loading efficiency with slow and pH-sensitive release kinetics. SNPs loaded with curcumin showed a better uptake performance by C6 cells compared with free curcumin (p-value < 0.01). Coated nanoparticles loaded with TMZ showed higher cytotoxicity, apoptosis (p-value < 0.0001), and down-regulation of MGMT, E2F6, and RELA and lower IC50 value (p-value < 0.0001) than free TMZ and control (p-value < 0.0001) groups. Using Cs as a targeting agent in Se@TMZ/Eud-Cs system improved the possibility for targeted drug delivery to C6 cells. This drug delivery system enhanced the apoptosis rate and decreased the expression of genes related to TMZ resistance. In conclusion, Se@TMZ/Eud-Cs may be an option for the enhancement of TMZ efficiency in GBM treatment

FLP/FRT Recombination from Yeast: Application of a Two Gene Cassette Scheme as an Inducible System in Plants

Phytosensors are plants that are genetically engineered for sensing and reporting the presence of a specific contaminant, including agriculturally important biological agents. Phytosensors are constructed by transforming plants to contain specific biotic- or abiotic-inducible promoters fused to a reporter gene. When such transgenic plants encounter the target biotic or abiotic agent, the specific inducible promoter is triggered and subsequently drives the expression of the reporter gene, which produces a signal for detection. However, several systems lack robustness, rapid induction and promoter strength. Here, we tested the FLP/FRT recombination system in a construct containing a two gene cassette organization and examined its potential in transgenic Arabidopsis and tobacco plants using a β-glucuronidase (GUS) reporter. In this model system, a heat-shock inducible promoter was employed to control the expression of the FLP recombinase gene. Upon heat induction and subsequent active FLP-mediated excision event, the GUS gene was placed in close proximity to the 35S promoter resulting in an active GUS reporter expression. Our results demonstrate that the two gene cassette scheme of inducible FLP/FRT recombination system is functional in tobacco and Arabidopsis, providing additional insights into its possible application in phytosensing such as creating strong readout capabilities

Field-Grown Transgenic Switchgrass (Panicum virgatum L.) with Altered Lignin Does Not Affect Soil Chemistry, Microbiology, and Carbon Storage Potential

Cell wall recalcitrance poses a major challenge on cellulosic biofuel production from feedstocks such as switchgrass (Panicum virgatum L.). As lignin is a known contributor of recalcitrance, transgenic switchgrass plants with altered lignin have been produced by downregulation of caffeic acid O-methyltransferase (COMT). Field trials of COMT-downregulated plants previously demonstrated improved ethanol conversion with no adverse agronomic effects. However, the rhizosphere impacts of altering lignin in plants are unknown. We hypothesized that changing plant lignin composition may affect residue degradation in soils, ultimately altering soil processes. The objective of this study was to evaluate effects of two independent lines of COMT-downregulated switchgrass plants on soils in terms of chemistry, microbiology, and carbon cycling when grown in the field. Over the first two years of establishment, we observed no significant differences between transgenic and control plants in terms of soil pH or the total concentrations of 19 elements. An analysis of soil bacterial communities via high-throughput 16S rRNA gene amplicon sequencing revealed no effects of transgenic plants on bacterial diversity, richness, or community composition. We also did not observe a change in the capacity for soil carbon storage: There was no significant effect on soil respiration or soil organic matter. After five years of establishment, δ13C of plant roots, leaves, and soils was measured and an isotopic mixing model used to estimate that 11.2 to 14.5% of soil carbon originated from switchgrass. Switchgrass-contributed carbon was not significantly different between transgenic and control plants. Overall, our results indicate that over the short term (two and five years), lignin modification in switchgrass through manipulation of COMT expression does not have an adverse effect on soils in terms of total elemental composition, bacterial community structure and diversity, and capacity for carbon storage

Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

<p>Abstract</p> <p>Background</p> <p>We aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.</p> <p>Results</p> <p>For rapid analysis, an <it>Agrobacterium</it>-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct <it>in vivo</it>. Tobacco (<it>Nicotiana tabacum </it>cv. Xanthi) leaves were infiltrated with <it>Agrobacterium </it>harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.</p> <p>Conclusions</p> <p>These observations demonstrate that the <it>Agrobacterium</it>-mediated transient expression is an efficient method for <it>in vivo </it>assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput <it>in planta </it>expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.</p

Transgenic miR156 Switchgrass in the Field: Growth, Recalcitrance and Rust Susceptibility

