Effects of irradiation on the cowpea weevil (Callosobruchus maculates F.) and moisture sorption isotherm of cowpea seed (Vigna unguiculata L. Walp)

Cowpeas during storage may be attacked by a number of biological agents (microorganisms, rodents, and insects) which results in losses in the quality and quantity of the stored seeds. One of the means of reducing these losses is through the application of radiation processing. The aim of this study was to determine the effects of gamma irradiation on a major storage insect pest, Callosobruchus maculatus F. and on moisture sorption isotherms of cowpea seeds in storage. The cowpeas were infested with adults C. maculatus and then irradiated at doses of 0.0, 0.25, 0.5, 0.75, 1.0 and 1.5 (kGy) at a dose rate of 1.074 kGyhr-1. Samples were stored for 1 month under controlled temperature (27.3-30 oC) and humidity (70-85 %) during which counting of the insects was done every 48 hours and those alive or dead noted. Moisture sorption isotherms of the cowpea samples were equally determined by establishing equilibrium relative humidity (ERH) of 55, 65, 75, 85 and 95 % using a formulation of glycerol-water mixture at temperature of 30+-1 and the weight (loss or gain) of the samples was determined every 2 days. Irradiation at a dose of 0.25 kGy killed the C. maculatus within eight days and therefore 0.25 kGy would be economically beneficial as a control dose. There was significant difference (p is less than 0.05) in the percent mortality between the irradiated and the non-irradiated weevils, and the percent mortality increased with increase in the radiation dose. At moisture content of 14 % the irradiated cowpea samples were safely stored for one month at an equilibrium relative humidity of 60-75 % at a temperature of 29+-2 oC

Evidence for adaptation of porcine Toll-like receptors

Naturally endemic infectious diseases provide selective pressures for pig populations. Toll-like receptors (TLRs) represent the first line of immune defense against pathogens and are likely to play a crucial adaptive role for pig populations. This study was done to determine whether wild and domestic pig populations representing diverse global environments demonstrate local TLR adaptation. The genomic sequence encoding the ectodomain, responsible for interacting with pathogen ligands of bacterial (TLR1, TLR2 and TLR6) and viral (TLR3, TLR7 and TLR8) receptors, was obtained. Mitochondrial D-loop region sequences were obtained and a phylogenetic analysis using these sequences revealed a clear separation of animals into Asian (n = 27) and European (n = 40) clades. The TLR sequences were then analyzed for population-specific positive selection signatures within wild boars and domesticated pig populations derived from Asian and European clades. Using within-population and between-population tests for positive selection, a TLR2-derived variant 376A (126Thr), estimated to have arisen in 163,000 years ago with a frequency of 83.33 % within European wild boars, 98.00 % within domestic pig breeds of European origin, 40.00 % within Asian wild boars, and 11.36 % within Asian domestic pigs, was identified to be under positive selection in pigs of European origin. The variant is located within the N terminal domain of the TLR2 protein 3D crystal structure and could affect ligand binding. This study suggests the TLR2 gene contributing to responses to bacterial pathogens has been crucial in adaptation of pigs to pathogens.</p

Abstract 2050: Porcine drug metabolism and toxicity <i>in vitro</i> model utilizing transformed hepatocyte cell lines (HepCre)

