A 3D structural model and dynamics of hepatitis C virus NS3/4A protease (genotype 4a,strain ED43) suggest conformational instability of the catalytic triad: implications in catalysis and drug resistivity

Egypt has the highest prevalence of hepatitis C virus (HCV) infection worldwide with a frequency of 15%. More than 90% of these infections are due to genotype 4, and the subtype 4a (HCV-4a) predominates. Moreover, due to the increased mobility of people, HCV-4a has recently spread to several European countries. The protease domain of the HCV nonstructural protein 3 (NS3) has been targeted for inhibition by several drugs. This approach has had marked success in inhibiting genotype 1 (HCV-1), the predominant genotype in the USA, Europe, and Japan. However, HCV-4a was found to resist inhibition by a number of these drugs, and little progress has been made to understand the structural basis of its drug resistivity. As a step forward, we sequenced the NS3 HCV-4a protease gene (strain ED43) and subsequently built a 3D structural model threaded through a template crystal structure of HCV-1b NS3 protease. The model protease, HCV-4a, shares 83% sequence identity with the template protease, HCV-1b, and has nearly identical rigid structural features. Molecular dynamics simulations predict similar overall dynamics of the two proteases. However, local dynamics and 4D analysis of the interactions between the catalytic triad residues (His57, Asp81, and Ser139) indicate conformational instability of the catalytic site in HCV-4a NS3 protease. These results suggest that the divergent dynamics behavior, more than the rigid structure, could be related to the altered catalytic activity and drug resistivity seen in HCV-4a.     

Long-term culture of primary porcine oviduct epithelial cells: Validation of a comprehensive in vitro model for reproductive science

Recently, we established a protocol for the cultivation of primary porcine oviduct epithelial cells (POEC), which promoted tissue-like morphology for a prolonged culture period. The present study focuses on developing this model into a comprehensive, standardized culture system, as a candidate tool for reproductive toxicity testing and basic research. We cultivated POEC isolated from 25 animals in our culture system for both 3 and 6 weeks and systematically analyzed effects of medium conditioning, supplementation with standardized sera, and culture duration in both freshly isolated and cryopreserved cells. The differentiation status was evaluated via histomorphometry, transepithelial electrical resistance (TEER) measurement, and expression analyses. The culture system possessed high reproducibility, more than 95% of cultures achieved a fully differentiated phenotype. Cells recapitulated in vivo–like morphology and ultrastructure from 3 to 6 weeks. Cryopreservation of the cells prior to cultivation did not affect culture quality of POEC. Employment of conditioned medium ensured optimal promotion of POEC differentiation, and different standardized sera induced fully differentiated phenotypes. Consistent TEER establishment indicated the presence and maintenance of cell type–specific intercellular junctions. The functionality of POEC was proven by consistent mucin secretion and stable expression of selected markers over the whole culture duration. We conclude that POEC are suitable for experiments from 3 weeks up to at least 6 weeks of culture. Therefore, this culture system could be used for in vitro estrous cycle simulation and long-term investigation of toxic effects on oviduct epithelium.     

Genetic Mapping and Genomic Selection Using Recombination Breakpoint Data

The correct models for quantitative trait locus mapping are the ones that simultaneously include all significant genetic effects. Such models are difficult to handle for high marker density. Improving statistical methods for high-dimensional data appears to have reached a plateau. Alternative approaches must be explored to break the bottleneck of genomic data analysis. The fact that all markers are located in a few chromosomes of the genome leads to linkage disequilibrium among markers. This suggests that dimension reduction can also be achieved through data manipulation. High-density markers are used to infer recombination breakpoints, which then facilitate construction of bins. The bins are treated as new synthetic markers. The number of bins is always a manageable number, on the order of a few thousand. Using the bin data of a recombinant inbred line population of rice, we demonstrated genetic mapping, using all bins in a simultaneous manner. To facilitate genomic selection, we developed a method to create user-defined (artificial) bins, in which breakpoints are allowed within bins. Using eight traits of rice, we showed that artificial bin data analysis often improves the predictability compared with natural bin data analysis. Of the eight traits, three showed high predictability, two had intermediate predictability, and two had low predictability. A binary trait with a known gene had predictability near perfect. Genetic mapping using bin data points to a new direction of genomic data analysis.     

Evidence that the Echinococcus granulosus G6 genotype has an affinity for the brain in humans

The present study investigates the molecular characteristics of cerebral Echinococcus cysts. A total of 10 specimens of cerebral Echinococcus cysts, including six formalin-fixed paraffin blocks and four intact cerebral cysts, were used for this study. The target DNA was successfully amplified from eight samples and sequenced. BLAST analysis indicated that sequenced isolates belong to the Echinococcus granulosus (G6) genotype. All of the eight sampled brain cysts belonged to the G6 genotype, while all of the eight liver cysts belonged to G1. This is a strong indication that G6 has a higher affinity for the human brain than G1.     

A case of primary amenorrhea with 46+XY genotype from Kashmir Valley

Primary amenorrhea is one of the common reproductive disorder affecting females. It leads to the absence of menarche in the reproductive age group in females and/or complete absence of reproductive organs. There are many causes which lead to PA, including genetic aberrations which are the leading factors.     

