Effect of the Feed Additive Fytera Start on Growth Performance and Stool Quality of Nursery Pigs Fed Nutritional and Pharmacological Copper and Zinc Diets

A total of 340 barrows (DNA 200 × 400; initially 13.4 ± 0.17 lb BW) were used in a 38-d growth study to determine the effect of Fytera Start (Selko, Indianapolis, IN) in diets with or without pharmacological levels of Zn and Cu on growth performance and stool quality of nursery pigs. Fytera Start is a blend of botanical extracts that has recently been introduced for use in nursery pig diets. Pigs were weaned at approximately 21 d of age, randomly allotted to pens based on initial BW, and then allotted to 1 of 4 dietary treatments in a completely randomized design. There were 5 pigs per pen and 17 pens per treatment across two barns. Treatment diets were formulated in three dietary phases and fed from d 0 to 10, d 10 to 21, and d 21 to 38, respectively. Treatments were arranged in a 2 × 2 factorial with main effects of Fytera Start (none or 100 ppm) and nutritional vs. pharmacological levels of Zn and Cu. The nutritional mineral concentrations were 110 ppm Zn and 16.5 ppm Cu throughout phases 1 to 3. The pharmacological mineral concentrations were 3,000, 2,000, and 110 ppm Zn in phases 1, 2, and 3, respectively, combined with 250 ppm Cu throughout phases 1 to 3. To achieve expected levels of Zn and Cu in the diet, Zn from zinc oxide and Cu from copper sulfate were added. On d 0, there was an unintentional main effect of Fytera Start (P = 0.008) on BW. As a result, d 0 BW was used as a covariate for all other growth performance responses. From d 0 to 21 and d 0 to 38, there was a Fytera Start × Zn/Cu interaction on ADG and ADFI (P \u3c 0.05) in which the addition of Fytera Start resulted in a numeric increase in ADG and ADFI in pigs not fed pharmacological levels of Zn/Cu; however, in pigs fed pharmacological levels of Zn/Cu, the inclusion of Fytera Start resulted in a numeric reduction in ADG and ADFI. There was a tendency for a main effect of Zn/Cu level on overall feed efficiency (P \u3c 0.10) where pharmacological levels of Zn/Cu improved feed efficiency. For fecal dry matter, there was a Zn/Cu × day interaction (P = 0.001) in which there was no difference in fecal DM regardless of Zn/Cu level on d 10 (P \u3e 0.10), but pigs fed pharmacological levels of Zn/Cu had lower fecal DM (P \u3c 0.001) compared to those not fed pharmacological levels of Zn/Cu on d 21. There was a main effect of day resulting in increased fecal DM (P \u3c 0.001) on d 10 compared to d 21. There was a main effect of day on fecal score (P = 0.010) resulting in a lower frequency of softer feces at d 10 compared to d 21. The lower frequency of softer feces observed on d 10 is consistent with the higher fecal DM on d 10 compared to d 21. In summary, feeding pharmacological levels of Zn and Cu resulted in increased BW, ADG, and ADFI. The inclusion of Fytera Start numerically increased BW, ADG, and ADFI in pigs fed nutritional levels of Zn/Cu and numerically decreased BW, ADG, and ADFI in pigs fed pharmacological levels of Zn/Cu. There was no impact of Fytera Start or Zn and Cu level on fecal DM on d 10. However, feeding pharmacological levels of Zn and Cu resulted in lower fecal DM at d 21. Fecal DM was higher on d 10 compared to d 21, and fecal score was numerically lower on d 10 compared to d 21

Effect of Sulfate or Hydroxychloride Forms of Zinc, Manganese, and Copper on Growth Performance, Weight Variation, Carcass Characteristics, and Economics of Grow-Finish Pigs

