Predoctoral Fellowship Research Articles

The marine natural product coibamide targets expression of HER family receptors

Natural products have historically been a significant source of inspiration for drug design and development, particularly in the areas of cancer and infectious disease. The utility of natural products as drug leads is attributed to the fact that these molecules have evolved to bind specific biological targets and have the potential to reveal new aspects of cell signaling. We previously reported the discovery of coibamide, a cytotoxic N‐methyl‐stabilized depsipeptide isolated from a marine cyanobacterium growing within Coiba National Park, Panama. Initial testing of coibamide in the National Cancer Institute in vitro 60 (NCI60) cancer cell line panel revealed pM to nM potency as a growth inhibitor, an unmatched selectivity profile and histological selectivity for breast, CNS‐derived tumors, ovarian and colon cancer cells.We have undertaken studies to determine the mechanism of action of coibamide and have determined that coibamide selectively targets the ER secretory pathway. To date, these relatively rare natural products are known for their ability to selectively inhibit biosynthesis receptor tyrosine kinases, growth factors and cell adhesion proteins. We studied the relative expression of human epidermal growth factor receptor (HER) proteins in human HER2‐amplified and triple negative breast cancer cells following exposure to coibamide. Coibamide inhibited expression of HER family members with differential sensitivity (EGFR=HER3<<HER2). Time‐ and concentration‐dependent decreases in HER protein expression were accompanied by a dampening in AKT/MAPK signaling. Coibamide promoted proteasomal degradation of HER family proteins consistent with the action of an ER secretory pathway inhibitor. These results suggest that coibamide preferentially inhibits the biosynthesis of a subset of ER secretory pathway proteins to induce cancer cell death rather than a total inhibition of cellular secretory function. The HER family of proteins are associated with drug resistance and treatment failure in response to many modern, targeted anti‐cancer medicines and thus this work has the potential to reveal a new way to target HER signaling.Support or Funding InformationThis work was supported by the Oregon State University (OSU) College of Pharmacy, the American Brain Tumor Association (JEI) and an American Foundation for Pharmaceutical Education (AFPE) Pre‐Doctoral Fellowship in the Pharmaceutical Sciences (JDS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Central Adiposity is a Strong Predictor of Non‐alcoholic Fatty Liver Disease (NAFLD) in South Asian Women

Incidence of non‐alcoholic fatty liver disease (NAFLD) is increasing with increasing obesity rates due to its relation with the metabolic syndrome. This is of concern in the South Asian population since they develop metabolic complications at lower levels of obesity compared to their Western counterparts. Furthermore, the relationship between NAFLD, insulin resistance and obesity is not well characterized in humans. To address this issue, we used a convenience sample of Sri Lankan adult females (n = 34) and collected anthropometric data, adipose tissue specimens (for histology), and fasted serum samples (for metabolic and inflammatory markers). Hepatic steatosis was assessed by ultrasound scanning and used to classify participants as NAFL 0, NAFL 1, and NAFL 2. Waist circumference significantly increased with increasing NAFLD grade. Participants with NAFLD had significantly higher body mass index, hip circumference, and fasting blood glucose, as well as a higher mean adipocyte area in both omental and abdominal areas, indicating a higher degree of adipocyte hypertrophy associated with fatty liver. There were, however, no differences in measures of dyslipidemia. Various adipokines were measured; however, resistin was the only pro‐inflammatory adipokine significantly elevated in NAFL 2. These findings indicate that measures of adiposity and fasting blood glucose may be important indicators of NAFLD in South Asian women.Support or Funding InformationThis research was funded by an International Research/Development Seed Grant from the Office of International Affairs at Texas Tech University, an International Research Seed Grant from the College of Human Sciences at Texas Tech University, and the Obesity Research Cluster at Texas Tech University. NMM is in part supported by the National Institutes of Health National Center for Complementary and Integrative Health (NIH NCCIH) award number 1 R15 AT008879‐01A1. KAS is a United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) Fellow supported by an Agriculture and Food Research Initiative's (AFRI) Food, Agriculture, Natural Resources, and Human Sciences Education and Literacy Initiative (ELI) Predoctoral Fellowship under award number 2017‐67011‐26029. NSK was funded by the International Research Center, University of Peradeniya, Sri Lanka (InRC/2015/01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional assignment of Structural Genomics proteins through computed chemical properties, graph representation of active sites, and biochemical validation

There are currently over 14,300 Structural Genomics (SG) protein structures deposited in the PDB by protein structure initiatives. However, most of these SG proteins have unknown or putative function annotations. This accumulated structural information represents a tremendous contribution to structural biology and genomics. Still, the addition of accurate functional annotations for these SG proteins would add substantial value to this information. Our approach to functional annotation and validation incorporates predicting functional assignments through structure‐based computed chemical properties and local structure matching followed by biochemical validation. This research focuses on four superfamilies: Crotonase, Ribulose Phosphate Binding Barrel, 6‐Hairpin Glycosidase, and Concanavlin A‐like Lectins and Glucanases. First, Partial Order Optimum Likelihood (POOL) is used to predict computationally the catalytically important residues in each protein structure. Next, Structurally Aligned Local Sites of Activity (SALSA) develops spatially‐localized consensus signatures for the proteins of known function in each functional family within each superfamily based on POOL‐predicted residues and functionally characterized residues of importance. Then, the POOL‐predicted residues for each SG protein are compared to each consensus signature and scored to determine their degree of similarity at the local active site. Finally, we introduce a new, computationally faster method for sorting protein superfamilies and annotating protein function using local structure matching in graph representation: Graph Representation of Active Sites for Prediction of Function (GRASP‐Func). Sets of tetrahedra are generated through Delaunay triangulation for each protein structure using the alpha carbon atoms of each residue. Then, sets of proteins with matched tetrahedra are grouped together and images are generated showing the relationship of each protein (node) and its neighbors (edges) with similar active sites. We compare SALSA and GRASP‐Func and show that both methods correctly sort the superfamilies into their respective functional families. Both methods also make similar functional predictions for the SG proteins, with GRASP‐Func performing in far less time. Thus GRASP‐Func enables large‐scale comparisons and functional assignments within and across superfamilies. Finally, we are able to test these predictions biochemically to confirm function. Biochemical data for the Crotonase Superfamily show that while proteins have some promiscuous functionality, our methods predict the correct dominant function for each protein tested. The goal of this project is to provide a validated approach to functional annotation to enable applications from drug target identification to green chemistry and biofuel production.Support or Funding InformationSupport from NSF‐CHE‐1305655, NSF‐MCB‐1158176, NSF‐MCB‐1517290, PhRMA Foundations (Predoctoral Fellowship in Informatics awarded to CLM), NSF‐GRFP (JSL), MathWorks, Inc., and American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC (PJB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MTORC2 Regulates the Maturation of Protein Kinase C by Folding and Phosphorylation