Transformation of Trichothecenes in Ileal Digesta and Faeces from Pigs

The capacity of pig gastrointestinal microflora to metabolise the trichothecenes 3-acetyl-deoxynivalenol (3-acDON) and nivalenol (NIV) was investigated. 3-acDON was deacetylated to DON in anaerobic incubations with pig faeces collected at different pig farms. Furthermore, both 3-acDON and NIV were metabolised to the corresponding de-epoxy metabolite in these incubates. Five pigs, in which the gastrointestinal microflora lacked the ability to transform 3-acDON and NIV to their corresponding de-epoxidated metabolites, were given low levels of DON in the feed for seven weeks. The gastrointestinal micro-organisms did not acquire the de-epoxidation ability during the seven week long exposure period. At the end of the exposure period, faeces from pigs with a known de-epoxidation ability was spread out in the pens and left for 24 hours. One week after the faeces had been spread out in the pens, the de-epoxidation ability was found in faecal incubations from four out of five experimental pigs. This change in metabolic ability of the intestinal de-epoxidation ability was not accompanied by any detectable changes in the DNA-profiles of the bacterial community composition. The results show that the intestinal de-epoxidation ability is common at pig farms in the Uppsala area, and that the ability may be transferred between pigs in a stock.     

Effect of Dietary Mono- and Polyunsaturated Fatty Acids on the Fatty Acid Composition of Pigs' Adipose Tissues

In two experiments with growing-finishing pigs six different dietary fats were added to a conventional diet (control - C) to study the effects of dietary monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) on the fatty acid composition of backfat and kidney fat at similar amounts of double bonds in feed (Exp. 1:7% pork fat - PF, 4.95% olive oil - OO, 3.17% soybean oil - SO) or a constant amount of 5% of processed fats (Exp. 2: partially hydrogenated fat - SAT, fractionated pork fats: olein - OLE, stearin - STE). Compared with the control, PUFA were only slightly increased in backfat of pigs fed PF, OLE, STE or OO, although dietary PUFA intake was up to 70% higher. With SO PUFA were significantly increased in adipose tissues, predominantly at the expense of MUFA. Consequently, a non-linear relationship was found between PUFA intake and proportion in backfat. MUFA were incorporated at the expense of SFA, therefore, adipose tissues of OO fed animals were lowest in SFA. Despite comparable amounts of double bonds in feed (Exp. 1), the degree of unsaturation measured as fat score (sum of double bonds) was in the order SO > OO > PF > C. In contrast, the proportion of SFA was C > PF = SO > OO. Regarding the decisive role of SFA for fat consistency it may be concluded that MUFA should also be considered in feeding recommendations for pigs. Furthermore, in case of a high dietary supply of MUFA, a simple index of double bonds might not be sufficiently conclusive to judge pig fat quality.     

Effect of avilamycin and tylosin on the metabolizable energy in growing and finishing pigs

In two metabolism trials with growing and finishing pigs the influence of the antibiotic feed additives Avilamycin and Tylosin on the metabolizable energy was investigated at different levels of dietary protein content. In the first experiment (growing pigs) the antibiotics were supplied at levels of 0 mg/kg, 40 mg/kg Avilamycin and 40 mg/kg Tylosin to diets containing 18.5%, 17.5%, 16.5% and 14.0% of crude protein. In the second experiment (finishing pigs) 0 mg/kg antibiotics, 20 mg/kg Avilamycin and 20 mg/kg Tylosin were used in diets containing 16.5% and 14.0% of crude protein. The body weight of the animals averaged 46 kg (growing pigs) and 68 kg (finishing pigs).

In growing pigs the supplementation of Tylosin increased the digestibility of dry matter and energy at 1 percentage unit each, while in finishing pigs no effects were observed. Since the urinary energy excretion was not affected by antibiotics, there was only in Tylosin treated growing pigs a slight rise in dietary contents of metabolizable energy by 1.6%. The reduction of the dietary protein content resulted in increased digestibility of dry matter and energy, reduced urinary energy excretions and increased dietary contents of metabolizable energy.     

On the toxicokinetics and the metabolism of deoxynivalenol (don) in the pig

Eleven castrated male pigs weighing 88.1?±?3.9?kg on average were adapted to a diet containing DON (4.2?mg DON/kg) over a period of 7 days. Feed was given restrictively with 1.1?kg per meal (two meals per day). On the day of measurement, all pigs were slaughtered at different time intervals following the morning meal containing DON (1, 2, 3, 4, 5, 6, 8, 15, 18 and 24?h after feeding), with the exception of one pig which was slaughtered unfed. DON and de-epoxy-DON were analysed in serum and digesta from consecutive segments of the digestive tract (stomach, small intestine divided into three parts of a similar length, caecum, colon, rectum). DON was rapidly and nearly completely absorbed while passing through the stomach and the proximal small intestine. Maximum serum concentration appeared 4.1?h after the DON-containing meal and half of the systemically absorbed DON was eliminated after 5.8?h. De-epoxy-DON appeared in increasing proportions from the distal small intestine and reached approximately 80% of the sum of DON plus de-epoxy-DON in faeces collected from the rectum. It was concluded that de-epoxydation of DON, which primarily occurs in the hindgut, probably does not contribute much to a detoxification in the pig.