Assessing Population Structure and Genetic Diversity in US Suffolk Sheep to Define a Framework for Genomic Selection

Long-term sustainability of breeds depends on having sufficient genetic diversity for adaptability to change, whether driven by climatic conditions or by priorities in breeding programs. Genetic diversity in Suffolk sheep in the United States was evaluated in four ways: 1) using genetic relationships from pedigree data [(n = 64 310 animals recorded in the US National Sheep Improvement Program (NSIP)]; 2) using molecular data (n = 304 Suffolk genotyped with the OvineHD BeadChip); 3) comparing Australian (n = 109) and Irish (n = 55) Suffolk sheep to those in the United States using molecular data; and 4) assessing genetic relationships (connectedness) among active Suffolk flocks (n = 18) in NSIP. By characterizing genetic diversity, a goal was to define the structure of a reference population for use for genomic selection strategies in this breed. Pedigree-based mean inbreeding level for the most recent year of available data was 5.5%. Ten animals defined 22.8% of the current gene pool. The effective population size (Ne) ranged from 27.5 to 244.2 based on pedigree and was 79.5 based on molecular data. Expected (HE) and observed (HO) heterozygosity were 0.317 and 0.306, respectively. Model-based population structure included 7 subpopulations. From Principal Component Analysis, countries separated into distinct populations. Within the US population, flocks formed genetically disconnected clusters. A decline in genetic diversity over time was observed from both pedigree and genomic-based derived measures with evidence of population substructure as measured by FST. Using these measures of genetic diversity, a framework for establishing a genomic reference population in US Suffolk sheep engaged in NSIP was proposed

Autophagy, but Not Proteolysis, May Aid in Muscle Protein Synthesis

For muscle growth to occur, protein synthesis must be greater than protein degradation. However, up to this point, anabolic pathways have garnered the brunt of investigations examining anabolic capacity with little investigation into the connectedness of catabolic signaling on these anabolic targets. PURPOSE: The purpose of this study was to elucidate the contributions of proteasomal-dependent and autophagic-dependent catabolic pathways on anabolism via analysis of fractional synthetic rates (FSR) in L6 myotubes. METHODS: Differentiated, cultured L6 myoblasts were treated with media containing 4% deuterium oxide (stable isotope label) and a corresponding pharmacological treatment (NSC 185058 [autophagic inhibitor; 100 μM], MG-262 [proteasomal inhibitor; 0.01 μM] or DMSO control; n=3/group) during the final 24-hours of the differentiation period prior to harvest. The myofibrillar pellet of the processed samples was used to determine FSR via mass-spectrometry analysis. DMSO-treated myotubes served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences among groups. RESULTS: Our results indicate that MG-262 had no impact on myofibrillar FSR when compared to DMSO control (MG-262 1.0993 %/day vs. control 1.239 %/day). However, NSC 185058 lowered myofibrillar FSR (NSC 185058 0.9009 %/day vs. control 1.239 %/day; P=0.0282). CONCLUSION: These data suggest that inhibition of autophagic machinery can impair anabolism. This may be due to autophagy’s role in increasing the amino acid pool within the cell. Further, the lack of inhibition seen from MG-262 suggests that there is a delineation of roles within the catabolic pathways in regard to their influence on anabolism in healthy, metabolically unchallenged myotubes

Insulin-induced Increase in Anabolic Capacity is Blunted by Autophagic Inhibition in L6 Myotubes

Insulin is an anabolic hormone that acts on skeletal muscle cells to stimulate protein synthesis, an effect that is enhanced by the availability of amino acids. While autophagy within the cell provides an intracellular source of amino acids to support anabolism, little is known about how this pathway impacts the insulin-induced increase in anabolic capacity within skeletal muscle cells. PURPOSE: The purpose of this study was to determine the impact of autophagic inhibition in cultured L6 myotubes in conjunction with insulin stimulation in vitro. METHODS: Differentiated, cultured L6 myotubes were treated for 24 hours with or without insulin (100 nM) and NSC 185058 (100 μM), a specialized inhibitor of the autophagic catabolic pathway, in media enriched with 4% deuterium. Cells were harvested from each treatment group (n=3/group) 24 hours post-deuterium enrichment and were processed for protein synthesis and western blot protein analysis. A one-way ANOVA was used to compare groups, and when significant F ratios were present, a Student’s Newman-Keuls post hoc procedure was used to test differences among group means. Alpha was set at p≤0.05 for all analyses. RESULTS: Cells treated with insulin (INS) had a higher ratio of phosphorylated to total P70S6K compared to untreated (CON) cells and those incubated with both insulin and NSC 185058 (INS+NSC; 1694% and 327%, respectively; p\u3c0.05). INS+NSC also decreased the ratio of phosphorylated to total 4EBP1 relative to CON (-51%) and INS (-49%), although these differences were not significant (p\u3e0.05). Myofibrillar protein synthesis was stimulated with INS compared to CON and INS+NSC (30.3% and 70.1% respectively; p\u3c0.05) but was lower in INS+NSC relative to CON (-23.4%; p\u3c0.05). CONCLUSION: Results from our study indicate that insulin (100 nM) stimulates anabolism in skeletal muscle cells, but that addition of the autophagic inhibitor NSC 185058 (100 μM) blunts this effect to a level similar to or less than control. Further, our data suggest that the reduction of protein synthesis is mediated through the downregulation of the mTORC1 signaling pathway. While it is widely recognized that insulin promotes anabolic activity through both the direct stimulation of mTOR signaling and extracellular amino acid uptake, our data strongly indicate that autophagic processes are necessary for full anabolic responses in muscle. This decrease in anabolic capacity supports previous literature indicating that the amino acid availability impacts the stimulatory impact of insulin on protein synthesis

Multiple model triangulation to identify factors associated with lameness in British sheep flocks

Identification of factors associated with an outcome can be challenging when the number of explanatory variables is large in relation to the number of observations. Multiple model triangulation, where results from several model types are combined, improves the likelihood of identifying true predictor variables. The aim of this study was to use triangulation to identify covariates likely to be truly associated with the prevalence of lameness in sheep flocks in Great Britain. Data were collected using a questionnaire sent to 3200 sheep farmers in Great Britain in 2018. The useable response rate was 14.1 %. The geometric mean prevalence of lameness was 1.4 % (95 % CI 1.2−1.7) for ewes, and 0.6 % (95 % CI 0.5−0.9) for lambs, however, approximately 60 % flocks had >2% prevalence of lameness in ewes. Four model types were investigated, two generalised linear models (negative binomial and quasi-Poisson) built using stepwise selection, and two elastic net models (Poisson and Gaussian distributions) refined with selection stability estimation. Triangulated covariates were those selected in three or all four models – 10 for ewes and 12 for lambs. Higher prevalence of lameness in ewes was associated with 5−100% feet bleeding during routine foot trimming compared with not foot trimming, footbathing the flock to treat severe footrot (SFR) and always using formalin in footbaths, both compared with not footbathing, using FootVax™ for 3 weeks compared with always. Lower prevalence of lameness in ewes was associated with vaccinating with FootVax™ for >5 years compared with not vaccinating, peat soil compared with no peat soil, and having no lame ewes to treat. Higher prevalence of lameness in lambs was associated with 5−100% feet bleeding during routine foot trimming, always foot trimming ewes with SFR, not knowingly selecting replacement ewes from ewes that were never lame compared with always, replacement sheep purchased and homebred compared with only homebred, treating lambs >3 days after recognition of lameness compared with 0-3 days and footbathing the flock to treat interdigital dermatitis compared with not footbathing at all. Lower prevalence of lameness in lambs was associated with peat soil, flocks in Scotland versus England, an altitude of >230−500 m compared with ≤230 m, never using antibiotic injection to treat lambs with SFR compared with always, and having no lame lambs to treat. We conclude triangulation identified reliable management practices for farmers to implement to minimise lameness in sheep