Sustainable utilization of lignocellulosic perennial grass feedstocks will be enabled by high biomass production and optimized cell wall chemistry for efficient conversion into biofuels. MicroRNAs are regulatory elements that modulate the expression of genes involved in various biological functions in plants, including growth and development. In greenhouse studies, overexpressing a microRNA (miR156) gene in switchgrass had dramatic effects on plant architecture and flowering, which appeared to be driven by transgene expression levels. Highexpressing lines were extremely dwarfed, whereas low and moderate-expressing lines had higher biomass yields, improved sugar release and delayed flowering. Four lines with moderate or low miR156 overexpression from the prior greenhouse study were selected for a field experiment to assess the relationship between miR156 expression and biomass production over three years. We also analysed important bioenergy feedstock traits such as flowering, disease resistance, cell wall chemistry and biofuel production. Phenotypes of the transgenic lines were inconsistent between the greenhouse and the field as well as among different field growing seasons. One low expressing transgenic line consistently produced more biomass (25%–56%) than the control across all three seasons, which translated to the production of 30% more biofuel per plant during the final season. The other three transgenic lines produced less biomass than the control by the final season, and the two lines with moderate expression levels also exhibited altered disease susceptibilities. Results of this study emphasize the importance of performing multiyear field studies for plants with altered regulatory transgenes that target plant growth and development

The TcEG1 beetle (Tribolium castaneum) cellulase produced in transgenic switchgrass is active at alkaline pH and auto-hydrolyzes biomass for increased cellobiose release

Background Genetically engineered biofuel crops, such as switchgrass (Panicum virgatum L.), that produce their own cell wall-digesting cellulase enzymes would reduce costs of cellulosic biofuel production. To date, non-bioenergy plant models have been used in nearly all studies assessing the synthesis and activity of plant-produced fungal and bacterial cellulases. One potential source for cellulolytic enzyme genes is herbivorous insects adapted to digest plant cell walls. Here we examine the potential of transgenic switchgrass-produced TcEG1 cellulase from Tribolium castaneum (red flour beetle). This enzyme, when overproduced in Escherichia coliand Saccharomyces cerevisiae, efficiently digests cellulose at optima of 50 °C and pH 12.0. Results TcEG1 that was produced in green transgenic switchgrass tissue had a range of endoglucanase activity of 0.16–0.05 units (µM glucose release/min/mg) at 50 °C and pH 12.0. TcEG1 activity from air-dried leaves was unchanged from that from green tissue, but when tissue was dried in a desiccant oven (46 °C), specific enzyme activity decreased by 60%. When transgenic biomass was “dropped-in” into an alkaline buffer (pH 12.0) and allowed to incubate at 50 °C, cellobiose release was increased up to 77% over non-transgenic biomass. Saccharification was increased in one transgenic event by 28%, which had a concurrent decrease in lignin content of 9%. Histological analysis revealed an increase in cell wall thickness with no change to cell area or perimeter. Transgenic plants produced more, albeit narrower, tillers with equivalent dry biomass as the control. Conclusions This work describes the first study in which an insect cellulase has been produced in transgenic plants; in this case, the dedicated bioenergy crop switchgrass. Switchgrass overexpressing the TcEG1 gene appeared to be morphologically similar to its non-transgenic control and produced equivalent dry biomass. Therefore, we propose TcEG1 transgenics could be bred with other transgenic germplasm (e.g., low-lignin lines) to yield new switchgrass with synergistically reduced recalcitrance to biofuel production. In addition, transgenes for other cell wall degrading enzymes may be stacked with TcEG1 in switchgrass to yield complementary cell wall digestion features and complete auto-hydrolysis

The glycolytic enzyme, phosphoglycerate mutase, has critical roles in stomatal movement, vegetative growth, and pollen production in Arabidopsis thaliana

Stomatal movements require massive changes in guard cell osmotic content, and both stomatal opening and stomatal closure have been shown to be energy-requiring processes. A possible role for glycolysis in contributing to the energetic, reducing requirements, or signalling processes regulating stomatal movements has not been investigated previously. Glycolysis, oxidization of glucose to pyruvate, is a central metabolic pathway and yields a net gain of 2 ATP and 2 NADH. 2,3-biphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is a key enzymatic activity in glycolysis and catalyses the reversible interconversion of 3-phosphoglycerate to 2-phosphoglycerate. To investigate functions of iPGAMs and glycolysis in stomatal function and plant growth, Arabidopsis insertional mutants in At1g09780 and At3g08590, both of which have been annotated as iPGAMs on the basis of sequence homology, were identified and characterized. While single mutants were indistinguishable from the wild type in all plant phenotypes assayed, double mutants had no detectable iPGAM activity and showed defects in blue light-, abscisic acid-, and low CO2-regulated stomatal movements. Vegetative plant growth was severely impaired in the double mutants and pollen was not produced. The data demonstrate that iPGAMs and glycolytic activity are critical for guard cell function and fertility in Arabidopsis