Protein Kinase C (PKC) is primed by a series of ordered phosphorylations and conformational transitions to yield an autoinhibited enzyme that is positioned to respond to second messengers. For most PKC isozymes, this maturation to produce a signaling‐competent enzyme depends on the mammalian Target of Rapamycin Complex 2 (mTORC2); however, the molecular mechanisms by which mTORC2 regulates PKC are unknown. Here we use a variety of Förster resonance energy transfer (FRET)‐based live cell imaging and pulse‐chase biochemical assays to examine the role of mTORC2 in the the folding, phosphorylation, and activity of overexpressed PKCbII. We show that in cells lacking Sin1, a critical component of mTORC2, PKC exists in an unprimed, unphosphorylated, and open conformation, with fully‐exposed membrane‐targeting modules. In contrast, overexpression of PKCbII in cells with functional mTORC2 results in accumulation of a fully‐phosphorylated and autoinhibited enzyme. Introduction of Glu at the two phosphorylated residues on the C‐terminal tail is insufficient to bypass the requirement for mTORC2 in the folding or rate‐limiting processing step of PKC. We also show that PDK‐1 is involved in the mTORC2‐regulated rate‐limiting step of PKC processing, as overexpression of PDK‐1 in the absence of mTORC2 results in phosphorylation of the hydrophobic motif, but not the turn motif. Finally, analysis of chimeric proteins of an mTORC2‐dependent (PKCbII) and mTORC2‐independent (PKCd) enzyme, coupled with peptide array experiments, identify two critical regions in the kinase domain and C‐terminal tail, respectively, that each confer mTORC2‐dependence. Taken together, our data support a model in which mTORC2 serves two independent functions in the processing of PKC: 1) as the direct kinase of the turn motif, and 2) in the regulation of the rate‐limiting conformational change that allows the enzyme to autophosphorylate at the hydrophobic motif and adopt an autoinhibited conformation.Support or Funding InformationThis work was funded by NIH GM43154 to AN, NIH Pharmacological Sciences Training Grant NIH T32GM007752 to TB (AN), and PhRMA Foundation Pre Doctoral Fellowship in Pharmacology/Toxicology to TB (AN).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatic Endoplasmic Reticulum Associated Degradation (ERAD) manages FGF21 levels and metabolism via CREBH during fasting‐feeding and growth

The liver regulates growth and systemic energy homeostasis through inter‐organ communication via the secretion of growth factors, hormones and peptides. Fibroblast growth factor 21 (FGF21) is a liver‐derived, fasting‐induced hormone with broad effects on growth, nutrient metabolism and insulin sensitivity, and huge potential as an anti‐diabetes/obesity therapeutic. Here, we report the discovery of a novel regulatory mechanism repressing FGF21 expression under growth and fasting‐feeding. The SEL1L‐HRD1 complex is the most conserved branch of mammalian endoplasmic reticulum (ER)‐associated degradation (ERAD) machinery, responsible for the recruitment, ubiquitination and retro‐translocation of ER proteins for cytosolic proteasomal degradation. Mice with liver‐specific deletion of SEL1L exhibit growth retardation and improved metabolism with markedly elevated circulating FGF21, phenocopying FGF21 transgenic mice or pharmacological models. Mechanistically, we show that the SEL1L‐HRD1 ERAD complex controls FGF21 transcription by regulating the turnover (and thus nuclear abundance) of ER‐resident transcription factor CREBH, while having no effect on the other well‐known FGF21 transcription factor PPARα. We further show a physiologically‐regulated, inverse correlation between SEL1L‐HRD1 ERAD and CREBH‐FGF21 levels under fasting‐feeding and growth. This study not only establishes the importance of ERAD in liver in the regulation of systemic energy metabolism, but also reveals a novel hepatic “ERAD‐CREBH‐FGF21” axis directly linking ER protein turnover to gene transcription and systemic metabolic regulation.Support or Funding InformationAHA predoctoral fellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of function RGS2 mutations augment vascular contractility ex vivo

ObjectiveRegulator of G protein signaling 2 (RGS2) plays a significant role in alleviating vascular contraction and promoting vascular relaxation due to its GTPase accelerating protein activity toward Gαq. Through a cell‐based Ca2+ mobilization assay, we identified 4 loss‐of‐function (LOF) mutations (Q2L, D40Y, R44H and R188H) out of 16 rare, missense mutations in RGS2 selected from various human exome sequencing projects. This study is to investigate whether these LOF RGS2 mutations disrupt the protein function in regulating vascular contractility.Approach and resultWe confirmed that RGS2‐deficient mice expressed a hypertensive phenotype by telemetric femoral artery blood pressure measurement in conscious mice (WT SBP 120 ± 3 vs RGS2−/−SBP 128 ± 2 mmHg, p<0.01). Isolated mesenteric arteries from RGS2−/− mice showed higher contractile response to 10 nM angiotensin II (AngII) stimulation in pressure myograph while isolated aortic rings from those animals generated more tension upon phenylephrine (PE) stimulation (Emax 238 mg vs 68 mg, p = 0.01) compared to aortic rings from RGS2+/+ mice. Reintroduction of wild‐type RGS2‐GFP plasmids into RGS2−/− mesenteric arteries by reversible permeabilization suppressed the vasoconstrictor response to 50 nM AngII (RGS2 WT plasmid −4% vs pcDNA control −35% of basal diameter, p = 0.01). Transfection with RGS2 LOF mutation plasmidss did not suppress Ang II constriction compared to those expressing WT‐RGS2 (Q2L −20%, D40Y, R44H and R188H −30% of basal diameter, p < 0.05).ConclusionsThis study is the first to investigate the function of human mutant RGS2 in resistance arteries in a near physiological condition. It demonstrates that RGS2 attenuates vasoconstriction in the mesenteric arteries and that RGS2 mutations disrupt this effect. Although these mutations are rare, knowledge about their function may provide insights into pathophysiology and treatment.Support or Funding InformationThis work was supported by the American Heart Association predoctoral fellowship [15PRE24680004] to H.P).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Lysyl Oxidase Activity Prevents Volume Overload Diastolic and Systolic Dysfunction