Autophagy is Required for the Anabolic Response to Muscle Contraction

Exercise is a key stimulus in regulating the behavior and metabolism of skeletal muscle, with exercise inducing muscular growth through activation of the anabolic mechanistic target of rapamycin kinase (mTOR). Separately, there is mounting evidence that exercise increases autophagy (one of the main routes by which intracellular proteins are degraded) and that the autophagic process may indeed be required for adaptations to exercise training. PURPOSE: To investigate the effects of autophagy inhibition on mTOR signaling and cellular anabolism after muscular contraction. METHODS: Cultured L6 myotubes were to exposed to electrical pulse stimulation using a stimulator set to deliver bipolar pulses of 30V at 100 Hz for 200 ms every fifth second for 60 minutes. Subsequently, cells received either vehicle control, or 100 μM NSC-185058, an antagonist of the key autophagy protein ATG4B and known inhibitor of autophagy. All groups were also exposed to 4% deuterium oxide, a stable isotopic tracer for measurements of protein synthesis. 24 hours post “exercise” bout, cells were lysed in ice-cold Norris buffer, and prepared for Western immunoblot of protein expression, or determination of protein fractional synthesis rate (FSR) of the myofibrillar fraction via mass-spectrometry analysis. Non-stimulated cells receiving vehicle control treatment served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences between groups. RESULTS: We found that phosphorylation of a key downstream target of mTOR, P70S6 kinase, was roughly seven times greater in cells subjected to EPS and vehicle control (710.3%) relative to control (p0.05). While there was a trend for EPS treatment to increase expression of ATG4B, along with a reduction of ATG4B content as a result of NSC-185058 treatment, this finding did not rise to the level of statistical significance. There were no differences in FSR between cells exposed to EPS; however, NSC-185058 treatment significantly reduced FSR in EPS treated cells relative to controls (0.8712 %/hr vs 1.193 %/hr). CONCLUSION: These findings present two conclusions: high-intensity EPS as an in vitro model of exercise elevates mTOR signaling through P70S6K 24 hours post exercise, and mTOR activation as a result of muscular contraction is reliant upon autophagy in skeletal muscle. Further work will be required to elucidate the dynamics of this relationship, and the interplay between skeletal muscle autophagy and anabolism

DNA Targeting as a Likely Mechanism Underlying the Antibacterial Activity of Synthetic Bis-Indole Antibiotics

We previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) organisms. Further, exposure of E. coli and S. aureus to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in S. aureus, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects

Converging on bladder health through design thinking: From an ecology of influence to a focused set of research questions

Lower urinary tract symptoms affect a substantial number of women in the United States (U.S.) and globally. In 2015, the Prevention of Lower Urinary tract Symptoms in women (PLUS) Research Consortium was funded to establish the scientific basis for prevention efforts by (1) understanding healthy bladder function and (2) identifying risk and protective factors for bladder health in women across the lifecourse. This transdisciplinary consortium generated a list of over 600 candidate risk and protective factors for bladder health in women and girls and refined and prioritized these into 29 focused research questions to inform a national longitudinal observational study in the U.S. This paper describes that process using design thinking, a human-centered set of principles and strategies by which innovations are developed, as a framework. Design thinking is an iterative process consisting of five stages: Empathizing with end-users of innovations, Defining core principles girding the work, Ideation of all possible solutions, and rapid-cycle Prototyping and Testing of solutions. Lessons learned are offered to inform future prevention science research endeavors that might benefit from such an approach

Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.Fil: LaRusch, Jessica. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Jung, Jinsei. Yonsei University College of Medicine; Corea del SurFil: General, Ignacio. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lewis, Michele D.. Mayo Clinic. Division of Gastroenterology and Hepatology; Estados UnidosFil: Park, Hyun Woo. Yonsei University College of Medicine; Corea del SurFil: Brand, Randall E.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Gelrud, Andres. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Anderson, Michelle A.. University of Michigan; Estados UnidosFil: Banks, Peter A.. Brigham and Women’s Hospital. Division of Gastroenterology; Estados UnidosFil: Conwell, Darwin. Brigham and Women’s Hospital. Division of Gastroenterology; Estados UnidosFil: Lawrence, Christopher. Medical University of South Carolina; Estados UnidosFil: Romagnuolo, Joseph. Medical University of South Carolina; Estados UnidosFil: Baillie, John. University of Duke; Estados UnidosFil: Alkaade, Samer. St. Louis University. School of Medicine; Estados UnidosFil: Cote, Gregory. Indiana University; Estados UnidosFil: Gardner, Timothy B.. Dartmouth-Hitchcock Medical Center; Estados UnidosFil: Amann, Stephen T.. North Mississippi Medical Center; Estados UnidosFil: Slivka, Adam. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Sandhu, Bimaljit. Virginia Commonwealth University Medical Center; Estados UnidosFil: Aloe, Amy. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Kienholz, Michelle L.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Yadav, Dhiraj. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Barmada, M. Michael. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Bahar, Ivet. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: Lee, Min Goo. Yonsei University College of Medicine; Corea del SurFil: Whitcomb, David C.. Univeristy of Pittsburgh. School of Medicine; Estados UnidosFil: North American Pancreatitis Study Group. No especifica

The Vehicle, Fall 1982

Vol. 24, No. 1 Table of Contents Winter SurveillanceB.L. Davidsonpage 3 The InvitationBecky Lawsonpage 4 Check In, Check OutSteve Sandstrompage 4 On The Front Porch StepKeila Tooleypage 5 Old Greek ManDevon Flesorpage 5 Exotic PassionsBecky Lawsonpage 6 PhotographLisa Owenspage 7 Beyond The ThornsBrook Wilsonpage 8 Ritual Of HeatB.L. Davidsonpage 11 The GamerBecky Lawsonpage 12 It\u27s OverKeila Tooleypage 13 DreamJohn Stockmanpage 14 Silver DollarGina J. Grillopage 15 The DancerJessica Lewispage 16 Snapshots Of Rural IllinoisIsabel M. Parrottpage 16 The Last SeasonTheresa Whitesidepage 17 DrawingKaren Haneypage 17 Rotary LuncheonJessica Lewispage 18 Factory TourLinda Fraembspage 18 The ImmigrantsD.L. Lewispage 19 At Shedd AquariumLinda Fraembspage 20 The GuardianBecky Lewispage 20 Digital LifeEverett Tackettpage 21 Full ServiceScott Graypage 22 Dust ShowLinda A. Brownpage 23 At SixMaureen Foertschpage 24 DrawingJean Imherrpage 24 ReflectionMaggie Kennedypage 25 Cat DefiningBecky Lawsonpage 26 Ode To An Unread NewspaperLinda Fraembspage 26 GumSteve Sandstrompage 27 The DancerChrystal Clarkpage 27 PoemD.L. Lewispage 28 For LucyStacey Flanniganpage 29 An AbortionDevon Flesorpage 29 ReveriesKeila Tooleypage 30 Sunday Morning After Tequila With LemonScott Graypage 33 Staging A Living Jewel BoxMichelle Mitchellpage 34 The Other WomanStacey Flanniganpage 35 The Natural LookMichelle Mitchellpage 35 Poem To A Girl Named SandalsJohn Stockmanpage 36 PhotographLisa Owenspage 37 In The Balcony Of The Bijou On A Saturday NightScott Graypage 38 The Canadian Soccer PlayerBecky Lawsonpage 39 The HealingJohn Stockmanpage 39 AppeasedDevon Flesorpage 40 CodaJohn Stockmanpage 40https://thekeep.eiu.edu/vehicle/1040/thumbnail.jp