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The expectation-maximisation algorithm is employed to perform maximum likelihood estimation in a wide range of situations, including regression analysis based on clusterwise regression models. A disadvantage of using this algorithm is that it is unable to provide an assessment of the sample variability of the maximum likelihood estimator. This inability is a consequence of the fact that the algorithm does not require deriving an analytical expression for the Hessian matrix, thus preventing from a direct evaluation of the asymptotic covariance matrix of the estimator. A solution to this problem when performing linear regression analysis through a multivariate Gaussian clusterwise regression model is developed. Two estimators of the asymptotic covariance matrix of the maximum likelihood estimator are proposed. In practical applications their use makes it possible to avoid resorting to bootstrap techniques and general purpose mathematical optimisers. The performances of these estimators are evaluated in analysing small simulated and real datasets; the obtained results illustrate their usefulness and effectiveness in practical applications. From a theoretical point of view, under suitable conditions, the proposed estimators are shown to be consistent.

How the size of female yellowfin tuna (Thunnus albacares) affects their spawning capability and fecundity is still an open and unresolved question due to the difficulties in investigating these complex effects in highly migratory pelagic marine fish species. However, this information is key to understanding the reproductive potential and resilience of the stock. We investigate how energetic resources are allocated for reproduction by female yellowfin tuna according to their size in the Gulf of Guinea (central-eastern Atlantic Ocean). Our results reveal that larger females have not only larger ovaries by virtue of their greater abdominal cavity, but also different fatty acid profiles in the gonads compared to smaller females, with potential effects on their spawning and recruitment patterns. This study contributes to the knowledge of size-dependent variation in female yellowfin tuna and paves the way for future studies on size-dependent effects on reproductive parameters in this species.

Protected designation of origin dry-cured hams are obtained from heavy pigs (slaughtered at about 160 kg of live weight). A specific breeding program designed to improve meat quality for this production has included as key traits the level of intermuscular fat between the leg muscles and ham weight loss during the seasoning period together with a balance between fat and lean cuts. In this study we carried out genome-wide association studies for seven traits used in the genetic merit of Italian Duroc heavy pigs, five related to meat and carcass quality traits (visible intermuscular fat, ham weight loss at first salting, backfat thickness, ham weight and lean cuts), and two related to performance and efficiency traits (average daily gain and feed : gain ratio). A total of 573 performance-tested pigs were genotyped with the Illumina PorcineSNP60 BeadChip and genome-wide association analyses were carried out using the Bayes B approach with the 1 Mb window option of GenSel and random residuals for each of the seven traits. Detected windows were supported by independent single nucleotide polymorphism analyses with a linear mixed model (LMM) approach on the same animals for the same traits. A total of 30 windows identifying different quantitative trait loci (QTL) were detected and among those, 27 were confirmed by LMM in one of these traits. Among the confirmed windows, three QTL were reported for visible intermuscular fat, seven for ham weight loss at first salting and five and four for backfat thickness and lean cut, respectively. A total of eight QTL were detected for the other production traits. No overlapping QTL were reported except for one window on porcine chromosome 10 between lean cuts and ham weight that contained the CACNB2 gene that has been already associated with loin marbling score in other Duroc pigs. Several regions contained genes that have been already associated with production traits in other pig breeds, including Duroc lines, related to fat deposition or muscle structure. This work reports, for the first time, genome-wide association study results for several traits in Italian Duroc heavy pigs. These results will be useful to dissect the genetic basis for dry-cured ham production traits that determine the total genetic merit index of Italian Duroc pigs.

Commercial single nucleotide polymorphism (SNP) arrays have been recently developed for several species and can be used to identify informative markers to differentiate breeds or populations for several downstream applications. To identify the most discriminating genetic markers among thousands of genotyped SNPs, a few statistical approaches have been proposed. In this work, we compared several methods of SNPs preselection (Delta, F st and principal component analyses (PCA)) in addition to Random Forest classifications to analyse SNP data from six dairy cattle breeds, including cosmopolitan (Holstein, Brown and Simmental) and autochthonous Italian breeds raised in two different regions and subjected to limited or no breeding programmes (Cinisara, Modicana, raised only in Sicily and Reggiana, raised only in Emilia Romagna). From these classifications, two panels of 96 and 48 SNPs that contain the most discriminant SNPs were created for each preselection method. These panels were evaluated in terms of the ability to discriminate as a whole and breed-by-breed, as well as linkage disequilibrium within each panel. The obtained results showed that for the 48-SNP panel, the error rate increased mainly for autochthonous breeds, probably as a consequence of their admixed origin lower selection pressure and by ascertaining bias in the construction of the SNP chip. The 96-SNP panels were generally more able to discriminate all breeds. The panel derived by PCA-chrom (obtained by a preselection chromosome by chromosome) could identify informative SNPs that were particularly useful for the assignment of minor breeds that reached the lowest value of Out Of Bag error even in the Cinisara, whose value was quite high in all other panels. Moreover, this panel contained also the lowest number of SNPs in linkage disequilibrium. Several selected SNPs are located nearby genes affecting breed-specific phenotypic traits (coat colour and stature) or associated with production traits. In general, our results demonstrated the usefulness of Random Forest in combination to other reduction techniques to identify population informative SNPs.

Intermuscular fat content in protected designations of origin dry-cured hams is a very important meat quality trait that affects the acceptability of the product by the consumers. An excess in intermuscular fat (defined as the level of fat deposition between leg muscles) is a defect that depreciates the final product. In this study we carried out a genome-wide association study for visible intermuscular fat (VIF) of hams in the Italian Large White pig breed. This trait was evaluated on the exposed muscles of green legs in 1122 performance-tested gilts by trained personnel, according to a classification scale useful for routine and cheap evaluation. All animals were genotyped with the Illumina PorcineSNP60 BeadChip. The genome-wide association study identified three QTL regions on porcine chromosome 1 (SSC1; accounting for ~79% of the SNPs below the 5.0E−04 threshold) and SSC2, two on SSC7 and one each on SSC3, SSC6, SSC9, SSC11, SSC13, SSC15, SSC16 and SSC17. The most significant SNP (ALGA0004143 on SSC1 at 77.3 Mb; PFDRÂ