Abstract To date, in vitro cytotoxicity assays are not highly predictive of in vivo toxicity. The adverse effects of new drugs are often not discovered until preclinical animal safety studies or even clinical trials; 40% of drugs drop out in preclinical animal studies and 89% of those that reach clinical trials fail. There is a critical need for more predictive and reliable in vitro testing methods. Due to its physiological similarities with humans, pigs have emerged as a suitable and reliable animal model for pharmacological and toxicological studies. To further the pigs’ suitability, we have developed and characterized a transformed porcine hepatocyte cell line to support drug toxicity and metabolism assessments. Primary porcine hepatocytes isolated by a modified procedure of Panda's method (Panda et al., 2015) express phase I and II drug metabolism, phase-III transport, and nuclear receptors involved in regulation of drug metabolism transcripts. However, primary hepatocytes have a limited life span in culture and usually within 8 days post culture more than 50% of cells undergo apoptosis. Moreover, normal gene expression declines from day 5 in culture. To overcome these limitations, we have generated and characterized transformed hepatocyte cell lines (HepCre) derived from the transgenic Oncopig. Hepatocytes were isolated from Oncopigs (Schook et at., 2015) and transformed by treatment with Cre recombinase. When cultured in the presence of a differentiating agent (DMSO), the HepCre cell lines maintained normal hepatic functions, as well as the potential to express all the main drug metabolism and regulation genes comparable to that of cultured primary hepatocytes. The effect of model CYP (cytochrome P450) inducers (3- methylcholanthrene, rifampicin and phenobarbital) on the expression of CYP enzymes in primary hepatocytes and HepCre were studied. Treatment of 3-methylcholanthrene caused a significant upregulation of CYPA1 and CYPA2 transcripts, but the transcript of other CYPs remain unchanged. Both rifampicin and phenobarbital exposure resulted in the upregulation of several CYP transcripts, including CYP2A19, CYP2B22, and CYP3A. The gene transcription patterns in HepCre were similar to those of primary hepatocytes and in vitro human models. Toxicity responses of HepCre cells to hepatotoxic drugs (i.e. aflatoxin B1, chlorpromazine, amiodarone, and acetaminophen) was evaluated. After 72 hr of exposure, cell viability decreased in a concentration-dependent manner with IC50 of 5 μm, 50 μm, 20 μm, and 70 μm for aflatoxin B1, amiodarone, chlorpromazine, and acetaminophen, respectively. These findings indicate that this porcine transformed HepCre cell line represents a useful and predictive model for high throughput screening of new drugs as well as studies on metabolism and hepatotoxicity of chemicals. References: Panda S et al., (2015) PLoS ONE 10 (3): e0118841. Schook, L.B et al (2015). PLoS One 10: e0128864. Citation Format: Arun K. De, Kwame A. Darfour-Oduro, Laurie Rund, Kyle M. Schachtschneider, Kuldeep Singh, Lawrence B. Schook. Porcine drug metabolism and toxicity in vitro model utilizing transformed hepatocyte cell lines (HepCre). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2050.</jats:p

Adaptive Evolution of Toll-Like Receptors (TLRs) in the Family Suidae.

Members of the family Suidae have diverged over extended evolutionary periods in diverse environments, suggesting that adaptation in response to endemic infectious agents may have occurred. Toll-like receptors (TLRs) comprise a multigene family that acts as the first line of defense against infectious microbes at the host-environment interface. We hypothesized that across the Suidae, positive selection mediated by infectious agents has contributed to the evolution of TLR diversity. Thus, we analyzed Sus scrofa, Sus barbatus, Sus verrucosus, Sus celebensis, Sus scebifrons, Babyrousa babyrussa, Potamochoerus larvatus, Potamochoerus porcus and Phacochoerus africanus genomes. Specifically, analyses were performed to identify evidence of positive selection using Maximum likelihood (ML) methods within a phylogenetic framework for bacterial and viral sensing Suidae TLR extracellular domains. Our analyses did not reveal evidence of positive selection for TLR3 and TLR7, suggesting strong functional conservation among these two genes for members of the Suidae. Positive selection was inferred for Suidae TLR1, TLR2, TLR6 and TLR8 evolution. ML methods identified amino acid sites of the bacterial sensing TLR1, TLR2, TLR6 and the viral sensing TLR8 to be under persistent positive selection. Some of these sites are in close proximity to functionally relevant sites, further strengthening the case for pathogen mediated selection for these sites. The branch leading to the genus Sus demonstrated evidence of episodic positive selection for TLR1, indicating selection mediated by infectious agents encountered within the specific geographic origin of the Sus. These results indicate that species of the Suidae have positively selected residues within functional domains of TLRs reflective of prior infections. Thus, TLR genes represent candidates for experimental validation to determine their functional role in antibacterial and antiviral activity within members of the Suidae