The cardiac extracellular matrix (ECM) is a structural framework which bridges cardiomyocytes to the cystoskeletal myofibril, preserving alignment, while providing tensile strength to the myocardium. Abnormalities in the architectural scaffolding of the ECM have been noted during various cardiac pathologies. The major structural components of the cardiac ECM are collagens, which are produced by fibroblasts. The relative expression of collagens and the degree of cross‐linking dictate the passive mechanical properties of the heart. Lysyl oxidase (LOX) is a copper‐dependent amine oxidase that plays a critical role in covalently cross‐linking fibrillar collagens, which determines the insolubility, stiffness and resistance to degradation of the resulting collagen fibrils. In our previous studies we have shown marked increases in LOX expression and activity in failing hearts relative to compensated and unstressed hearts. Our goal of this study was to determine whether LOX inhibition, initiated during the early stages of cardiac remodeling, could attenuate fibrosis and cardiac dysfunction.We used the aortocaval fistula (ACF) surgical model on Sprague‐Dawley rats to induce biventricular volume overload (VO). Two weeks after surgery, both sham‐operated and VO rats were treated with the LOX inhibitor, beta‐aminoproprionitrile (BAPN; 100 mg/kg/d), for 12 weeks. BAPN was administered via an osmotic pump surgically inserted into the abdominal cavity. After a total of 14 weeks of VO, left ventricular (LV) pressure‐volume catheterization was used to assess alterations in cardiac function. Western blot protein analyses were performed to assess alterations in LOX, collagens I and III, matrix metalloproteinases (MMPs), and their inhibitors (TIMPs). Further, LV strain and stiffness were calculated to determine alterations in diastolic function.Our data demonstrate that chronic VO caused a significant increase in LV wall stress at both diastole and systole, and produced significant LV hypertrophy (183%, 60%, and 92% increase, respectively vs. control; p<0.05). Further, VO induced significant increases in LV strain and stiffness (126% and 77% increase, respectively vs. control), as well as significant increases in the expression of fibrotic proteins including LOX, collagen I, collagen III, MMP2, and TIMP1 (135%, 58.4%, 26.7%, 39%, and 63% increase, respectively vs. control; p<0.05). Finally, VO also caused systolic dysfunction, as evident by decreases in cardiac contractility and ejection fraction (51% and 26% decrease, respectively vs. control; p<0.05). Further, our data highlight the cardioprotective role of LOX inhibition against adverse cardiac remodeling due to VO. LOX inhibition completely prevented VO‐induced increases in LV wall stress and ventricular hypertrophy. Further, LOX inhibition prevented VO‐induced fibrosis and diastolic dysfunction, as evident by normalized levels of collagens, LOX, MMPs, TIMPs, and LV stiffness. Finally, LOX inhibition prevented the VO‐induced decline in cardiac contractility. Overall, our data indicate that LOX inhibition prevented adverse ECM remodeling and cardiac dysfunction in the volume overloaded heart.Support or Funding InformationSupport for this project provided by the American Heart Association Southeast Affiliate and the NIH Heart, Lung and Blood Institute: Grant‐in‐Aid (#16GRNT30440008‐JDG), AHA Predoctoral Fellowship (# 16PRE29150010‐EEH), and F31 (#1F31HL134263‐ 01A1‐EEH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gut dysbiosis in the development of cerebral small vessel disease

The importance of a healthy gut microbiota on host physiology is becoming increasingly evident. Recent studies suggest that alterations to the microbiota (i.e., dysbiosis) can have adverse effects beyond the GI tract, and has been linked to hypertension and stroke. We have demonstrated that the microbiota of the spontaneously hypertensive stroke prone rat (SHRSP), a model of cerebral small vessel disease (CSVD), is significantly different than that of WKY controls. Thus we hypothesized that gut dysbiosis contributes to the development of CSVD. Phenotypically, CSVD includes hypertension, vessel remodeling, BBB breakdown and neuroinflammation. If our hypothesis is valid, then creating a gut microbiota in a WKY rat that resembles one from an SHRSP rat should produce CSVD phenotypes. Conversely, creating a gut microbiota in a SHRSP rat that resembles one from a healthy WKY strain should abolish or attenuate the onset of CSVD phenotypes, including hypertension. Given that rat pups acquire their microbiota from their mother and the immediate environment, we used foster mothers to shape the microbiota of rat pups. WKY and SHRSP pups were removed from their birthing mother at 2–3 days of age and fostered on mothers of the same or opposite strain. Upon weening, fostered animals were co‐housed with animals of the same strain as their fostered mother. Systolic blood pressure (SBP) was measured using tail cuff plethysmography from 6–20 weeks of age. At 20 weeks of age, rats were sacrificed and brain and gut tissue were collected to assess for CSVD phenotypes and gene expression for inflammatory markers and toll‐like receptor signaling. Feces were collected at each BP measurement to evaluate the composition of the gut microbiota using 16s rRNA sequencing. Repeated measures two‐way ANOVA of SBP from week 6–20 showed a 15 mmHg increase in WKY rats fostered by an SHRSP mother (sWKY)compared to WKY fostered on WKY mothers (wWKY; p<.001), and a 7 mmHg decrease in SHRSPs fostered by a WKY mother (wSHRSP) compared to SHRSPs fostered by a SHRSP mother (sSHRSP) (p=.027). Independent of genotype, rats nursed by SHRSP mothers had evidence of brain and gut inflammation including increases in TLR2, TLR4, NF‐kB1, and Il‐1α expression in the brain and proximal colon mucosa, compared to animals fostered by WKYs (p<.01). Furthermore, by using IgG extravasation into the brain parenchyma as a marker for blood brain barrier integrity, we discovered that wSHRSP animals had improved blood brain barrier integrity as compared to sSHRSP (p<.05). Through sequencing of fecal 16s rRNA it was determined that cross‐fostering animals on opposite strains lead to significant alterations in the gut microbiota composition. Most striking was that animals nursing on SHRSP mothers led to significant decreases in Akkermansia, a genus associated with improving gut barrier integrity (p<.05). This study supports the hypothesis that gut dysbiosis contributes to the onset and development of CSVD, possibly by creating an inflammatory environment in the colon. Furthermore, maintaining a healthy gut microbiota in a CSVD strain has the potential to reduce CSVD phenotypes, gut inflammation and attenuate hypertension. These findings support the idea that the gut is a possible therapeutic target to treat CSVD and other cardiovascular diseases.Support or Funding InformationAHA Predoctoral Fellowship 16PRE29640005This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cytokine Responses to Cyclical Blood Flow Restriction Exercise