Haematological and clinical-biochemical parameters are considered indicators of the physiological/health status of animals and might serve as intermediate phenotypes to link physiological aspects to production and disease resistance traits. The dissection of the genetic variability affecting these phenotypes might be useful to describe the resilience of the animals and to support the usefulness of the pig as animal model. Here, we analysed 15 haematological and 15 clinical-biochemical traits in 843 Italian Large White pigs, via three genome-wide association scan approaches (single-trait, multi-trait and Bayesian). We identified 52 quantitative trait loci (QTLs) associated with 29 out of 30 analysed blood parameters, with the most significant QTL identified on porcine chromosome 14 for basophil count. Some QTL regions harbour genes that may be the obvious candidates: QTLs for cholesterol parameters identified genes (ADCY8, APOB, ATG5, CDKAL1, PCSK5, PRL and SOX6) that are directly involved in cholesterol metabolism; other QTLs highlighted genes encoding the enzymes being measured [ALT (known also as GPT) and AST (known also as GOT)]. Moreover, the multivariate approach strengthened the association results for several candidate genes. The obtained results can contribute to define new measurable phenotypes that could be applied in breeding programs as proxies for more complex traits.

Infectious diseases have large economic impacts on the pig breeding industry worldwide. A few genetic markers associated to disease resistance have been recently identified and used in marker-assisted selection (MAS) in a few pig populations, as part of disease control programs. Neonatal Diarrhea (ND), Post-weaning Diarrhea (PWD), caused by enterotoxigenic E. coli, and Porcine Reproductive and Respiratory Syndrome, caused by PRRS virus (PRRSV), are diseases with high priority for the pig industry. Since interactions or antagonism between growth, innate immunity and disease resistance traits could exist, this work investigated if four disease resistance gene markers [mucin 4 (MUC4), rs338992994 associated with resistance to ND; fucosyltransferase 1 (FUT1), rs335979375 associated with resistance to PWD; and guanylate binding protein 1 (GBP1), rs340943904 and rs80800372 (also known as WUR10000125) associated with resistance to PRRS] could affect seven production traits (average daily gain, back fat thickness, lean meat cuts, feed gain ratio, ham weight, visible intermuscular fat and ham weight loss at first salting) and 15 haematological parameters in about 550 performance tested Italian Large White pigs. We also monitored allele frequencies of the same markers on Italian Large White boars sampled under the national selection breeding program over a 20 years-period. Moreover, we evaluated allele frequencies of these polymorphisms in four Italian local pig breeds (Apulo-Calabrese, Casertana, Cinta Senese and Nero Siciliano). The frequency of the resistance-associated alleles for the four polymorphisms was usually higher in all local pig breeds, indirectly supporting a higher rusticity of autochthonous breeds, compared to commercial populations. The two GBP1 polymorphisms were not in complete linkage disequilibrium in all breeds, except in Apulo-Calabrese. No significant allele frequency change for the investigated markers occurred over 20 years in the Italian Large White boar population in three of these markers. Only FUT1 showed a modest but significant change of allele frequencies over this period. Association analyses carried out in Italian Large White pigs for production traits, meat quality or haematological parameters under investigation showed no significant effect of any genotyped polymorphisms. Our results indicate that implementing MAS programs in Italian Large White pigs with polymorphisms associated with disease resistance have no direct effects on production traits. The selection program running for this heavy pig breed might not negatively impact disease resistance derived by the investigated major genes.

Summary Calcium, magnesium and phosphorus are essential electrolytes involved in a large number of biological processes. Imbalance of these minerals in blood may indicate clinically relevant conditions and are important in inferring acute or chronic pathologies in humans and animals. In this work, we carried out a genome-wide association study (GWAS) for the level of these three electrolytes in the serum of 843 performance-tested Italian Large White pigs. All pigs were genotyped with the Illumina PorcineSNP60 BeadChip, and GWAS was carried out using genome-wide efficient mixed-model association. For the level of Ca2+, eight single nucleotide polymorphisms (SNPs) were significant, considering a false discovery rate (FDR)

This letter illustrates simple assumptions for proving consistency of the maximum likelihood estimator under multivariate Gaussian and Student’s t linear cluster-weighted models, which allow density estimation, clustering and linear regression analysis with continuous random predictors in presence of unobserved heterogeneity.

We genotyped 58 single nucleotide polymorphisms (SNPs) in 25 candidate genes in about 800 Italian Holstein sires. Fifty-six (minor allele frequency >0.02) were used to evaluate their association with single traits: milk yield (MY), milk fat yield (FY), milk protein yield (PY), milk fat percentage (FP), milk protein percentage (PP), milk somatic cell count (MSCC); and complex indexes: longevity, fertility and productivity-functionality type (PFT), using deregressed proofs, after adjustment for familial relatedness. Thirty-two SNPs were significantly associated (proportion of false positives

Melanocortin 4 receptor (MC4R) plays a key role in controlling energy homeostasis. Several studies have already reported effects on production traits of polymorphisms identified in the porcine MC4R gene. In this study we analysed data on 6 MC4R polymorphisms (c.-780C>G; c.-135C>T; c.175C>T; c.707G>A or p.Arg236His; and c.892G>A or p.Asp298Asn; c.*430A>T) genotyped from (1) two groups of Italian Large White pigs (280+280 animals) with extreme estimated breeding values (EBVs) for back fat thickness (BFT), selected among a performance tested population of about 12,000 pigs, and from (2) 19 Italian Duroc pigs. Two haplotypes, differentiated by the c.892G>A, were identified in the Duroc populations. Four haplotypes were identified in the Italian Large White population, one of which (haplotype 4) was identified for the first time in this study. Single marker and haplotype association analyses for BFT were obtained by comparing allele and haplotype frequency differences from the two extreme tails using χ2 and Cochran-Armitage trend tests. Results confirmed the effects of the c.892G>A single nucleotide polymorphism (SNP) on BFT, as also defined by different distributions in the two tails of haplotypes carrying the alternative nucleotides at this polymorphic site (P

Valproate (VPA) is an anti-epileptic and mood-stabilizing drug with a broad range of action and which mechanism of action still remains in part elusive. Recently the discovery that VPA modifies the epigenome increasing the transcriptional rate of target genes raises the issue of understanding the exact role of this mechanism. In this work we tested the possibility that VPA could modify the epigenome of lymphomonocytes (PBMC) obtained from epileptic patients chronically treated in monotherapy with VPA and phenobarbital. Acetyl-histone H3 expression was assessed by western blotting and global DNA methylation by incorporation of [(3)H]dCTP. A significant increase in histone acetylation and a correlated decrease of global DNA methylation were shown at VPA therapeutically relevant plasma concentrations. This effect was drug-related, since it was not demonstrated in PBMC obtained from phenobarbital-treated patients. Moreover, a VPA dose-response curve was performed on PBMC obtained from healthy controls, demonstrating an increase of acetyl-histone H3 content. We suggest that the epigenetic properties of VPA expressed on PBMC at these concentrations might be operative in different tissues, with possible implications for the field of neuropsychiatric disorders.

Many-body interactions with the underlying bulk electrons determine the properties of confined electronic states at the surface of a metal. Using momentum resolved nonlinear photoelectron spectroscopy we show that one can tailor these many-body interactions in graphite, leading to a strong renormalization of the dispersion and linewidth of the image potential state. These observations are interpreted in terms of a basic self-energy model, and may be considered as exemplary for optically induced many-body interactions.

In this study, we investigated at the genome-wide level if 20 years of artificial directional selection based on boar genetic evaluation obtained with a classical BLUP animal model shaped the genome of the Italian Large White pig breed. The most influential boars of this breed (n = 192), born from 1992 (the beginning of the selection program of this breed) to 2012, with an estimated breeding value reliability of >0.85, were genotyped with the Illumina Porcine SNP60 BeadChip. After grouping the boars in eight classes according to their year of birth, filtered single nucleotide polymorphisms (SNPs) were used to evaluate the effects of time on genotype frequency changes using multinomial logistic regression models. Of these markers, 493 had a PBonferroni

Using time-resolved reflectivity measurements on unaligned and aligned bundled single-wall carbon nanotubes with a pump energy of 1.55 eV, quasi-resonant with the second Van Hoye singularity of semiconducting tubes, a positive sign of the transient reflectivity is detected in unaligned nanotubes. In contrast a negative sign is detected in aligned nanotubes. This discovery addresses a long-standing question showing that in unaligned nanotubes the stronger intertube interactions favor the formation of short-lived free charge carriers in semiconducting tubes. A detailed analysis of the transient reflectivity spectral response shows that the free carriers in the photo-excited state of semiconducting tubes move towards metallic tubes in about 400 fs. (C) 2011 Elsevier Ltd. All rights reserved.

A pilot study using Affymetrix Gene Chip(R) Porcine Genome Arrays was set up to evaluate the impact of time lags from death on gene expression profiling of porcine skeletal muscle at four post mortem times (up to 24 h) during the routine processing of fresh thighs. All post chip parameters and data analyses (Average background, Scale Factors, Percent Presence, 3'/5' ratios of beta-actin and glyceraldehyde-3-phosphase dehydrogenase control genes, RNA degradation diagnostics, principal component analysis, hierarchical clustering, mixed regression models with time effects) did not show any effect of post mortem time. Therefore, microarray data obtained from muscle specimens collected in a processing plan over a quite long period have the potential to identify treatments or pre mortem conditions without any potential bias derived from subtle RNA degradation. These results open new perspectives to develop and analyse gene expression biomarkers for pig production and product authentication.

Single-layer graphene supported on a metal surface has shown remarkable properties relevant for novel electronic and optoelectronic devices. However, the nature of the electronic states derived from unoccupied surface states and quantum well states, lying in the real-space gap between the graphene and the solid surface, has not been explored and exploited yet. Herein, we use ultraviolet nonlinear angle-resolved photoemission spectroscopy to unveil the coexistence at the graphene/Cu(111) interface of a highest occupied Shockley surface state (HOSS) and the two lowest unoccupied surface states (LUSS). The experimental results and electronic structure calculations, based on one-dimensional model potential, indicate that the two unoccupied states originate from the hybridization of an n = 1 image potential state with a quantum well state. The hybridized nature of these unoccupied states is benchmarked by a similar experiment done on single-layer graphene grown on copper polycrystalline foil where only the image state survives being the quantum well state at this interface inhibited.

Metabolomics has opened new possibilities to investigate metabolic differences among animals. In this study, we applied a targeted metabolomic approach to deconstruct the pig sex metabolome as defined by castrated males and entire gilts. Plasma from 545 performance-tested Italian Large White pigs (172 castrated males and 373 females) sampled at about 160 kg live weight were analyzed for 186 metabolites using the Biocrates AbsoluteIDQ p180 Kit. After filtering, 132 metabolites (20 AA, 11 biogenic amines, 1 hexose, 13 acylcarnitines, 11 sphingomyelins, 67 phosphatidylcholines, and 9 lysophosphatidylcholines) were retained for further analyses. The multivariate approach of the sparse partial least squares discriminant analysis was applied, together with a specifically designed statistical pipeline, that included a permutation test and a 10 cross-fold validation procedure that produced stability and effect size statistics for each metabolite. Using this approach, we identified 85 biomarkers (with metabolites from all analyzed chemical families) that contributed to the differences between the 2 groups of pigs ( < 0.05 at the stability statistic test). All acylcarnitines and almost all biogenic amines were higher in castrated males than in gilts. Metabolites involved in tryptophan catabolism had the largest differences (i.e., delta = 20% for serotonin) between castrated males (higher) and gilts (lower). The level of several AA (Ala, Arg, Gly, His, Lys, Ser, Thr, and Trp) was higher in gilts (delta was from approximately 1.0 to approximately 4.8%) whereas products of AA catabolism (taurine, 2-aminoadipic acid, and methionine sulfoxide) were higher in castrated males (delta was approximately 5.0-6.0%), suggesting a metabolic shift in castrated males toward energy storage and lipid production. Similar general patterns were observed for most sphingomyelins, phosphatidylcholines, and lysophosphatidylcholines. Metabolomic pathway analysis and pathway enrichment identified several differences between the 2 sexes. This metabolomic overview opened new clues on the biochemical mechanisms underlying sexual dimorphism that, on one hand, might explain differences in terms of economic traits between castrated male pigs and entire gilts and, on the other hand, could strengthen the pig as a model to define metabolic mechanisms related to fat deposition.