A total of 1,026 grow-finish pigs (337 × 1050 PIC; initially 57.2 ± 0.73 lb) were used in a 124-d trial to compare sulfate and hydroxychloride forms of Zn, Mn, and Cu on growth performance, carcass characteristics, weight variation, and economics of grow-finish pigs. Pigs were housed in mixed gender pens with 27 pigs per pen and 19 pens per treatment. The treatments were structured as a completely randomized design and consisted of a control diet containing 150, 16, and 110 ppm of Cu, Mn, and Zn, respectively, from sulfate sources or the same inclusion provided by hydroxychloride sources. Experimental diets were corn-soybean meal-DDGS-based and fed in meal form in phase 1 from 57 to 110 lb, phase 2 from 110 to 165 lb, phase 3 from 165 to 220 lb, and phase 4 from 220 to 300 lb. In the grower period (57 to 173 lb), there was a tendency (P = 0.052) to improve F/G when sulfate Mn, Zn, and Cu were fed. In the finisher period (d 61 to 124), pigs fed hydroxychloride mineral sources had improved (P = 0.041) ADG. For pig body weight variability, there was no evidence of differences (P ≥ 0.10) on the coefficient of variation between treatments. Pigs marketed at the end of the study which were fed hydroxychloride sources tended to have greater HCW (P = 0.054) compared to sulfate sources, but no evidence for differences (P ≥ 0.10) were found in any other carcass trait at any marketing event. There was a tendency (P = 0.088) to reduce feed cost per lb of gain when using sulfate sources compared to hydroxychloride forms; however, IOFC was not impacted by mineral source (P \u3e 0.10). In conclusion, these data suggest there were no differences in pig weight variability, overall pig growth performance, or carcass characteristics between mineral sources

Long-term culture of genome-stable bipotent stem cells from adult human liver.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.This work was supported by grants to MH (EU/236954) and to HC (The United European Gastroenterology Federation (UEGF) Research Prize 2010, EU/232814-StemCellMark and NWO/116002008). MH is supported by The Wellcome Trust Sir Henry Dale fellowship. The Rspo cell line was kindly provided by Dr. Calvin Kuo.This is the final published version. It first appeared at http://www.cell.com/abstract/S0092-8674%2814%2901566-9

Novel Missense Mutation A789V in IQSEC2 underlies X-Linked intellectual disability in the MRX78 family

Disease gene discovery in neurodevelopmental disorders, including X-linked intellectual disability (XLID) has recently been accelerated by next-generation DNA sequencing approaches. To date, more than 100 human X chromosome genes involved in neuronal signaling pathways and networks implicated in cognitive function have been identified. Despite these advances, the mutations underlying disease in a large number of XLID families remained unresolved. We report the resolution of MRX78, a large family with six affected males and seven affected females, showing X-linked inheritance. Although a previous linkage study had mapped the locus to the short arm of chromosome X (Xp11.4-p11.23), this region contained too many candidate genes to be analyzed using conventional approaches. However, our X-chromosome exome resequencing, bioinformatics analysis and inheritance testing revealed a missense mutation (c.C2366T, p.A789V) in IQSEC2, encoding a neuronal GDP-GTP exchange factor for Arf family GTPases (ArfGEF) previously implicated in XLID. Molecular modeling of IQSEC2 revealed that the A789V substitution results in the insertion of a larger side-chain into a hydrophobic pocket in the catalytic Sec7 domain of IQSEC2. The A789V change is predicted to result in numerous clashes with adjacent amino acids and disruption of local folding of the Sec7 domain. Consistent with this finding, functional assays revealed that recombinant IQSEC2A789V was not able to catalyze GDP-GTP exchange on Arf6 as efficiently as wild-type IQSEC2. Taken together, these results strongly suggest that the A789V mutation in IQSEC2 is the underlying cause of XLID in the MRX78 family