IntroductionWe developed a novel adaptation of blood flow restriction exercise, cyclical blood flow restriction exercise (C‐BFRE), as a method to potentially augment the cardio‐protective effects associated with conventional exercise (CE) and remote ischemic preconditioning (RIPC). As the cytokines IL‐6 and IL‐10 have been implicated in mediating the acute cardio‐protection associated with both exercise training and RIPC when performed independently, we hypothesized that C‐BFRE would augment the systemic release of these factors compared to an acute bout of either CE or RIPC alone.Methods13 young healthy subjects (6M/7F, age 28±2 y) completed 3 experimental sessions separated by at least 1 month each. Aerobic exercise was performed via 40‐min of treadmill walking at 65–70% HRmax with (C‐BFRE) and without (CE) application of bilateral thigh cuffs inflated to 220 mmHg (4 × 5‐min inflation periods followed by 5‐min reperfusion periods). RIPC consisted of 4 × 5‐min thigh cuff inflation periods (220 mmHg) followed by 5‐min reperfusion periods in the absence of exercise. Venous blood samples (via an antecubital vein) were collected at baseline and immediately following completion of each protocol for analysis of IL‐6 and IL‐10 via multiplex ELISA.ResultsPlasma IL‐6 increased from baseline following CE (Pre, 0.26 ± 0.03 vs. Post, 0.40 ± 0.07 pg/ml; P=0.03) but not with C‐BFRE (Pre, 0.28 ± 0.05 vs. Post, 0.34 ± 0.07 pg/ml; P=0.75) or RIPC (Pre, 0.31 ± 0.04 vs. Post, 0.32 ± 0.03 pg/ml; P=1.0). There were no changes in plasma IL‐10 from pre‐ to post‐intervention for any of the three conditions (P=0.42 for main effect of time).DiscussionContrary to our hypothesis, we observed no change in plasma IL‐10 or IL‐6 with application of acute C‐BFRE. These findings suggest that the model of C‐BFRE utilized in this study does not augment the release of these cytokines in young healthy individuals. This may be due to the timing of the blood samples, the use of a standard occlusive pressure across all subjects, and the young, healthy subjects tested in this study. Future work should delineate the inflammatory response induced by C‐BFRE at more extended recovery time points, with use of individualized occlusive pressures, and in more clinically relevant populations in both acute and long term settings.Support or Funding InformationFunding for this study was provided, in part, by training fellowships awarded to Justin Sprick through a NIH‐supported Neurobiology of Aging Training Grant (T32 AG020494, PI: Singh, M), and a Ruth L. Kirchstein NRSA F31 Predoctoral Fellowship (1F31HL134242‐01A1; PI: Sprick, JD), a UNTHSC Faculty Research Pilot Grant (PI: Rickards, CA), and a Texas Chapter of the American College of Sports Medicine (TACSM) Student Research Development Award (PI: Sprick, JD).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Infant Birthweight and Postpartum Hypertension in African Green Monkeys

Postpartum hypertension (PPHT) is characterized by significantly elevated arterial pressure developing within 48 hours following delivery. PPHT affects 2% of all pregnancies and the etiology remains unclear. We have previously reported that the African Green Monkey (AGM; Chlorocebus aethiops sabaeus) develops gestational hypertension with pathophysiological features of preeclampsia. In this study, we report characteristics of PPHT in the AGM that occurs in the absence of hypertension before or after the onset of pregnancy. Systolic blood pressures (SBP) were collected using forearm plethysmography under ketamine sedation (15 mg/kg i.m.). Animals were characterized normotensive (NT; SBP < 120 mmHg) and postpartum hypertensive (PPHT; SBP ≥ 140 mmHg developing within 48 hours following delivery). All PPHT were NT pre‐ and during pregnancy. Twenty‐four hour urine was collected in the third trimester and two weeks postpartum for three consecutive days. SBP for nonpregnant, NT was 98.7 ± 3.7 mmHg (n=18), third trimester SBP for NT was 107.0 ± 4.4 mmHg (n=5), and postpartum day 14 (PP14) SBP for NT was 94.0 ± 12.3 mmHg (n=4; p>0.05). Nonpregnant SBP for PPHT was 115.8 ± 17.6 mmHg (n=8), third trimester SBP was 112.1 ± 13.3 mmHg (n=6), and PPHT, PP14 SBP was 137.2 ± 14.3 mmHg (n=6; p <0.05 PP14 vs. third trimester). Nonpregnant plasma osmolality for NT was 306.3 ± 3.6 mOsm/kg (n=13) and third trimester NT plasma osmolality was 298.7 ± 4.6 mOsm/kg (n=7; p>0.05). Third trimester plasma osmolality for PPHT was 288.8 ± 4.6 mOsm/kg (n=6) and 312.5 ± 5.7 mOsm/kg at PP14 (n=6; p<0.05 vs. third trimester). Nonpregnant protein excretion for NT was 472.5 ± 58.0 mg/day (n=13) and 501.8 ± 104.6 mg/day in third trimester (n=5; p>0.05). In PPHT animals, nonpregnant protein excretion was 684.5 ± 152.1 mg/day (n=1), 472.7 ± 62.1 mg/day in third trimester (n=6), and 171.7 ± 68.0 mg/day at PP14 (n=5; p<0.05 PP14 vs. third trimester). Nonpregnant sodium excretion for NT was 2.4 ± 0.4 mmol/day (n=13) and third trimester for NT was 1.8 ± 0.6 mmol/day (n=5; p>0.05). Nonpregnant sodium excretion for PPHT was 0.3 ± 1.4 mmol/day (n=1), third trimester for PPHT was 2.2 ± 0.5 mmol/day (n=6), and PP14 sodium excretion for PPHT was 3.4 ± 0.6 mmol/day (n=5; p >0.05). Babies born to mothers who ultimately developed PPHT were of a higher birthweight than those born to NT mothers (NT 338.2 ± 25.5 g, n=7; PPHT 404.5 ± 14.1 g, n=6; p<0.05). This data demonstrates that postpartum hypertension in the AGM is associated with decreased protein excretion, and increased SBP after delivery when compared to the third trimester of pregnancy. The mechanisms of this PPHT are largely unknown. Ongoing studies will evaluate changes in kidney function, plasma vasopressin concentrations, and plasma renin activity. The AGM has many physiological and behavioral similarities to humans, making the species an optimal model for the study of pregnancy disorders such as PPHT.Support or Funding InformationBiomedical Science Research Group, American Heart Association Predoctoral Fellowship 17PRE33670127This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pervasive, coordinated protein level changes driven by transcript isoform switching