The development of effective feeding strategies to reduce the detrimental effect of enterotoxigenic F4ac (ETEC) plays a crucial role in reducing the occurrence of therapeutic intervention with antibiotics in livestock. The ability of CNCM I-4407 (SCC), supplied in different patterns to counteract ETEC infection in weaned pigs, was evaluated. Fifty pigs weaned at 24 d were then divided into 5 groups: control (CO), CO + colistin (AB), CO + 5 × 10(10) cfu of SCC/ kg feed, from d 0 to 21 (PR), CO + 5 × 10(10) cfu of SCC/ kg feed from d 7 to 11 (CM), and CO + 1 shot of 2 × 10(11) cfu of SCC when the first diarrhea appeared (CU). On d 7 postweaning, all the pigs were orally challenged with 10(8) cfu of ETEC. Blood samples were taken from the pigs (d 7, 8, 12, and 21) while the fecal excretion of ETEC was assessed on d 7 and 10. Fecal consistency was scored from 12 h before infection to 144 h postinfection (p.i.). On d 21, the pigs were sacrificed. The in vitro adhesion test on the intestinal villi confirmed individual susceptibility to ETEC, excluding the presence of resistant pigs. Growth performance did not differ between the treatments. Mortality was reduced in the AB group (P< 0.01) and, marginally, in the PR group (P = 0.089) when compared to the CO group. The CO group had a higher fecal score than AB in the period of observation (from P = 0.01 to P< 0.001). Yeast administration reduced the fecal score when compared to the CO group 12 and 48 h p.i. (P = 0.04). Total IgA never differed among the treatments, but the ETEC-specific IgA concentration was lower in the AB group than in CO (P = 0.04) at d 12. Four days p.i., the pigs fed live yeast had reduced ETEC excretion compared with the CO pigs (P = 0.05). Blood concentrations of dodecenoyl-L-carnitine (P < 0.01), glutaryl-L-carnitine/hydroxyhex¬anoyl-L-carnitine, phosphatidylcholine diacyl and phosphatidylcholine diacyl (P = 0.01 and P< 0.01, respectively), and α-amino adipic acid (P < 0.01) were reduced in the AB group compared to the CO group; PR + CM reduced the concentration of sphingomyelin-ceramide (P = 0.02) and increased the concentration of decadienyl-L-carnitine (C10:2; P= 0.02) vs. CO. The CM group had an increased concentration of C10:2 (P < 0.01) compared to the PR group. In conclusion, the administration of live yeast, even in concomitance with ETEC infections, reduces pig illness and mortality. The strain of SCC tested did not show a therapeutic effect.

The non-linear photoelectron spectra obtained by short laser pulses from a Ag(100) surface show a high-energy electron emission due to an athermal electron distribution created by the laser pulse. By comparing the photoemission at normal and non-normal emission geometry it is possible to evidence the independence of the hot electron photoemission on the parallel momentum and on different final-state configurations. A photoemission correlation measurement evidences that non-photoelectric effects, as tunneling or thermally assisted photoemission, do not contribute to the electron yield. Various theoretical models are discussed on the basis of the present data.

Summary The genetic identification of the population of origin of individuals, including animals, has several practical applications in forensics, evolution, conservation genetics, breeding and authentication of animal products. Commercial high-density single nucleotide polymorphism (SNP) genotyping tools that have been recently developed in many species provide information from a large number of polymorphic sites that can be used to identify population-/breed-informative markers. In this study, starting from Illumina BovineSNP50 v1 BeadChip array genotyping data available from 3711 cattle of four breeds (2091 Italian Holstein, 738 Italian Brown, 475 Italian Simmental and 407 Marchigiana), principal component analysis (PCA) and random forests (RFs) were combined to identify informative SNP panels useful for cattle breed identification. From a PCA preselected list of 580 SNPs, RFs were computed using ranking methods (Mean Decrease in the Gini Index and Mean Accuracy Decrease) to identify the most informative 48 and 96 SNPs for breed assignment. The out-of-bag (OOB) error rate for both ranking methods and SNP densities ranged from 0.0 to 0.1% in the reference population. Application of this approach in a test population (10% of individuals pre-extracted from the whole data set) achieved 100% of correct assignment with both classifiers. Linkage disequilibrium between selected SNPs was relevant (r2 > 0.6) only in few pairs of markers indicating that most of the selected SNPs captured different fractions of variance. Several informative SNPs were in genes/QTL regions that affect or are associated with phenotypes or production traits that might differentiate the investigated breeds. The combination of PCA and RF to perform SNP selection and breed assignment can be easily implemented and is able to identify subsets of informative SNPs useful for population assignment starting from a large number of markers derived by high-throughput genotyping platforms.

SummaryIn this study, we investigated whether a selection programme based onboar genetic evaluation obtained with a classical BLUP animal model canchange allele frequencies in a pig population. All Italian Large White boarsborn from 1992 to 2012 with estimated breeding value reliability >0.85(n = 200) were selected among all boars of this breed. Boars were geno-typed with markers in major genes (IGF2 intron3-g.3072G>A, MC4Rp.D298N, VRTN PRE1 insertion, PRKAG3 p.I199V and FTO g.276T>G).Genotyping data were analysed grouping boars in eight classes accordingto their year of birth. To evaluate the influence of time on allele frequen-cies of the genotyped markers, multinomial logistic regression modelswere computed. Four of five polymorphic sites (IGF2, MC4R, VRTN andFTO) showed significant (p < 0.01) changes in allele frequencies over timedue to a progressive and continuous increase of one allele (associated withhigher lean meat content, higher average daily gain and favourable feed:gain ratio) and, consequently, decrease of the other one, following thedirectional selection of the selection programme of this pig breed. The ret-rospective analysis that was carried out in Italian Large White boars sug-gests that selection based on methodologies assuming the infinitesimalmodel is able to modify in a quite short period of time allele frequencies inmajor genes, increasing the frequency of alleles explaining a relevant(non-infinitesimal) fraction of the overall genetic variability for produc-tion traits.IntroductionSelection in livestock populations has been mainlybased on methodologies assuming the infinitesimalmodel that describes quantitative traits as determinedby an infinite number of genes, each of them with avery small effect. By this approach, genetic improve-ment is not expected to significantly change allelefrequencies at any particular locus in a population(Barton & Keightley 2002). However, experimentsdissecting genetic variability in livestock genomeshave substantially challenged the assumptions of theinfinitesimal model showing that some loci explain arelevant part of the genetic variance of quantitativetraits under selection. These loci have been termedquantitative trait loci (QTL) or major genes when thecausative genes and mutations are known and theireffects are particularly relevant. Therefore, directional

