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High quality pork is consumed as fresh meat, whereas other carcasses are used in the processing industry. Meat quality is determined measuring technical muscle variables. The objective of this research was to investigate the molecular regulatory mechanisms underlying meat quality differences of pork originating from genetically different Pietrain boars. Pietrain boars were approved for high meat quality using a DNA marker panel. Other Pietrain boars were indicated as average. Both groups produced litters in similar Pietrain sows. The LM were sampled from 9 carcasses produced by approved boars and 8 carcasses of average boars. Total RNA was isolated, and an equal portion of each sample was pooled to make a reference sample representing the mean of all samples. Each sample was hybridized on microarrays against the reference in duplicate using a dye swaps design. After normalization and subtraction of 2 times the background, only genes expressed in at least 5 carcasses were analyzed. For all analyses the mean of the M-values relative to the reference (i.e., fold change), were used. Sixteen genes showed significant linear or quadratic associations between gene expression and meat color (Minolta [a.sup.*] value, Minolta [L.sup.*] value, reflection, pH 24 h) after Bonferroni correction. All these genes had expression levels similar to the reference in all carcasses. Studying association between gene expression levels and meat quality using only genes with expression statistically differing from the reference in at least 5 carcasses revealed 29 more genes associating with the technological meat quality variables, again with meat color as a main trait. These associations were not significant after Bonferroni correction and explained less of the phenotypic variation in the traits. Bioinformatics analyses with The Database for Annotation, Visualization and Integrated Discovery (DAVID) using the list of genes with more than 2-fold changed expression level revealed that these genes were mainly found in muscle-specific processes, protein complexes, and oxygen transport, and located to muscle-specific cellular localizations. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed pathways related to protein metabolism, cellular proliferation, signaling, and adipose development differing between the 2 groups of carcasses. Approved meat carcasses showed less variation in gene expression. The results highlight biological molecular mechanisms underlying the differences between the high meat quality approved and average boars. Key words: association study, bioinformatics analysis, high fresh meat quality tested boar, longissimus, microarray, Sus scrofa Pietrain doi: 10.2527/jas.2010-2952

The objectives of this study were to evaluate the influence of different pure pig breeds and muscle types on the expression of muscle proteins, as well as their interactions, and second, to find biomarkers for breed and muscle types. A total of 126 male pigs, including 43 Landrace, 21 Duroc, 43 Large White, 13 Pietrain, and 6 Belgian Landrace, were slaughtered at the age of 174 [+ or -] 6 d. Samples from the semimembranosus muscle (SM) and LM were collected 24 h postmortem. Proteomic spectra were generated on an anion exchanger (Q10), a cation exchanger (CM10), and on immobilized metal affinity capture (IMAC30) ProteinChip arrays and analyzed using surface-enhanced laser desorption/ ionization time-of-flight mass spectrometry ProteinChip techniques. Breed and muscle type did not affect the number of peaks per spectrum but, interestingly, affected the average intensity of the peaks. Of these peaks, a total of 4 proved to be potential protein biomarkers to differentiate LM or SM muscles, and 2 to classify specific breed types. Additionally, several peaks influenced by the interaction between muscle and breed types could correctly classify pig muscles according to their breed. Further studies need to be carried out to validate and identify these potential protein biomarkers for breed and muscle types in finishing pigs. Key words: biomarker, breed, muscle, pig, proteomic, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry doi: 10.2527/jas.2009-2256

Cancer vaccines have emerged as a potent strategy to improve cancer immunity, with or without the combination of checkpoint blockade. In our investigation, liposomal formulations containing synthetic long peptides and α-Galactosylceramide, along with a DC-SIGN-targeting ligand, Lewis Y (Le Y ), were studied for their anti-tumor potential. The formulated liposomes boosted with anti-CD40 adjuvant demonstrated robust invariant natural killer (iNKT), CD4 + , and CD8 + T-cell activation in vivo. The incorporation of Le Y facilitated the targeting of antigen-presenting cells expressing DC-SIGN in vitro and in vivo. Surprisingly, mice vaccinated with Le Y -modified liposomes exhibited comparable tumor reduction and survival rates to those treated with untargeted counterparts despite a decrease in antigen-specific CD8 + T-cell responses. These results suggest that impaired induction of antigen-specific CD8 + T-cells via DC-SIGN targeting does not compromise anti-tumor potential, hinting at alternative immune activation routes beyond CD8 + T-cell activation., Competing Interests: H.J.v.d.V. is employed by and acts as chief scientific officer of LAVA Therapeutics. M.A. and G.S. are from LIPOSOMA BV. L.B. is from JJP Biologics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Induction of tumor-specific cytotoxic CD8 + T cells (CTLs) via immunization relies on the presentation of tumor-associated peptides in major histocompatibility complex (MHC) class I molecules by dendritic cells (DCs). To achieve presentation of exogenous peptides into MHC class I, cytosolic processing and cross-presentation are required. Vaccination strategies aiming to induce tumor-specific CD8 + T cells via this exogenous route therefore pose a challenge. In this study, we describe improved CD8 + T cell induction and in vivo tumor suppression of mono-palmitic acid-modified (C16:0) antigenic peptides, which can be attributed to their unique processing route, efficient receptor-independent integration within lipid bilayers, and continuous intracellular accumulation and presentation through MHC class I. We propose that this membrane-integrating feature of palmitoylated peptides can be exploited as a tool for quick and efficient antigen enrichment and MHC class I loading. Importantly, both DCs and non-professional antigen-presenting cells (APCs), similar to tumor cells, facilitate anti-tumor immunity by efficient CTL priming via DCs and effective recognition of tumors through enhanced presentation of antigens., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Background: Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts., Results: We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal., Conclusion: This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism., Competing Interests: Competing interestsPdW is employed at the DSM Biotechnology Center, part of the company Royal DSM. Royal DSM owns intellectual property rights and commercializes aspects of the technology discussed in this paper. PdW, DBJ, and ML are listed on a patent application on xylose isomerase (WO2018073107)., (© The Author(s) 2020.)

Meat quality traits have low heritability and large environmental influences. To predict, improve and manage meat quality, proteomic biomarkers are superior to genetic markers. The objectives of this research were (1) to find associations between proteome profiles of longissimus muscle at slaughter and meat quality accuracies of prediction of traits ranged from 20 up to 80%. Differentially expressed proteins related to drip loss and ultimate pH were identified by NanoLC-FTMSMS. The proteins highlight biological mechanisms that may explain how these traits develop biologically and how they are related to each other., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Xenopus laevis Connexin-41 (Cx41) mRNA contains three upstream open reading frames (uORFs) in the 5'-untranslated region (UTR). We analyzed the translation efficiency of constructs containing the Cx41 5'-UTR linked to the green fluorescent protein reporter after injection of transcripts into one-cell stage Xenopus embryos. The translational efficiency of the wild-type Cx41 5'-UTR was only 2% compared with that of the beta-globin 5'-UTR. Mutation of each of the three uAUGs into AAG codons enhanced translation 82-, 9-, and 4-fold compared with the wild-type Cx41 5'-UTR. Based on these increased translation efficiencies, the percentages of ribosomes that recognized the uAUGs were calculated. Only 0.03% of the ribosomes that entered at the cap structure scanned the entire 5'-UTR and translated the main ORF. The results indicate that all uAUGs are recognized by the majority of the scanning ribosomes and that the three uAUGs strongly modulate translation efficiency in Xenopus laevis embryos. Based on these data, a model of ribosomal flow along the mRNA is postulated. We conclude that the three uORFs may play an important role in the regulation of Cx41 expression.