Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

Expression of the genes for arylamine N-acetyltransferases in mice

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes involved in the metabolism of arylamine and hydrazine xenobiotics. Murine NAT isoenzymes are encoded by three Nat genes. Only Nat2 was previously known to be polymorphic, a single nucleotide substitution causing the slow acetylator phenotype in the A/J strain. The present study (Chapter 3) describes novel polymorphisms in all three Nat genes of the wild-derived inbred strains Mus spretus and Mus musculus castaneus. Functional analysis of hepatic and heterologously expressed NAT variants from the two strains demonstrated that M. m. castaneus is a fast and M. spretus a slow acetylator. A previously isolated 14.3kb Nat-positive mouse genomic clone of 129/Ola strain origin (clone A) was sequenced (Chapter 4). A 8.6kb HindIII fragment, containing the entire Nat2 coding region, was subcloned from clone A and used to generate a targeting construct for the production of Nat2 knock-out mice. Probes and appropriate PCR methodologies were developed for screening of embryonic stem cells for targeted incorporation of the construct (Chapter 4). Computational analysis of clone A sequence revealed a number of important elements around the Nat2 gene, including four microsatellite markers which were found to be polymorphic among different mouse strains. Combined with preliminary physical mapping work (Chapter 4), these markers will assist accurate localisation of the Nat genes on mouse chromosome 8. A non-coding exon was mapped 6.4-6.1kb upstream of the intronless Nat2 coding region. A functional polyadenylation signal was also identified 0.45kb downstream of the mouse Nat2 coding region. The genomic structure of other genes for mammalian NAT was also analysed, by comparison of ESTs and genomic sequences deposited in electronic databases. Reverse transcription PCR confirmed that Nat2 is expressed in many tissues, while Nat1 and Nat3 are expressed in the liver and spleen, respectively. The Nat2 transcript was also detected in mouse embryonic stem cells, suggesting a possible involvement of murine NAT2 early in development (Chapter 5). The elements constituting the core promoter of Nat2 were characterised, and a preliminary search for other transcriptional regulatory sequences was carried out, using reporter gene assays and electrophoretic mobility shift assays (Chapter 6)

Comparative analysis of xenobiotic metabolising N-acetyltransferases from ten non-human primates as in vitro models of human homologues

Xenobiotic metabolising N-acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M. sylvanus, the NAT2 of Erythrocebus patas, and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates

Continuous relaxation of MINLP problems by penalty functions: a practical comparison

A practical comparison of penalty functions for globally solving mixed-integer nonlinear programming (MINLP) problems is presented. The penalty approach relies on the continuous relaxation of the MINLP problem by adding a specific penalty term to the objective function. A new penalty algorithm that addresses simultaneously the reduction of the error tolerances for optimality and feasibility, as well as the reduction of the penalty parameter, is designed. Several penalty terms are tested and different penalty parameter update schemes are analyzed. The continuous nonlinear optimization problem is solved by the deterministic DIRECT optimizer. The numerical experiments show that the quality of the produced solutions are satisfactory and that the selected penalties have different performances in terms of efficiency and robustness.This work has been supported by COMPETE: POCI-01-0145-FEDER-007043 and FCT - Fundação para a Ciência e Tecnologia, within the projects UID/CEC/00319/2013 and UID/MAT/00013/2013.info:eu-repo/semantics/publishedVersio

Approaches to modelling the shape of nanocrystals.

Unlike in the bulk, at the nanoscale shape dictates properties. The imperative to understand and predict nanocrystal shape led to the development, over several decades, of a large number of mathematical models and, later, their software implementations. In this review, the various mathematical approaches used to model crystal shapes are first overviewed, from the century-old Wulff construction to the year-old (2020) approach to describe supported twinned nanocrystals, together with a discussion and disambiguation of the terminology. Then, the multitude of published software implementations of these Wulff-based shape models are described in detail, describing their technical aspects, advantages and limitations. Finally, a discussion of the scientific applications of shape models to either predict shape or use shape to deduce thermodynamic and/or kinetic parameters is offered, followed by a conclusion. This review provides a guide for scientists looking to model crystal shape in a field where ever-increasingly complex crystal shapes and compositions are required to fulfil the exciting promises of nanotechnology

Wulff-Based Approach to Modeling the Plasmonic Response of Single Crystal, Twinned, and Core-Shell Nanoparticles.

The growing interest in plasmonic nanoparticles and their increasingly diverse applications is fuelled by the ability to tune properties via shape control, promoting intense experimental and theoretical research. Such shapes are dominated by geometries that can be described by the kinetic Wulff construction such as octahedra, thin triangular platelets, bipyramids, and decahedra, to name a few. Shape is critical in dictating the optical properties of these nanoparticles, in particular their localized surface plasmon resonance behavior, which can be modeled numerically. One challenge of the various available computational techniques is the representation of the nanoparticle shape. Specifically, in the discrete dipole approximation, a particle is represented by discretizing space via an array of uniformly distributed points-dipoles; this can be difficult to construct for complex shapes including those with multiple crystallographic facets, twins, and core-shell particles. Here, we describe a standalone user-friendly graphical user interface (GUI) that uses both kinetic and thermodynamic Wulff constructions to generate a dipole array for complex shapes, as well as the necessary input files for DDSCAT-based numerical approaches. Examples of the use of this GUI are described through three case studies spanning different shapes, compositions, and shell thicknesses. Key advances offered by this approach, in addition to simplicity, are the ability to create crystallographically correct structures and the addition of a conformal shell on complex shapes

Analysis of the PRNP gene polymorphisms in healthy Greek sheep during 2012 – 2016

Η τρομώδης νόσος (scrapie) αποτελεί μια βραδέως εξελισσόμενη λοιμώδης νόσος των προβάτων και των αιγών, η οποία οδηγεί σε εκφυλισμό του κεντρικού νευρικού συστήματος. Η scrapie είναι μια μορφή των μεταδιδόμενων σπογγωδών εγκεφαλοπαθειών (TSEs) όπως και η σπογγώδη εγκεφαλοπάθεια των βοειδών (BSE). Στα πρόβατα είναι γνωστό ότι οι πολυμορφισμοί στα κωδικόνια 136, 154 και 171 του γονιδίου PRPN που κωδικοποιεί τη πρωτεΐνη PrP σχετίζονται άμεσα με την ευαισθησία/ανθεκτικότητα της φυσικής και πειραματικής κλασικής scrapie. Σε πολλές χώρες, αλλά όχι στην Ελλάδα έχουν εφαρμοστεί προγράμματα με στόχο την αύξηση της ανθεκτικότητας στη νόσο. Η παρούσα εργασία στηρίχτηκε κυρίως στην ιδιωτική πρωτοβουλία παραγωγών που ήταν πρόθυμοι να βελτιώσουν την εκτροφή τους αυξάνοντας την ανθεκτικότητα στην τρομώδη νόσο. Έτσι μελετήθηκαν οι γονότυποι του γονιδίου PrP (των τριών προαναφερθέντων κωδικονίων) σε 5815 δείγματα αίματος από υγιή κριάρια προερχόμενα από 160 υγιείς εκτροφές, κατά τη χρονική περίοδο 2012-2016. Επιπρόσθετα σε 1399 δείγματα αίματος αναλύθηκαν μόνο οι πολυμορφισμοί του κωδικονίου 171. Οι γονοτυπικές αναλύσεις πραγματοποιήθηκαν με τη χρήση της Real Time PCR (TaqMan ανιχνευτές) χρησιμοποιώντας ειδικά σημασμένους ανιχνευτές. Τα αποτελέσματά μας παρουσιάζουν αυξημένα ποσοστά των δύο γονοτύπων ARR/ARR και ARR/ARQ, οι οποίοι συνδέονται με ανθεκτικότητα έναντι της νόσου (27.29% και 34.6%, αντίστοιχα) και σχετικά μειωμένο το ποσοστό του γονότυπου ARQ/ARQ (24.23%) που συνδέεται με ευαισθησία έναντι της νόσου και ο οποίος είναι ο πιο κοινός γονότυπος στις Ελληνικές εκτροφές. Αυτή η κοινή προσπάθεια οδήγησε στην καθιέρωση ενός σημαντικού αριθμού εκτροφών που διαθέτουν αυξημένο πληθυσμό κριαριών γενετικά ανθεκτικών στην κλασική scrapie.Scrapie is a slowly progressive infectious disease of sheep and goats that causes degeneration of the central nervous system. Scrapie is one of several transmissible spongiform encephalopathies (TSEs), like the bovine spongiform encephalopathy (BSE). In sheep, polymorphisms at codons 136, 154 and 171 of the host gene PRPN that encodes the PrP protein, are known to be closely linked to susceptibility or resistance to natural and experimental classical scrapie. In many countries, but not in Greece, breeding programs have been implemented to increase genetic resistance. This study was supported mainly by the private initiatives of farmers willing to improve their flocks by increasing the resistance to scrapie. Thus, the PrP genotypes (of the three mentioned codons) from 5815 blood samples of clinically healthy rams from 160 healthy flocks during the period 2012 – 2016 were determined. Additionally, 1399 blood samples were genotyped only for the 171 codon. Samples were analyzed by Real Time PCR (TaqMan probes) with specific labeled probes. Our results showed an increased percentage of the two genotypes, ARR/ARR and ARR/ARQ linked with resistance to the disease (27.29% and 34.6%, respectively) and relatively reduced percentage of the genotype ARQ/ARQ (24.23%) which is associated with susceptibility to disease and is the most common genotype in the Greek flocks. This joined effort has resulted in the establishment of an important number of farms with an increased population of genetically resistant rams to classical scrapie