Application of genomics (next generation sequencing and high throughput genotyping) and metabolomics technologies in farm animals are opening new opportunities for the identification of genetic factors affecting traits of economic relevance. In this study we combined several resources and data with the final aim to identify DNA polymorphisms and metabolites associated with production traits in Italian heavy pigs. Two genome wide association studies were carried out using a selective genotyping approach in Italian Large White pigs based on extreme and divergent estimated breeding values (EBVs) for average daily gain (ADG) and back fat thickness (BFT). Next generation sequencing was carried out using the Ion Torrent technology to identify single nucleotide polymorphisms (SNPs) from two reduced representation libraries constructed from pigs with extreme BFT EBVs. Metabolomics information was obtained using a mass spectrometry (MS/MS) analytical pipeline in a performance tested population. Integration of these data made it possible to identify markers (SNPs, copy number variation and metabolites) associated with ADG, BFT and several other correlated traits.

AUTHORS: Marco De Giorgi1, Alessandro Cinti2, Maria Grazia Cerrito2, Claudia Arena2, Elisa Chisci2, Gloria Galimberti2, Cristina Bugarin3, Giuseppe Gaipa3, Roberto Giovannoni2, Marialuisa Lavitrano2, and Ryszard Tom Smolenski1,2 AFFILIATIONS: 1Department of Biochemistry, Medical University of Gdansk, Gdansk, Poland. 2Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; 3Pediatric Department, Laboratory of Cell Therapy Stefano Verri, University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy TITLE: Simultaneous expression of novel combinations of graft protective human genes in porcine endothelial cells BACKGROUND AND AIM. Porcine vascular endothelium is the first cell type that is in direct contact with human blood in xenotransplantation settings. Endothelial activation and injury has been demonstrated to be the basis of several inflammatory and immunological mechanisms occurring during the rejection of xenogeneic tissues. Our aim was to investigate the potential protective roles of new combinations of human genes against endothelial activation, inflammation and procoagulant changes in a relevant in vitro model of xenotransplantation. METHODS AND RESULTS. A porcine endothelial cell line (Porcine Iliac Artery Endothelial cells, PIEC) have been transfected with F2A technology-based multicistronic plasmids in order to obtain the simultaneous expression of up to three human genes or a combination thereof. PIEC cells over-expressing Heme Oxygenase 1 (hHO1), Ecto 5’ Nucleotidase (hE5’N or hCD73) and Ecto Nucleoside Triphosphate Diphosphohydrolase 1 (hENTPD1 or hCD39) or hCD73 and hCD39 were produced. As a control, PIEC cells have been mock-transfected or transfected with single gene expressing vectors. PIEC stably transfected cell lines (hHO1/hCD73/hCD39-, hCD73/hCD39-, hCD73-, hCD39- transgenic cell lines) have been enriched for the expression of hCD73 and/or hCD39 markers by fluorescence activated cell sorting (FACS). On the other hand, to enrich by FACS, transfected cells expressing only hHO1, which has a cytoplasmic localization, an EGFP coding sequence had been added to the transgenic construct. For each transgenic PIEC cell line it was obtained a range of 96% - 98% cells positive for the selected transgenic marker(s), as evaluated by post-sorting FACS analyses. The expression of hHO1 in hHO1/hCD37/hCD39- and hHO1- transfected cells has been verified by immunoblotting. The correct subcellular localization of hCD73 and hCD39 in the plasma membrane or the cytosolic and perinuclear distribution of hHO1 have been confirmed by immunofluorescence and confocal microscope analyses. Evaluation of apoptotic cell death induced by 10ng/ml human TNF alpha treatment showed that the expression of human genes protects PIEC cells from caspase 3 and 7 activity. In fact, the hHO1/hCD73/hCD39 transfected cells has a 35% reduction in caspase activity after 16 hours of TNF treatment as compared to wild type cells. CONCLUSIONS. PIEC cells over-expressing selected combinations of human genes were produced by using F2A-based technology and characterized. This porcine endothelial cells model that co-express two to three human genes will be used to investigate the protective role, at molecular level, of the combination of genes against the endothelial activation, inflammation and coagulation dysregulation following xenogeneic injuries.

In the framework of model-based cluster analysis, finite mixtures of Gaussian components represent an important class of statistical models widely employed for dealing with quantitative variables. Within this class, we propose novel models in which constraints on the component-specific variance matrices allow us to define Gaussian parsimonious clustering models. Specifically, the proposed models are obtained by assuming that the variables can be partitioned into groups resulting to be conditionally independent within components, thus producing component-specific variance matrices with a block diagonal structure. This approach allows us to extend the methods for model-based cluster analysis and to make them more flexible and versatile. In this paper, Gaussian mixture models are studied under the above mentioned assumption. Identifiability conditions are proved and the model parameters are estimated through the maximum likelihood method by using the Expectation-Maximization algorithm. The Bayesian information criterion is proposed for selecting the partition of the variables into conditionally independent groups. The consistency of the use of this criterion is proved under regularity conditions. In order to examine and compare models with different partitions of the set of variables a hierarchical algorithm is suggested. A wide class of parsimonious Gaussian models is also presented by parameterizing the component-variance matrices according to their spectral decomposition. The effectiveness and usefulness of the proposed methodology are illustrated with two examples based on real datasets.

The development of high throughput genomics (next generation sequencing and high throughput genotyping) and metabolomics platforms has opened new perspectives for the identification of the genetics factors affecting traits of biological relevance in all species, including production traits in farm animals. In pigs, benefits derived from the recent sequencing of the pig genome can be fully exploited by combining advanced genomics and metabolomics approaches. In this work we integrated several resources, experiments and data with the final aim to identify markers (DNA polymorphisms and metabolites) associated with production traits in Italian Large White pigs. High throughput genotyping was carried out using the Illumina Porcine60SNP BeadChip array and customized Golden Gate system on extreme and divergent pigs for back fat thickness (BFT) estimated breeding values (EBVs) (300-560 animals) and average daily gain (ADG) EBVs (360 pigs), chosen among a population of about 12,000 performance tested pigs. Next generation sequencing was carried out using the Ion Torrent PGM machine to identify single nucleotide polymorphisms (SNPs) from two reduced representation libraries developed from pooled genomic DNA constructed from 50 pigs with most positive and 50 pigs with most negative BFT EBVs, respectively. A total of 7,510,918 reads were produced and 447,031 SNPs were called, using stringent criteria. Genome wide association studies made it possible to identify a quite large number of significant SNPs affecting BFT, ADG and correlated traits. In addition, several genome regions containing significant SNPs for BFT were enriched of SNPs identified from the Next Generation Sequencing experiment. Metabolomics information was obtained from 800 performance tested pigs using a mass spectrometry (MS/MS) analytical pipeline to measure 180 blood plasma metabolites. Estimated heritability and correlation among all these parameters and production traits indicated that a few metabolites could be useful predictors of EBVs for production traits. All these data will be used to develop a first systems biology platform to understand the fine biological mechanisms affecting production traits in pigs.

This paper introduces rpartScore (Galimberti, Soffritti, and Di Maso 2012), a new R package for building classification trees for ordinal responses, that can be employed whenever a set of scores is assigned to the ordered categories of the response. This package has been created to overcome some problems that produced unexpected results from the package rpartOrdinal (Archer 2010). Explanations for the causes of these unexpected results are provided. The main functionalities of rpartScore are described, and its use is illustrated through some examples.

"Signal" alignments play critical roles in many clinical setting. This is the case of mass spectrometry data, an important component of many types of proteomic analysis. A central problem occurs when one needs to integrate (mass spectrometry) data produced by different sources, e.g., different equipment and/or laboratories. In these cases some form of "data integration'" or "data fusion'" may be necessary in order to discard some source specific aspects and improve the ability to perform a classification task such as inferring the "disease classes'" of patients. The need for new high performance data alignments methods is therefore particularly important in these contexts. In this paper we propose an approach based both on an information theory perspective, generally used in a feature construction problem, and on the application of a mathematical programming task (i.e. the weighted bipartite matching problem). We present the results of a competitive analysis of our method against other approaches. The analysis was conducted on data from plasma/ethylenediaminetetraacetic acid (EDTA) of "control" and Alzheimer patients collected from three different hospitals. The results point to a significant performance advantage of our method with respect to the competing ones tested.

Background Carcass fatness is an important trait in most pig breeding programs. Following market requests, breeding plans for fresh pork consumption are usually designed to reduce carcass fat content and increase lean meat deposition. However, the Italian pig industry is mainly devoted to the production of Protected Designation of Origin dry cured hams: pigs are slaughtered at around 160 kg of live weight and the breeding goal aims at maintaining fat coverage, measured as backfat thickness to avoid excessive desiccation of the hams. This objective has shaped the genetic pool of Italian heavy pig breeds for a few decades. In this study we applied a selective genotyping approach within a population of ~ 12,000 performance tested Italian Large White pigs. Within this population, we selectively genotyped 304 pigs with extreme and divergent backfat thickness estimated breeding value by the Illumina PorcineSNP60 BeadChip and performed a genome wide association study to identify loci associated to this trait. Results We identified 4 single nucleotide polymorphisms with P≤5.0E-07 and additional 119 ones with 5.0E-07 Conclusions Further investigations are needed to evaluate the effects of the identified single nucleotide polymorphisms associated with backfat thickness on other traits as a pre-requisite for practical applications in breeding programs. Reported results could improve our understanding of the biology of fat metabolism and deposition that could also be relevant for other mammalian species including humans, confirming the role of neuronal genes on obesity.

We investigated the possible involvement of vascular damage in the pathogenesis of Alzheimer's disease (AD), by assessment of plasma levels of tissue factor pathway inhibitor (TFPI), a serine protease inhibitor induced by endothelial injury, and homocysteine (Hcy), a known risk factor for cerebrovascular disorders, folate levels were also measured. 110 probable AD, 38 mild cognitive impairment, 31 patients affected by idiopathic Parkinson's disease (without dementia) and 100 healthy controls, who displayed no vascular disorders were enrolled. TFPI and Hcy were significantly higher in AD patients with respect to other groups. The levels of TFPI and Hcy were positively correlated in hyperhomocysteinemic AD and mild cognitive impairment subjects, and were negatively correlated with folate levels. Our findings suggest that an impairment of endothelial function associated with high Hcy levels may occur in AD patients, despite the absence of manifest cerebrovascular lesions. Therefore, TFPI may represent a candidate marker of endothelial damage in AD and might be used for the identification and monitoring of patients that would benefit from folate supplementation treatment.

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

A 68-year-old man presented with a 4-month history of progressive memory loss and mood disorders. Neurologic examination revealed severe impairment of attention and verbal skills, without motor and sensory deficits. His medical history included mild arterial hypertension, idiopathic partial epilepsy, and obsessive compulsive disorder. Brain MRI showed the presence of bilateral, asymmetric, swollen white matter lesions in the cerebral hemispheres, hyperintense in T2-weighted images, that partially involved the left frontal cortex (figure). On diffusion-weighted sequences, the white matter abnormalities were consistent with vasogenic edema. No pathologic contrast enhancement was present. Figure MRI of cerebral amyloid angiopathy–related inflammation (CAA-ri) and levels of anti-Aβ 1-40 and 1-42 autoantibodies in the CSF Axial fluid-attenuated inversion recovery brain MRI shows bilateral hyperintense lesions of the subcortical white matter (A), which are reduced after 20 days of steroid treatment (B). Axial T2*-weighted gradient-echo MRI (C) obtained 33 days later shows further reduction of white matter lesions and multiple, scattered, hypointense cortical lesions due to microhemorrhages (arrows). (D) Reiber's graph. X- and y-axes show, respectively, albumin (QAlb) and immunoglobulin G quotient (QIgG), obtained by the ratio between the level of the protein in the CSF from the first lumbar puncture and in the plasma. The QAlb indicates the permeability of the blood–brain barrier (BBB) to water-soluble molecules. The QIgG (total IgG including specific anti-Aβ autoantibodies) plotted into the graph discriminates between intrathecal production of IgG and …

Acetyl-cholinesterase inhibitors (AChEI) are drugs frequently prescribed for the treatment of Alzheimer's disease (AD), exerting an effect on cognition, as well as on behavioural and psychological symptoms of dementia and activities of daily living. The efficacy of AChEI may be ascribed not only to the activation of cholinergic transmission, but also to other mechanisms, among which a putative regulation of the immune response has already been hypothesized. In the present study, we evaluated, in a cross-sectional sample of 66. AD patients and 48 healthy controls, the putative influence of AChEI on anti-Abeta 1-42 antibody plasma levels by ELISA assay. AD patients receiving AChEI therapy showed increased plasma levels of anti-Abeta 1-42 antibodies respect to untreated AD patients and antibodies levels similar to those of healthy controls, both before and after normalization by total IgG values. Our results support a potential role of AChEI in the modulation of the immune response against Abeta. We suggest that a strategy aimed at increasing the endogenous response against this peptide might represent an interesting therapeutic target to be further investigated. © 2010 Elsevier Ireland Ltd.