Mosaic structural variation in children with developmental disorders

Delineating the genetic causes of developmental disorders is an area of active investigation. Mosaic structural abnormalities, defined as copy number or loss of heterozygosity events that are large and present in only a subset of cells, have been detected in 0.2–1.0% of children ascertained for clinical genetic testing. However, the frequency among healthy children in the community is not well characterized, which, if known, could inform better interpretation of the pathogenic burden of this mutational category in children with developmental disorders. In a case–control analysis, we compared the rate of large-scale mosaicism between 1303 children with developmental disorders and 5094 children lacking developmental disorders, using an analytical pipeline we developed, and identified a substantial enrichment in cases (odds ratio = 39.4, P-value 1.073e − 6). A meta-analysis that included frequency estimates among an additional 7000 children with congenital diseases yielded an even stronger statistical enrichment (P-value 1.784e − 11). In addition, to maximize the detection of low-clonality events in probands, we applied a trio-based mosaic detection algorithm, which detected two additional events in probands, including an individual with genome-wide suspected chimerism. In total, we detected 12 structural mosaic abnormalities among 1303 children (0.9%). Given the burden of mosaicism detected in cases, we suspected that many of the events detected in probands were pathogenic. Scrutiny of the genotypic–phenotypic relationship of each detected variant assessed that the majority of events are very likely pathogenic. This work quantifies the burden of structural mosaicism as a cause of developmental disorders

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.Fil: Markolovic, Suzana. University of Oxford; Reino UnidoFil: Zhuang, Qinqin. University Of Birmingham; Reino UnidoFil: Wilkins, Sarah E.. University of Oxford; Reino UnidoFil: Eaton, Charlotte D.. University Of Birmingham; Reino UnidoFil: Abboud, Martine I.. University of Oxford; Reino UnidoFil: Katz, Maximiliano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: McNeil, Helen E.. University Of Birmingham; Reino UnidoFil: Leśniak, Robert K.. University of Oxford; Reino UnidoFil: Hall, Charlotte. University Of Birmingham; Reino UnidoFil: Struwe, Weston B.. University of Oxford; Reino UnidoFil: Konietzny, Rebecca. University of Oxford; Reino UnidoFil: Davis, Simon. University of Oxford; Reino UnidoFil: Yang, Ming. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Ge, Wei. University of Oxford; Reino UnidoFil: Benesch, Justin L. P.. University of Oxford; Reino UnidoFil: Kessler, Benedikt M.. University of Oxford; Reino UnidoFil: Ratcliffe, Peter J.. University of Oxford; Reino Unido. The Francis Crick Institute; Reino UnidoFil: Cockman, Matthew E.. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Fischer, Roman. University of Oxford; Reino UnidoFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Chowdhury, Rasheduzzaman. University of Stanford; Estados Unidos. University of Oxford; Reino UnidoFil: Coleman, Mathew L.. University Of Birmingham; Reino UnidoFil: Schofield, Christopher J.. University of Oxford; Reino Unid

A high-quality human reference panel reveals the complexity and distribution of genomic structural variants

Structural variation (SV) represents a major source of differences between individual human genomes and has been linked to disease phenotypes. However, the majority of studies provide neither a global view of the full spectrum of these variants nor integrate them into reference panels of genetic variation. Here, we analyse whole genome sequencing data of 769 individuals from 250 Dutch families, and provide a haplotype-resolved map of 1.9 million genome variants across 9 different variant classes, including novel forms of complex indels, and retrotransposition-mediated insertions of mobile elements and processed RNAs. A large proportion are previously under reported variants sized between 21 and 100 bp. We detect 4 megabases of novel sequence, encoding 11 new transcripts. Finally, we show 191 known, trait-associated SNPs to be in strong linkage disequilibrium with SVs and demonstrate that our panel facilitates accurate imputation of SVs in unrelated individuals

Long-term culture of genome-stable bipotent stem cells from adult human liver

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy

Mutation p.R356Q in the Collybistin Phosphoinositide Binding Site Is Associated With Mild Intellectual Disability

The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR a2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3-R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3-R356Q and CB3SH3-R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding