Detecting inbreeding depression for reproductive traits in Iberian pigs using genome-wide data

Background

The current availability of genotypes for very large numbers of single nucleotide polymorphisms (SNPs) is leading to more accurate estimates of inbreeding coefficients and more detailed approaches for detecting inbreeding depression. In the present study, genome-wide information was used to detect inbreeding depression for two reproductive traits (total number of piglets born and number of piglets born alive) in an ancient strain of Iberian pigs (the Guadyerbas strain) that is currently under serious danger of extinction.

Methods

A total of 109 sows with phenotypic records were genotyped with the PorcineSNP60 BeadChip v1. Inbreeding depression was estimated using a bivariate animal model in which the inbreeding coefficient was included as a covariate. We used two different measures of genomic inbreeding to perform the analyses: inbreeding estimated on a SNP-by-SNP basis and inbreeding estimated from runs of homozygosity. We also performed the analyses using pedigree-based inbreeding.

Results

Significant inbreeding depression was detected for both traits using all three measures of inbreeding. Genome-wide information allowed us to identify one region on chromosome 13 associated with inbreeding depression. This region spans from 27 to 54 Mb and overlaps with a previously detected quantitative trait locus and includes the inter-alpha-trypsin inhibitor gene cluster that is involved with embryo implantation.

Conclusions

Our results highlight the value of high-density SNP genotyping for providing new insights on where genes causing inbreeding depression are located in the genome. Genomic measures of inbreeding obtained on a SNP-by-SNP basis or those based on the presence/absence of runs of homozygosity represent a suitable alternative to pedigree-based measures to detect inbreeding depression, and a useful tool for mapping studies. To our knowledge, this is the first study in domesticated animals using the SNP-by-SNP inbreeding coefficient to map specific regions within chromosomes associated with inbreeding depression.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-014-0081-5) contains supplementary material, which is available to authorized users.     

Effect of water extraction on sugars recovery from steam exploded olive tree pruning

Biomass of olive tree pruning can be considered a suitable raw material for the production of ethanol due to its high content of potentially fermentable carbohydrates. However its high extractives content could cause condensation reactions between extractives and acid insoluble lignin during pretreatment, hindering the enzymatic hydrolysis of pretreated material. In this work, the effect of extractives removal before steam explosion of olive tree pruning was evaluated. The objectives are to recover as much glucose as possible in the extraction stage and to avoid the condensation reactions. The effect of temperature and time of water extracted material on sugars recovery was studied using a response surface method according to a central composite design. Extractive removal previous to steam explosion resulted in 20% more total sugars recovery in comparison to a material without water extraction stage.     

Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates

Several bifunctional chelates (BFCs) were investigated as carriers of (64)Cu for PET imaging. The most widely used chelator for (64)Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N'-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with (64)Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H(2)N-Aoc-[d-Tyr(6),βAla(11),Thi(13),Nle(14)]bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the (64)Cu/Oxo-DO3A complex remained intact after 20 h while the (64)Cu/DOTA-BBN complex was completely demetalated. In contrast, both (64)Cu/NOTA- and (64)Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that (64)Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.     

Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages

A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists.     

Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in?vitro and in?vivo. This α helix participates in?a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.     

Bacterial extract cantastim activates macrophages via TLR-2

CANTASTIM is a second generation bacterial immunomodulator. The aim of this study was to examine the mechanism by which bacterial immunomodulator CANTASTIM induces production of inflammatory cytokines in monocytes/macrophages. Proinflammatory cytokines were induced in PMA-differentiated THP-1 cells by stimulation with TLR agonists and CANTASTIM in the presence or absence of anti-TLR blocking antibodies or isotype matched control antibodies. Also, RNA interference was used to knockdown TLR2 or TLR4 expression in PMA-differentiated THP-1 cells before stimulation. As expected, induction of TNF-alpha and IL-6 by TLR4 agonist LPS was inhibited in a significant manner by anti-TLR4 but not by anti-TLR2 antibody. Unexpectedly, treatment with anti-LR2 blocking antibody inhibited only IL-6 production induced by Pam3CSK4 while the level of TNF-alpha was unchanged. When cells were stimulated by TLR2 agonist heat-killed Listeria monocytogenes the release of TNF-alpha was significantly attenuated by anti-TLR2 antibodies. Silencing of TLR2 led to a statistically significant inhibition of TNF-alpha secretion induced by TLR2 agonist while siRNA silencing of TLR4 did not affect the response to TLR2 agonist. Cells exposed to CANTASTIM produced significant levels of pro-inflammatory cytokines but the levels were lower than LPS-stimulated cells. Production of both cytokines was inhibited by treatment with anti-TLR2 blocking antibody and not by anti-TLR4 antibody. Silencing of TLR2 led to a statistically significant inhibition of TNF-a secretion induced by CANTASTIM while silencing of TLR4 had no effect on the response to CANTASTIM. These results support the hypothesis that CANTASTIM may exert its immunomodulatory and adjuvant activities through interaction of its bacterial components with TLR2.     

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.