We report on experimental results of the fast electron transport in compressed plasmas, created by laser-induced shock propagation in both cylindrical and planar geometry. Two experiments were carried out. The first one was based on the compression of a polyimide cylinder filled with foams of three different initial densities (rho(0)). X-ray and proton radiographies of the target during the compression coupled with hydrodynamic simulations show that the obtained core densities and temperatures range from 2 to 11 g/cm(3) and from 30 to 120 eV, respectively. By studying the K-shell fluorescence from dopant atoms inside the target and from tracer layers situated at both front and rear side of the target it has been possible to investigate the fast electron propagation. The results show that Cu K(alpha) yield emitted by the target rear side foil decreases with increasing compression, independently of rho(0). An electron collimation can also be observed for certain experimental conditions where a convergent resistivity gradient interacts with the fast electron beam. The second experiment was performed in a planar geometry with a compressing shock counter-propagative to the fast electron beam. In this case the areal density rho z seen by the electrons is constant during the compression in such a way that changes in the fast electron range should be ascribed to collective mechanisms. The study of the K(alpha) fluorescence, from buried fluorescent layers of different atomic numbers, shows that the electrons with energy

We identified, combining different approaches (sequencing, literature mining, and in silico porcine expressed sequence tag mining), a few hundreds of single nucleotide polymorphisms (SNPs; n. = 667) in candidate genes for fat deposition traits and genotyped these markers in Italian Large White pigs using a selective genotyping approach based on estimated breeding value for back fat thickness. Significant association (P nominal value

Finite mixture models are useful tools for clustering two-way datasets within a sound statistical framework which can assess some important questions, such as how many clusters are there in the data. Models that can also be used for clustering multilevel data have been proposed, with the intent to produce clusterings of units at every level on the basis of all the available variables, considering the hierarchical structure of the dataset. This paper introduces a new class of mixture models for datasets with two levels that makes it possible to discover a clustering of level 2 units and different clusterings of level 1 units corresponding to different subsets of the variables (multiple cluster structures). This new class is obtained by adapting a mixture model proposed to identify multiple cluster structures in a data matrix to the multilevel situation. The usefulness of the new method is shown using simulated data and a real example.

Down syndrome (DS) is the most common genetic disorder characterized by an extra copy of chromosome 21. DS subjects show signs of progressive cognitive decline, and most of them develop Alzheimer's type dementia at the age of 50 to 55 years. The aim of this study was to evaluate amyloid precursor protein (APP) metabolites and anti-Abeta 1-42 antibodies plasma levels in DS as possible biomarkers of Abeta accumulation potentially leading to neurodegeneration. We investigated plasma levels of sAPPα, Abeta 1-42, and anti-Abeta 1-42 antibodies by enzyme-linked immunosorbent assay in 24 DS subjects, 10 non-DS mentally retarded subjects and 18 age-matched controls. We found that sAPPα levels were about 1.5-fold higher and Abeta 1-42 levels were about 6-fold higher in DS respect to mentally retarded patients and to controls. DS patients showed Abeta 1-42 antibodies levels 4-fold higher than non-DS mentally retarded group and 2-fold higher than controls. Moreover, anti-Abeta 1-42 antibodies levels were inversely correlated with age in DS subjects. Our results suggested sAPPα as a possible peripheral marker for the alteration in APP metabolism in DS and highlighted an alteration in anti-abeta antibodies, for the first time evaluated in plasma from DS subjects. © 2010 by Lippincott Williams & Wilkins.

Aim The aim of this study was to investigate the effects of frequency modulated electromagnetic neural stimulation (FREMS), a recently developed safe and effective treatment of painful diabetic neuropathy, on cutaneous microvascular function. Methods Thirty-one patients with painful neuropathy were enrolled in a randomised, double-blind, crossover FREMS vs. placebo study; each received two series of 10 treatments of either FREMS or placebo in random sequence within no more than 3 weeks. Patients were studied at baseline, end of FREMS and placebo series, and after 4 months of follow-up. Cutaneous blood flow was measured by laser doppler flowmetry and partial tissue tension of oxygen (TcPO 2 ) and carbonic anhydride (TcPCO 2 ) by oxymetry at the lower extremities in basal resting conditions and as incremental response after thermal stimulation. Results Crossover analysis showed no consistent differences between FREMS and placebo. After 4-month follow-up, a 52% increase of cutaneous blood flow was observed in resting conditions ( P =.0086 vs. baseline), while no differences were observed as incremental flow after warming; compared with baseline, no significant differences were observed for TcPO 2 and TcPCO 2 , both in resting conditions and as incremental response to warm. Conclusion These results indicate that 10 treatments with FREMS may induce an enhancement of microvascular blood flow measurable at 4 months of follow-up. The findings of this study will need to be confirmed in a larger, adequately powered study (ClinicalTrial.gov Id: NCT00337324).

Clinical data alignment plays a critical role in identifying important features for significant experiments. A central problem is data fusion i.e., how to correctly integrate data provided by different labs. This integration is done in order to increase ability of inferring target classes of controls and patients. Our paper proposes an approach based both on a information theoretic perspective, generally used in a feature construction problem [3] and on the approximated solution for a mathematical programming task (i.e. the weighted bipartite matching problem [6]). Numerical evaluations with two competitive approaches show the improved performance of the proposed method. For this evaluation we used data sets from plasma / ethylenediaminetetraacetic acid (EDTA) of controls and Alzheimer patients collected in three different hospitals.

The mechanics of two pendulums coupled by a stressed spring is discussed, and the behavior for small oscillations is described. When the system is in its highest symmetry configuration, the pendulums are independent and the normal frequencies are degenerate.

Aims The aim of this case–control study was to compare quality of life (QoL) and treatment satisfaction in adults with Type 1 diabetes (T1DM) treated with either continuous subcutaneous insulin infusion (CSII) or multiple daily injections (MDI). Methods Consecutive patients aged between 18 and 55 years, and attending diabetes clinics for a routine visit, completed the Diabetes-Specific Quality-of-Life Scale (DSQOLS), the Diabetes Treatment Satisfaction Questionnaire (DTSQ) and the SF-36 Health Survey (SF-36). Case (CSII) and control subjects (MDI) were recruited in a 1 : 2 ratio. Results Overall, 1341 individuals were enrolled by 62 diabetes clinics; 481 were cases and 860 control subjects. Cases had a longer diabetes duration and were more likely to have eye and renal complications. Age, school education, occupation and HbA 1c were similar. Of control subjects, 90% followed glargine-based MDI regimens and 10% used NPH-based MDI regimens. On multivariate analysis, after adjusting for socioeconomic and clinical characteristics, scores in the following areas of the DSQOLS were higher in cases than control subjects: diet restrictions ( β = 5.96; P < 0.0001), daily hassles ( β = 3.57; P = 0.01) and fears about hypoglycaemia ( β = 3.88; P = 0.006). Treatment with CSII was also associated with a markedly higher DTSQ score ( β = 4.13; P < 0.0001) compared with MDI. Results were similar when CSII was compared separately with glargine- or NPH-based MDI regimens. Conclusions This large, non-randomized, case–control study suggests quality of life gains deriving from greater lifestyle flexibility, less fear of hypoglycaemia, and higher treatment satisfaction, when CSII is compared with either glargine-based or NPH-based MDI regimens. Diabet. Med. 25, 213–220 (2008)

Fat deposition in pigs is a complex trait for which several studies in experimental populations have identified a large number of QTL, some of which segregating in outbred commercial populations. As pigs and humans have numerous physiological and phenotypic similarities for fat deposition, in order to identify the genetic factors affecting this trait in both species, it could be possible to take advantage of the results obtained in one species and, applying a comparative approach, transfer biological information to the other one, and vice versa. Therefore, such approach, on one hand, might provide useful information to improve selection efficiency in pig breeding and, on the other hand, could contribute to understand the genetic basis of human obesity and related diseases. With these aims, we designed a project based on a selective genotyping approach in commercial pig populations for which extreme and divergent groups of pigs have been chosen according to the estimated breeding value (EBV) for fat deposition traits. A candidate gene approach has been applied in order to identify markers associated with fatness in pigs. DNA markers are chosen and identified using different approaches: i) mutations already reported in pig genes affecting fat deposition; ii) discovery of new mutations in candidate genes by resequencing; iii) database mining. For the latter approach, a bioinformatic pipeline has been designed to mine pig sequences available in DNA databases. In the start up phase, about 560 Italian Large White pigs, selected according to their fatness EBV, are genotyped for more than 700 SNPs. Some preliminary results are reported. The project portal is: http://fagenomich.biocomp.unibo.it/

We compared the levels of serum folate from Alzheimer’s disease (AD) patients and from age-matched healthy subjects and used primary cultures of fibroblasts, obtained from the two groups, to assess possible differences in their ability to bind folate. The results show that the levels of circulating folate are significantly (p < 0.01; n = 30) lower in AD patients than in controls (4.91±2.44 and 7.56±2.5 ng/mL, respectively). Moreover, the folate binding of AD fibroblasts is significantly (p < 0.01; n = 8) higher (2–4- fold) with respect to controls. RT-PCR experiments suggest that the higher folate binding could be due to an enhanced expression in AD fibroblasts of folate receptor α We compared the levels of serum folate from Alzheimer's disease (AD) patients and from age-matched healthy subjects and used primary cultures of fibroblasts, obtained from the two groups, to assess possible differences in their ability to bind folate. The results show that the levels of circulating folate are significantly (p < 0.01; n = 30) lower in AD patients than in controls (4.91 +/- 2.44 and 7.56 +/- 2.5 ng/mL, respectively). Moreover, the folate binding of AD fibroblasts is significantly (p < 0.01; n = 8) higher (2-4-fold) with respect to controls. RT-PCR experiments suggest that the higher folate binding could be due to an enhanced expression in AD fibroblasts of folate receptor

Transcriptional profile of post mortem skeletal muscle tissue and the changes in time course are unexplored topics in molecular biology applied to meat quality studies. This issue is of particular interest considering the fact that the availability of post mortem muscles at commercial abattoirs is usually associated with substantial delays. On the other hand, the possibility of using post mortem samples for gene expression analysis opens new perspectives for the transcriptome analysis with potential applications for product authentication, quality assessment and forensics. The recovery of high quality RNA has been considered a prerequisite for gene expression investigation. However, it is commonly asserted that RNA is subjected to irreversible damages starting from the death of the animals. Therefore, it is usually considered that RNA cannot be efficiently used for expression analysis of tissues at different post mortem stages, even if several experiments with human and laboratory animal specimens have demonstrated that, depending on the tissue and pre mortem and post mortem conditions, RNA can remain stable for a long period after death. In this study, for the first time, we investigated the porcine skeletal muscle transcriptional profile in a post mortem time course. RNA extracted from porcine Semimembranosus muscles sampled at 20 minutes, 2, 6 and 24 hours post mortem was assessed by agarose gel and microfluidic capillary electrophoresis. Similar RIN values were obtained for all samples indicating their suitability for downstream gene expression evaluations. Then, Affymetrix GeneChip porcine genome microarrays were hybridised with RNA extracted at all post mortem time points. The results indicated that most of the genes did not change their level, confirming that RNA degradation might not play a major role during the first 24 hours post mortem. However, two groups of genes showing different trends of expression during the post mortem stages were identified. These results open new perspectives for the analysis of hypoxic/anoxic skeletal muscle gene expression and the possibility to identify meat quality predictors.

Objective: To identify the real number of hyperhomocysteinemic Alzheimer's patients who may benefit from homocysteine-lowering therapy. Methods: Basal and post-methionine load homocysteine levels were assessed by rp-HPLC system. Results: PML test revealed twice as many hyperhomocysteinemic AD subjects with respect to the fasting analysis. Conclusion: PML test resulted useful in detecting higher number of hyperhomocysteinemic AD patients who may have the chance of an early folate treatment.