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous, genome‐wide measurements of mRNA, translation and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti‐correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases—or over 8% of all measured genes—involves temporally regulated switching between production of a canonical, translatable transcript and a 5′ extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether an mRNA is induced or not, but rather based on the type of transcript produced.Support or Funding InformationThis work was supported by NIH funding to G.A.B. (DP2‐GM‐119138), investigator awards from the Alfred P. Sloan Foundation and Pew Charitable Trusts to G.A.B., UC‐Berkeley start‐up funding to G.A.B. G.M.O. is supported by an NIH training grant (GM0007232) to UCB MCB. E.P. is supported by an NSF predoctoral fellowship. M.J. was supported by the Marie Sklodowska‐Curie IOF and he and A.K. are supported by Columbia start‐up funding.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative Analysis of Bacterial Cytochromes P450 Involved in the Biosynthesis of 16‐ Membered Ring Macrolide Antibiotics

Members of the cytochrome P450 superfamily of monooxygenases (P450s) are some of nature's most ubiquitous and versatile enzymes for performing oxidative metabolic transformations. Their unmatched ability to selectively functionalize C–H bonds has led to their growing employment in academic and industrial settings for the production of fine and commodity chemicals. Many of the most interesting and potentially biocatalytically useful P450s come from microorganisms, where they catalyze key tailoring reactions in natural product biosynthetic pathways. While most of these enzymes act on structurally complex pathway intermediates with high selectivity, they often exhibit narrow substrate scope, thus limiting their broader application. In the present work, we biochemically and structurally characterized diverse bacterial P450s involved in the biosynthesis of 16-membered ring macrolide antibiotics with significant potential for development into robust biocatalysts for the late-stage functionalization of complex molecules. Following exploratory efforts to probe the reactivity properties of the P450 MycCI from the mycinamicin biosynthetic pathway, we discovered that the enzyme exhibits appreciable activity on several 16-membered ring macrolactones independent of their glycosylation state. These results were corroborated by performing equilibrium substrate binding and kinetics experiments along with X-ray crystallographic analysis of MycCI bound to its native substrate. We also characterized TylHI, a homologous P450 from the tylosin pathway, and showed that its substrate scope is severely restricted compared with that of MycCI. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI exhibits a strict preference for 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. These results were partially explained through analysis of the X-ray crystal structure of TylHI in complex with its native substrate together with biochemical characterization of several sitedirected mutants. Comparative analysis of the MycCI/TylHI homolog ChmHI from the chalcomycin biosynthetic pathway provided a basis for constructing MycCI/TylHI chimeras in order to gain further insight into the features dictating the differences in the reactivity profiles of these two related P450s. These experiments unveiled the central role of the BC loop region in influencing the binding of 16-membered ring substrates to MycCI and TylHI. Overall, our studies have shed light on the molecular-level details underpinning the unique catalytic divergence of two homologous biosynthetic P450s and point toward specific regions in each protein that may be targeted in future efforts to rationally engineer novel P450-based biocatalysts. Support or Funding Information NSF under the CCI Center for Selective C–H Functionalization (CHE-1205646 and CHE-1700982), NIH (R01-GM078553, R35-GM118101, and T32-GM008353), University of Michigan Rackham Predoctoral Fellowship Physiological reactions catalyzed by P450s MycCI and TylHI in their respective biosynthetic pathways. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inducible Deletion of Endothelial Hba1 Significantly Reduces Exercise Fitness in Mice

Hemoglobin (Hb) alpha is uniquely expressed in the endothelial cells of resistance arteries. The same protein is generated by both Hba1 and Hba2 genes. We have shown that siRNA knockdown of endothelial Hba1 decreases Hb alpha protein, and increases NO signaling in the vascular wall. Furthermore, pharmacologic inhibition of the interaction between endothelial Hb alpha and endothelial nitric oxide synthase reduces blood pressure. To further study the role of endothelial Hb alpha, we have produced a novel genetically modified mouse in which exons 2 and 3 of the Hba1 gene are flanked by loxP sites (Hba1fl/fl). We have crossed the Hba1fl/fl mice with a Cdh5‐Cre/ERT2+ mice to produce a tamoxifen‐inducible genetic model in which Hba1 can be temporally deleted from endothelial cells (EC Hba1fl/fl). We compared EC Hba1fl/fl with Hba1−/− (global deletion of Hba1) and found both mice exhibit reduced expression of Hba protein in skeletal muscle endothelial arteries and arterioles. However, while Hba1−/− mice showed significant decreases in several red blood cell parameters including hemoglobin concentration, hematocrit, erythrocyte count, red cell volume, and red cell hemoglobin, EC Hba1fl/fl red blood cell parameters were all unchanged. In order to test roles of endothelial alpha globin in hypoxic blood flow control, we used a treadmill running test to measure acute endurance capacity, as well as long‐term measurement of voluntary running volume in running wheel‐equipped cages. We found that in both experimental conditions, male EC Hba1fl/fl mice had a significant reduction in exercise capacity, mirroring the exercise phenotype of Hba1−/− mice. We conclude that endothelial Hba1 is an important regulator of vascular function and skeletal muscle performance.Support or Funding InformationAmerican Heart Predoctoral Fellowship #16PRE30240007, NIH R01 HL088554This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypertension in serotonin‐deficient male rats is associated with reduced sympathetic drive to the vasculature

