Comparative Proteomic Analysis of Aedes aegypti Larval Midgut after Intoxication with Cry11Aa Toxin from Bacillus thuringiensis

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes

In-Service Performance of Airport Flexible Pavements Constructed Following State Specifications for Highway Pavement Materials

692M15-19-T-00026The Federal Aviation Administration (FAA) Reauthorization Act of 2018, Section 136 requires the FAA to allow the use of state highway specifications for airfield pavement construction at non-primary airports serving aircraft with a gross weight less than 60,000 lb if requested by the state. To confirm that state highway specifications provide an acceptable level of performance when used on airport pavements, the FAA initiated this project to compare the performance of airports constructed using state highway specifications with those constructed using FAA specifications. Performance data and specifications from 40 airport projects in five states were analyzed, with 21 using FAA specifications and 19 using state highway specifications. Based on the summarized pavement condition index (PCI) ratings from those projects (which are based on visual condition surveys and do not consider structural or functional performance), it was determined that the performance of airport asphalt pavements constructed using state highway specifications is statistically equivalent to asphalt pavements constructed using FAA specifications. The evaluations encompassed performance periods ranging from 1 to 15 years. Performance trends for the statistical analysis conducted showed an approximate PCI rating of 60 at year 14 for pavements constructed with both types of specifications. It was also determined that climate-based distresses were the predominant mode of distress for both FAA and state highway specification projects, with longitudinal and transverse cracking and weathering as the most prevalent types of distresses. The number of distresses that were load related was relatively minor, as only 8 of the 40 projects evaluated had load-related distresses. Of those projects, five used state highway specifications, and three used FAA specifications

Reduced Levels of Membrane-Bound Alkaline Phosphatase Are Common to Lepidopteran Strains Resistant to Cry Toxins from Bacillus thuringiensis

Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests

Global Functional Analyses of Cellular Responses to Pore-Forming Toxins

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs

Transformation of formaldehyde aminals into diaminocarbenes and carbenium salts

grantor: University of TorontoThe reactivities of the two stable diaminocarbenes 1,3-di-'tert '-butyl-imidazole-2-ylidene 1 and 1,3-di-'tert '-butyl-imidazolidine-2-ylidene 2 toward hydrogen, oxygen, water and carbon monoxide and SO2 were investigated. Contrary to common belief the deterioration of 1 and 2 is not due to oxidation by triplet oxygen, but is the result of hydrolysis. The hydrolysis rate at room temperature is very low for the aromatically stabilized 1 but very rapid for the non-aromatic 2. The reactions are not acid or base catalyzed. Computational data show that the reaction with triplet oxygen is only kinetically hindered and that the oxidation of 1 and 2 to the respective ureas is in fact strongly exothermic. Both carbenes insert into dihydrogen only in the presence of hydrogenation catalysts. Carbene 2 forms a 1:1 adduct with SO2 that is best described as a Lewis adduct. The aromatic ring protons in 1 undergo rapid deuterium - hydrogen exchange in DMSO-'d'6, CD3OD and D2O. The oxidation of animals by oxidizing agents like NBS, NCS, CCl4, CBr4, I2 or CuCl2 leads to diaminocarbeniurn salts in high yields. These oxidations can be extended to 1,3,5-triazacyclohexane R3TAC systems, to give the monocationic salts [R 3TAC][X]. The carbene, 1,3,5-tri-'tert'-butyl-1,3,5-triazacyclohexane-2-ylidene 9, derived through deprotonation of the [R3TAC][Br] has been observed in benzene-'d'6 solutions. Reaction of R3TAC with CuCl2 leads to metal-1,3,5-triazacyclohexane complexes.M.Sc

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity