Detection of quantitative trait loci affecting serum cholesterol, LDL, HDL, and triglyceride in pigs

<p>Abstract</p> <p>Background</p> <p>Serum lipids are associated with many serious cardiovascular diseases and obesity problems. Many quantitative trait loci (QTL) have been reported in the pig mostly for performance traits but very few for the serum lipid traits. In contrast, remarkable numbers of QTL are mapped for serum lipids in humans and mice. Therefore, the objective of this research was to investigate the chromosomal regions influencing the serum level of the total cholesterol (CT), triglyceride (TG), high density protein cholesterol (HDL) and low density protein cholesterol (LDL) in pigs. For this purpose, a total of 330 animals from a Duroc × Pietrain F2 resource population were phenotyped for serum lipids using ELISA and were genotyped by using 122 microsatellite markers covering all porcine autosomes for QTL study in QTL Express. Blood sampling was performed at approximately 175 days before slaughter of the pig.</p> <p>Results</p> <p>Most of the traits were correlated with each other and were influenced by average daily gain, slaughter date and age. A total of 18 QTL including three QTL with imprinting effect were identified on 11 different porcine autosomes. Most of the QTL reached to 5% chromosome-wide (CW) level significance including a QTL at 5% experiment-wide (GW) and a QTL at 1% GW level significance. Of these QTL four were identified for both the CT and LDL and two QTL were identified for both the TG and LDL. Moreover, three chromosomal regions were detected for the HDL/LDL ratio in this study. One QTL for HDL on SSC2 and two QTL for TG on SSC11 and 17 were detected with imprinting effect. The highly significant QTL (1% GW) was detected for LDL at 82 cM on SSC1, whereas significant QTL (5% GW) was identified for HDL/LDL on SSC1 at 87 cM. Chromosomal regions with pleiotropic effects were detected for correlated traits on SSC1, 7 and 12. Most of the QTL identified for serum lipid traits correspond with the previously reported QTL for similar traits in other mammals. Two novel QTL on SSC16 for HDL and HDL/LDL ratio and an imprinted QTL on SSS17 for TG were detected in the pig for the first time.</p> <p>Conclusion</p> <p>The newly identified QTL are potentially involved in lipid metabolism. The results of this work shed new light on the genetic background of serum lipid concentrations and these findings will be helpful to identify candidate genes in these QTL regions related to lipid metabolism and serum lipid concentrations in pigs.</p

Mirs-138 and-424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia

Key Points APTs as miRNA targets provide a novel molecular mechanism for how primary CLL cells escape from CD95-mediated apoptosis. Palmitoylation as a novel posttranslational modification in CLL might also impact on survival signaling, proliferation, and migration.</jats:p

The ciliary membrane-associated proteome reveals actin-binding proteins as key components of cilia

Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell-derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions

Injured Podocytes Are Sensitized to Angiotensin II-Induced Calcium Signaling

Background Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of Angll on podocytes has not been demonstrated in vivo. Methods To study the effects of AngII on podocyte calcium levels in vivo, we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3. Results In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocinencoding gene Nphs2, the consequent podocyte damage and proteinuria rendered the cells responsive to Angll and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease. Conclusions Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker

Mitochondrial membrane proteins and VPS35 orchestrate selective removal of mtDNA

Understanding the mechanisms governing selective turnover of mutation-bearing mtDNA is fundamental to design therapeutic strategies against mtDNA diseases. Here, we show that specific mtDNA damage leads to an exacerbated mtDNA turnover, independent of canonical macroautophagy, but relying on lysosomal function and ATG5. Using proximity labeling and Twinkle as a nucleoid marker, we demonstrate that mtDNA damage induces membrane remodeling and endosomal recruitment in close proximity to mitochondrial nucleoid sub-compartments. Targeting of mitochondrial nucleoids is controlled by the ATAD3-SAMM50 axis, which is disrupted upon mtDNA damage. SAMM50 acts as a gatekeeper, influencing BAK clustering, controlling nucleoid release and facilitating transfer to endosomes. Here, VPS35 mediates maturation of early endosomes to late autophagy vesicles where degradation occurs. In addition, using a mouse model where mtDNA alterations cause impairment of muscle regeneration, we show that stimulation of lysosomal activity by rapamycin, selectively removes mtDNA deletions without affecting mtDNA copy number, ameliorating mitochondrial dysfunction. Taken together, our data demonstrates that upon mtDNA damage, mitochondrial nucleoids are eliminated outside the mitochondrial network through an endosomal-mitophagy pathway. With these results, we unveil the molecular players of a complex mechanism with multiple potential benefits to understand mtDNA related diseases, inherited, acquired or due to normal ageing. Mitochondrial quality control mechanisms prevent damage accumulation, including in mitochondrial DNA (mtDNA). Here, Sen et al. show that altered mtDNA elicits local rearrangements in mitochondrial membrane potential and cristae structure, with mtDNA eliminated through VPS35 endosomes

Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss of Nigral Dopaminergic Neurons

Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of dopaminergic neurons (DaNs) in the substantia nigra (SNc), whereas DaNs in the neighboring ventral tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage calcium channels to generate action potentials, which lead to oxidant stress by yet unknown pathways. To determine the molecular mechanisms linking calcium load with selective cell death in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the mitochondrial membrane potential in mice of both sexes with genetically induced, severe mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox-sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all DaNs, exclusively SNc neurons showed an oxidized redox-system, i.e., a low reduced/oxidized glutathione (GSH-GSSG) ratio. This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of reactive oxygen species rather unlikely. Surprisingly, a high mitochondrial inner membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into the cell and into mitochondria, respectively, rescued the disturbed redox ratio and induced further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs to mitochondrial defects

A genetical genomics approach reveals new candidates and confirms known candidate genes for drip loss in a porcine resource population

In this study lean meat water-holding capacity (WHC) of a Duroc x Pietrain (DuPi) resource population with corresponding genotypes and transcriptomes was investigated using genetical genomics. WHC was characterized by drip loss measured in M. longissimus dorsi. The 60K Illumina SNP chips identified genotypes of 169 F-2 DuPi animals. Whole-genome transcriptomes of muscle samples were available for 132 F-2 animals using the Affymetrix 24K GeneChipA (R) Porcine Genome Array. Performing genome-wide association studies of transcriptional profiles, which are correlated with phenotypes, allows elucidation of cis- and trans-regulation. Expression levels of 1,228 genes were significantly correlated with drip loss and were further analyzed for enrichment of functional annotation groups as defined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. A hypergeometric gene set enrichment test was performed and revealed glycolysis/glyconeogenesis, pentose phosphate pathway, and pyruvate metabolism as the most promising pathways. For 267 selected transcripts, expression quantitative trait loci (eQTL) analysis was performed and revealed a total of 1,541 significant associations. Because of positional accordance of the gene underlying transcript and the eQTL location, it was possible to identify eight eQTL that can be assumed to be cis-regulated. Comparing the results of gene set enrichment and the eQTL detection tests, molecular networks and potential candidate genes, which seemed to play key roles in the expression of WHC, were detected. The alpha-1-microglobulin/bikunin precursor (AMBP) gene was assumed to be cis-regulated and was part of the glycolysis pathway. This approach supports the identification of trait-associated SNPs and the further biological understanding of complex traits.In this study lean meat water-holding capacity (WHC) of a Duroc&nbsp;&times;&nbsp;Pietrain (DuPi) resource population with corresponding genotypes and transcriptomes was investigated using genetical genomics. WHC was characterized by drip loss measured in M. longissimus dorsi. The 60K Illumina SNP chips identified genotypes of 169 F2 DuPi animals. Whole-genome transcriptomes of muscle samples were available for 132 F2 animals using the Affymetrix 24K GeneChip&reg; Porcine Genome Array. Performing genome-wide association studies of transcriptional profiles, which are correlated with phenotypes, allows elucidation of cis- and trans-regulation. Expression levels of 1,228 genes were significantly correlated with drip loss and were further analyzed for enrichment of functional annotation groups as defined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. A hypergeometric gene set enrichment test was performed and revealed glycolysis/glyconeogenesis, pentose phosphate pathway, and pyruvate metabolism as the most promising pathways. For 267 selected transcripts, expression quantitative trait loci (eQTL) analysis was performed and revealed a total of 1,541 significant associations. Because of positional accordance of the gene underlying transcript and the eQTL location, it was possible to identify eight eQTL that can be assumed to be cis-regulated. Comparing the results of gene set enrichment and the eQTL detection tests, molecular networks and potential candidate genes, which seemed to play key roles in the expression of WHC, were detected. The &alpha;-1-microglobulin/bikunin precursor (AMBP) gene was assumed to be cis-regulated and was part of the glycolysis pathway. This approach supports the identification of trait-associated SNPs and the further biological understanding of complex traits.&nbsp;</p