Serotonin (5‐hydroxytryptamine, 5‐HT) neurons have widespread projections to autonomic regions of the brainstem and spinal cord. However, the specific role of central 5‐HT in the autonomic regulation of arterial blood pressure (BP) is unclear. We have previously shown that in quiet wakefulness (QW) and rapid‐eye‐movement (REM) sleep, adult male rats deficient in tryptophan hydroxylase 2 (TPH2−/−), and hence lacking central 5‐HT, have increased BP compared to wild‐type (WT) controls. The BP of female TPH2−/− rats is the same as WT. The current study sought to clarify the mechanism by which a loss of central 5‐HT leads to increased BP. We hypothesized that a loss of central 5‐HT increases sympathetic vascular tone. Using femoral arterial catheters along with electroencephalography and electromyography, we confirmed the elevated BP of adult (5–8 month old) male TPH2−/− rats (n=14) compared to male WT littermates (n=17), during QW (TPH2−/−: 123 vs WT: 115 mmHg; p=0.006) and REM sleep (TPH2−/−: 128 vs WT: 117 mmHg; p=0.002), but not during non‐rapid‐eye movement sleep (NREM) (TPH2−/−: 120 vs WT: 115 mmHg; p=0.068). In contrast, the BP of female TPH2−/− rats (n=15) was the same as WT littermates (n=12) (TPH2−/−: 119, 117, 123 vs WT: 112, 113, 115 mmHg in QW, NREM and REM, respectively) (p>0.05). To determine the influence of central 5‐HT on cardiac vagal drive and sympathetic vasculature tone, we injected atropine methyl nitrate (1 mg/kg; i.v.) and hexamethonium (HEX, 30 mg/kg; i.v.) into a subset of rats (n=5 male TPH2−/− and 5 WT; n= 9 female THP2−/− and 8 WT), and measured the maximum increase in heart rate (HR) and nadir in BP, respectively, during subsequent QW. The atropine‐induced increase in HR and BP experienced by male and female TPH2−/− rats was not significantly different than in WT littermates. Unexpectedly, male TPH2−/− rats had a reduced drop in BP following HEX compared to male WT (p=0.004), a phenotype not observed in female TPH2−/− rats. Our data suggest that a loss of central 5‐HT decreases sympathetic vascular tone in males only. While the mechanism(s) that contribute to the hypertension of male TPH2−/− rats remains unresolved, the potential interaction between central 5‐HT and components of the renin‐angiotensin‐aldosterone and vasopressin systems is worth exploring.Support or Funding InformationFunding for this research was provided by an American Heart Association Scientist Development Grant (14SDG18560022; PI: KJC) and a Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (F31) to JLM.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nutrient sensing and mitochondrial Coenzyme Q biosynthesis: Are they connected by a phosphatase?

Coenzyme Q (also termed ubiquinone or Q) is an electron carrier in the mitochondrial respiratory chain that functions as an essential component in energy metabolism processes, while also serving as a vital lipid soluble antioxidant. Sufficient de novo Q biosynthesis is crucial for human health. Saccharomyces cerevisiae (baker's yeast) serves as an excellent model for studies on Q because of its powerful molecular genetics and the functional identity of yeast and human COQ genes. Coenzyme Q biosynthesis in S. cerevisiae occurs in the mitochondria and, currently, fourteen known mitochondrial proteins are responsible for facilitating this process—Coq1–Coq11, Yah1 (ferredoxin), Arh1 (ferredoxin reductase), and Hfd1 (aldehyde dehydrogenase). Many of the Coq proteins associate in a high molecular weight complex (“CoQ‐Synthome”) that localizes in the inner mitochondrial membrane. Previous work indicated that the stability of the Q biosynthetic complex (as well as the phosphorylation status of several Coq polypeptides, including Coq3, Coq5, Coq7, and potentially Coq6) relied on the presence of Coq8, the putative kinase of the system. Thus, the role of phosphorylation may provide novel information as to how the CoQ‐synthome is regulated. Indeed, intriguing links have been discovered that relate Coq polypeptides and phosphorylation status with nutrient availability and regulatory genes. Most recently, a link has been found between a nutrient sensing gene, SNF2, and the spicing efficiency of PTC7, the phosphatase responsible for controlling a key regulation site in Q biosynthesis. Our objectives are to further investigate these regulatory links. Such studies will aid our understanding and treatment of multiple diseases caused by CoQ‐deficiencies and provide novel insight in longevity and issues related to aging in humans.Support or Funding InformationThis work was supported by the National Science Foundation, Grant MCB‐1330803 and the National Institute of General Medical Sciences (GM‐085474), Whitcome Pre‐doctoral Fellowship in Molecular Biology, and the Ruth L. Kirschstein National Service Award GM‐007185.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic blockade stabilizes the breathing pattern of serotonin‐deficient rat pups during active sleep

Infant rats deficient in tryptophan hydroxylase 2 (TPH2−/−) and hence lacking central serotonin (5‐hydroxytryptamine; 5‐HT) display abnormally destabilized breathing during active sleep; their breathing pattern is punctuated with periods of apnea and tachypnea. Given that acetylcholine is a key driver of active sleep, as well as the fact that muscarinic receptors are abundantly expressed within key respiratory patterning regions, we hypothesized that increased central muscarinic drive contributes to the unstable breathing pattern of infant TPH2−/− rats during active sleep. To test this hypothesis, we conducted two separate experiments using whole‐body plethysmography coupled with nuchal EMG to monitor the breathing pattern of 2‐week‐old TPH2−/− and WT pups during active sleep. Experiment 1: following ~1 hr settling period to allow for the appearance of active sleep, we injected atropine methyl nitrate (Ap‐M), a CNS‐impermeant form of atropine, followed ~30 min later by an injection of atropine sulfate (Ap‐S), a CNS‐permeant form (both 1mg/kg, 10 ul bolus i.v.) (n= 8 and 10 TPH2−/− for Ap‐M and Ap‐S, respectively; n=8 WT for both drugs). Each drug was injected during a period of active sleep. For most animals, arousal did not occur and the effect of the drugs on the co‐efficient of variation in the respiratory period (CV‐P%) could be determined within one episode of active sleep. If arousal occurred, CV‐P% prior to injection was compared to CV‐P% in the next episode of active sleep. Experiment 2: the breathing pattern of TPH2−/− pups was monitored over 2 hrs before and after the administration of Ap‐S (1 mg/kg, 200 ul bolus i.p; n=17) or vehicle (saline; n=16). Tachypnea and apnea indices (# events/hr) were calculated for the active sleep episodes before and after Ap‐S. Administration of Ap‐M increased heart rate (~30–50 beats/min) and decreased heart rate variability for the duration of the experiment. In WT pups, CV‐P% tended to increase after i.v. administration of Ap‐M, but was not significantly changed following Ap‐S. In contrast, the CV‐P% of TPH2−/− pups was significantly increased after Ap‐M (p<0.01) and significantly reduced following Ap‐S (p=0.01). Ap‐S significantly reduced the tachypnea index of TPH2−/− pups relative to TPH2−/− pups given vehicle alone (p=0.04), but had no significant effect on apnea index. The reduced CV‐P% of TPH2−/− pups after Ap‐S relative to values after Ap‐M suggest that muscarinic blockade in the CNS by Ap‐S reverses the destabilizing effect of 5‐HT deficiency on the respiratory pattern. This occurs in spite of a destabilizing peripheral effect of Ap‐M on the breathing pattern. These data also suggest that during active sleep, 5‐HT signaling helps stabilize the breathing pattern of infant rats mainly by inhibiting muscarinic drive to patterning neurons promoting tachypnea.Support or Funding InformationFunding for this research was provided by an American Heart Association Scientist Development Grant (14SDG18560022; PI: KJC) and a Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (F31) to JLM.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Type 1 Phosphodiesterse Confers Therapeutic Benefit to Proteinopathy‐based HFpEF in Mice

Heart Failure with preserved Ejection Fraction (HFpEF) has overtaken Heart Failure with reduced Ejection Fraction (HFrEF) and represents more than 50% of heart failure cases, yet no pharmacological therapy with mortality benefits has been identified. Targeted clearance of misfolded proteins by the ubiquitin‐proteasome system (UPS) plays a pivotal role in protein quality control. UPS function insufficiency contributes to HF genesis, making UPS priming a potential therapeutic strategy for treatment of HF. Previously we reported that protein kinase G (PKG) positively regulates cardiac UPS functioning while others showed protein kinase A (PKA) stimulates the proteasome. Thus, we hypothesize that simultaneous activation of PKG and PKA improves cardiac UPS function and thereby benefits HF treatment. We found that myocardial protein and mRNA levels of phosphodiesterase 1 (PDE1), a major cardiac PDE that degrades both PKG and PKA, were markedly elevated in CryABR120G transgenic (tg) mice which are a classic model of cardiac proteinopathy with HFpEF as the major manifestation of its cardiac dysfunction. To test whether PDE1 inhibition primes cardiac UPS and thereby ameliorates cardiac proteotoxicity, we performed cell cultures and mouse in vivo experiments taking advantage of a proven surrogate UPS substrate (GFPdgn) and a bona fide misfolded protein (CryABR120G), which demonstrates that pharmacological PDE1 inhibition significantly improves cardiac UPS performance and enhances degradation of misfolded proteins. We then treated CryABR120G tg mice with PDE1 inhibitor LSN2790158 (3 mg/kg/day × 28 days) or vehicle control via osmotic mini‐pumps, starting at 4 months of age when HFpEF has clearly developed in the tg mice as reflected by significant decreases in left ventricular (LV) end‐diastolic volume, stroke volume, and cardiac output and increases in end‐diastolic LV posterior wall thickness along with unchanged EF (>50%), compared to littermate non‐tg mice. Echocardiography revealed that the PDE1 inhibitor treatment resulted in striking improvement of HFpEF. And Kaplan‐Meier survival analysis shows a significant delay of premature death in the PDE1 inhibition group vs. vehicle‐treated group. Additionally, PDE1 inhibition group showed significant reduction of NP‐40 insoluble but SDS soluble fractions of CryAB that represent a misfolded species of CryAB degraded by the proteasome, suggesting that the cardioprotective effect of PDE1 inhibition is, at least in part, due to enhanced removal of misfolded proteins. Our data provide compelling evidence for the first time that PDE1 inhibition shall be explored as a new therapeutic strategy for HFpEF and heart disease with increased proteotoxic stress.Support or Funding InformationThis work was supported in part by NIH grants HL072166, HL085629, and HL131667 and American Heart Association Predoctoral Fellowship grant 16PRE27790059; the PDE1 inhibitor LSN2790158 was provided by Eli Lilly and CompanyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Can Daytime Measures of Respiratory Sinus Arrhythmia and Breathing Stability Serve as Biomarkers for OSA?

Obstructive sleep apnea (OSA) is by definition a disorder of nighttime breathing however, there is good evidence that impairments in cognitive and cardiovascular function persist in the day and that adults with OSA exhibit daytime CO2 retention and hypoxemia. Although a diagnosis of OSA requires in‐home or in‐laboratory overnight polysomnography, these types of tests are expensive and, due to health insurance quotas, may be limited in availability. Here, we outline simple methods for assessing daytime respiratory sinus arrhythmia (RSA) and respiratory stability that show promise as potential diagnostic tools to identify adults at risk for OSA. We recorded heart rate (finger oxy‐photoplethysmography) and respiration (inductance plethysmography) in adults with obstructive sleep apnea (N=6) and age and BMI matched healthy adults (N=8). Subjects completed five minutes of eupneic breathing, five minutes of paced breathing (7.5 breaths/minute) and after equilibration, five minutes of hyperoxic, hypercapnia (5% FICO2). Using de‐identified data, we obtained estimates of RSA amplitude, RSA stability, and respiratory stability for each subject in each condition and performed a regression analysis to determine the relationship between each of these variables and subjects' apnea hypopnea index (0–25 AHI). Our preliminary results indicate that daytime measures of respiratory stability and RSA can differentiate healthy adults from adults diagnosed with OSA (p<0.006). Importantly, the analysis requires minimal instrumentation and short sampling periods (~5 minutes). Summary schematics arising from the analysis may prove useful as preliminary clinical/diagnostic biomarkers of RSA and cardio‐respiratory coupling in adults at risk for OSA.Support or Funding InformationThis work was supported by start‐up monies (Department of Physiology, University of Arizona) awarded to EFB and by The Finley and Florence Brown Predoctoral Fellowship (Sarver Heart Center, University of Arizona) awarded to JRV.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Matrix Metalloproteinase 2 (MMP2) In Diabetes‐induced Loss of PECAM‐1 in The Retina: Direct and Indirect Mechanisms

ObjectiveMatrix metalloproteinases (MMPs) are calcium‐dependent, zinc‐containing endopeptidases that are responsible for degrading extracellular matrix proteins, and for cleavage of various substrates such as cell surface receptors, proteoglycans, chemokines and cytokines. The aim of this study was to investigate the molecular mechanisms by which MMP2 may lead to PECAM‐1 loss in the diabetic retina, either directly through cleavage, or indirectly by modulating tumor necrosis factor‐α (TNF‐α) levels and activity.BackgroundPlatelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has major roles in barrier function, endothelial cell‐endothelial cell adhesion, and cell signaling. We have previously found a significant decrease (50–90%, P<0.05) in PECAM‐1 levels in the diabetic retina, and in cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions (30–40%, P<0.05). The decrease in PECAM‐1 levels was accompanied by a significant increase in MMP2 levels and activity in the diabetic plasma (72% and 78.5% respectively, P<0.05), and in RRMECs grown under high glucose conditions (12‐fold increase, P<0.05).Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with treatments added 6–24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. GM6001, a broad spectrum MMP inhibitor, was started at week six post‐STZ injection for two weeks (4 mM, 25 μl/eye, in sterile eye drops), and retinas were collected eight weeks post‐STZ injection for analysis. PECAM‐1, MMP2, and TNF‐α levels and interactions were assessed using western blotting, zymography, immunofluorescence, immunoprecipitation, or ELISA assays. In silico analysis for possible protease cleavage sites was done using PROSPER, provided by Monash University, Australia.ResultsSeveral PECAM‐1 cleavage sites by MMP2 were identified using in silico analysis. Moreover, we confirmed PECAM‐1/MMP2 interactions using immunoprecipitation. PECAM‐1 levels were significantly decreased in RRMECs treated with MMP2 (81%, P<0.05), with a significant increase in TNF‐α levels (118.2%, P<0.05). Moreover, RRMECs treated with TNF‐α had a significant decrease in PECAM‐1 levels (34%, P<0.05). Furthermore, TNF‐α levels were significantly increased in diabetic STZ plasma, and in RRMECs grown under HG conditions (31%, 74.4% respectively, P<0.05). To investigate the protective effect of MMP2 inhibition on PECAM‐1 levels, we treated diabetic rats with GM6001 eye drops, and saw an increase of PECAM‐1 levels (77.2%, P<0.05) when compared to untreated rats.ConclusionThese results indicate a possible role of MMP2 in the diabetes‐induced loss of PECAM‐1 in the retina, which can be via direct cleavage of PECAM‐1, or indirectly via upregulation of TNF‐α, with the two mechanisms evaluated in future studies.Support or Funding InformationNIH EY025632, and AHA Predoctoral Fellowship 16PRE30080003This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetically Engineering a Sheep Model of Hypophosphatasia

Hypophosphatasia (HPP) is a rare inherited disorder that affects the development of bones and teeth. The disease is caused by mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene (ALPL) and accompanied by a highly variable clinical presentation. Although HPP patient studies have advanced our understanding of HPP, as well as documented disease severity, the rarity of the disease combined with a lack of sufficient animal models has significantly delayed mechanistic understanding and therapeutic development. Similar to other human disease models, current HPP models have been engineered virtually exclusively in rodents – specifically mice harboring null or loss‐of‐function mutations. Although useful for modeling some features of HPP, these murine models do not faithfully represent the broad spectrum of human HPP clinical bone, muscle and tooth (odonto‐HPP) phenotypes. However, in sheep both Haversian bone remodeling and tooth development are analogous to humans with sheep TNSALP amino acid sequence sharing 89% identity with human. Thus, our objective was to generate a sheep model of HPP and we posit that ALPL mutation‐specific replacements in the sheep genome using CRISPR/Cas9 will produce a model that accurately phenocopies the bone and tooth pathophysiology of HPP. To genetically engineer a sheep model of HPP, a total of 52 in vitro genetically manipulated embryos were generated targeting the Exon 10 c.1077G>A mutation using CRISPR/Cas9. Three embryos were implanted per recipient ewe and implantations were performed in a total of 17 recipient ewes. Nine ewe pregnancies were confirmed by the measurement of Pregnancy‐Associated Protein on post‐implantation day 35 and further validated by ultrasound. From 9 live newborn lambs, four were heterozygous for our specific point mutation (4/6), one compound heterozygous (1/6) and one homozygous (1/6) for a total mutation efficiency rate of 66.6% (6/9). Similar to HPP patients with the same mutation, mutant sheep have variable clinical expression accompanied with decreased bone formation and mineralization in tails observed by DXA as well as significantly and appropriately decreased serum alkaline phosphatase activity. Additionally, an apparent “shell tooth” phenotype is observed in mutant lambs, further suggesting a mutant exon 10 genotype/phenotype that more completely recapitulates human skeletal and odonto‐HPP.Support or Funding Information1. William Townsend Porter Pre‐doctoral Fellowship2. TAMU/Texas Heart Institute Center for Cell and Organ Biotechnology Innovation Kitchen GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.