Journal articles on the topic '06a061'

Abstract Introduction Previous studies have shown that obese patients with obstructive sleep apnea (OSA) have a significantly greater percentage of fat tissue in soft palate than normal subjects. However, the influence of soft palate fat is not clear in non-obese adults with OSA. This study compared the volume of fat in the soft palate between lean adults with OSA and lean controls. Methods We examined soft palate fat in 21 lean OSA cases and 16 lean controls with body mass index (BMI) <25 kg/m2. All subjects underwent a magnetic resonance imaging (MRI) with three-point Dixon scan. We used volumetric reconstruction algorithms to quantify the amount of soft palate fat, which was compared between apnecis and controls. Analysis reproducibility was quantified using intraclass correlation coefficients (ICC) from repeated analyses of 20 randomly-chosen MRIs. Results Analysis of soft palate fat was highly reproducible, with an ICC (95% confidence interval) of 0.968 (0.923, 0.987). Lean apneics were younger than lean controls (45.3±13.0 vs. 62.1±10.4 years; p<0.0001). No significant differences between apneics and controls were observed in the average BMI (23.4±2.2 vs. 23.5 ± 2.6 kg/m2; p=0.824), the fat pads volume (4198±1728 vs. 3880±1544 mm3; p=0.646), and the proportion of males (61.9% vs. 68.8%; p=0.666). In unadjusted analyses, the lean OSA group showed significantly higher soft palate fat volume than lean controls (7605±2109 vs. 5327±1783 mm3; p=0.003). When adjusting for age, gender and BMI, no differences was observed between groups in soft palate fat volume (p=0.122) and fat pads volume (p=0.702). Conclusion Analysis of soft palate fat volume from Dixon MRI is highly reproducible. Our results indicate no significant difference in deposition of fat at soft palate between lean patients with OSA and lean controls when accounting for age, gender and BMI. Support This study is supported by National Institutes of Health Grant: 2P01HL094307-06A1. LX is supported by Young Elite Scientists Sponsorship Program of China Association for Science and Technology.

Abstract Introduction Studies measuring objective sleep duration in rural/indigenous populations are limited, showing sleep duration similar to that of industrialized countries. Little is known about sleep duration in women of reproductive age, and children within these populations. Our study is the first to objectively characterize sleep in mothers and children in an agrarian community with limited access to electricity, utilizing data from the Ghana Randomized Air Pollution and Heath Study (GRAPHS). Methods The GRAPHS cohort, a cluster-randomized trial, evaluated the efficacy of clean fuels on birthweight and infant pneumonia incidence in central Ghana. The study initially recruited pregnant women and newborns in 2013. This study is now utilizing wrist-actigraphy to analyze sleep-wake patterns among mothers and children of GRAPHS. We have thus far analyzed actigraphy in 39 mothers and 49 children (including 25 mother-child pairs), using the Motionlogger-MicroWatch with the Cole-Kripke algorithm to assess total sleep time (TST). We report baseline characteristics and sleep-wake patterns of our sample. Results Mean age of mothers was 33.5 years, (range 22-48), and mean age of children was 3.9 years (3-4). Average nights recorded were 4 (standard deviation [SD] 2.1). For mothers, average median time-in-bed was 7.9 (SD 1.2) hours, TST was 6.4 (SD .9) hours, and sleep efficiency was 82% (SD 7.9). Median bedtime was 9:33pm (SD 1.5 hours), and median wake-time was 5:56am (SD 1.4 hours). For children, average median time-in-bed was 9.9 (SD 1.0) hours, TST was 8.2 (SD 0.9) hours, and sleep efficiency was 83% (SD 6). Median bedtime was 8:03pm (SD 0.8 hours), and median wake-time was 6:06am (SD 0.6 hours). There was no correlation between sleep measures in mother-child pairs. Conclusion In an agrarian Ghanaian community with limited access to electricity, objective sleep measures in women were similar to prior studies conducted in indigenous/rural populations of developing African countries (Ndiaye et.al.2007, Samson et.al.2016), though data in children is lacking for comparison. When compared with post-industrialized countries, objective sleep measures for this age group of non-gravid women are sparse. In toddlers however, TST was lower in our cohort when compared with objective sleep amongst toddlers in industrialized nations. Support 2K24HL109156-06A1

Abstract Background: Sickle cell nephropathy (SCN) is one of the most common complications of SCD, leading in most cases to chronic kidney disease (CKD) and end-stage renal disease (ESRD). Despite the high prevalence of CKD in sickle cell disease (SCD) patients, there remains a poor understanding of the pathophysiological mechanism of SCN and a lack of biomarkers for early detection of SCD-associated CKD. Soluble urokinase-type plasminogen activator receptor (suPAR) is an emerging biomarker of CKD. suPAR is a member of the fibrinolytic system, which is dysregulated in SCD patients. Objective: To evaluate suPAR as a biomarker of SCD-associated nephropathy and identify plasma proteases responsible for its increase in SCD. Methods: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior to the sample collection. Whole blood and urine samples were collected from 77 SCD patients and 10 healthy individuals, and plasma was isolated. Levels of creatinine and cystatin C in plasma and albumin and creatinine in urine were measured by ELISA. eGFR was calculated using CKD-EPI creatinine-cystatin equation, and CKD stages were assigned. Plasma suPAR was measured by ELISA and was correlated with CKD stages. The activities of candidates uPAR proteases: Neutrophile elastase (NE), urokinase-type plasminogen activator (uPA) and plasmin in plasma samples from SCD patients were measured and compared to healthy participants. Results: The average age of SCD patients was 42.5 years (range 18-67 years). Most patients had HbSS genotype (67.5%),19.5% of patients were HbSC (hemoglobin C sickle cell compound heterozygous), and 13% had HbS β-thalassemia. More than half (53.2 %) were females. We observed an increased level of plasma suPAR (>3ng/ml) in more than 60% of SCA patients without renal disease, representing a risk factor for CKD progression. Plasma suPAR levels further increased in the patients with CKD and positively correlated with stages of CKD (r=0.419, R2=0.1696). Analysis of plasma proteases that cleaved uPAR producing soluble peptides (suPAR) demonstrated increased urokinase-type plasminogen activator (uPA) activity without significant changes in neutrophile elastase. Conclusion: This study validated plasma suPAR as a potential marker of CKD in SCD patients and identified plasma uPA as a uPAR protease that may increase circulating suPAR in SCD. Future longitudinal analysis of suPAR levels in patients with SCA is needed. Acknowledgments: We thank Drs. Namita Kumari and Xiaomei Niu for their help in samples identification. This work was supported by NIH Research Grants 1R01HL125005-06A1, 5U54MD007597, 1P30AI117970-06,1UM1AI26617, and 1SC1HL150685. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The gut incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), may play a pivotal role in mediating adverse effects of high glycemic index (GI) foods on metabolic diseases. GIP exerts its effects by binding to a receptor, GIPR; genome-wide association studies have identified variants in the GIPR region related to type 2 diabetes (T2D) and glycemic/GIP responses to glucose tolerance tests. We examined whether GIPR variants were related to impaired glycemic responses during oral glucose tolerance tests (OGTTs) after consuming 4 diets (each for 5 weeks) with different carbohydrate (Carb) content and GI levels in the OmniCarb randomized crossover feeding trial. A genetic risk score (GRS) was calculated by counting T2D-risk alleles of SNPs rs1800437, rs11671664, and rs2238689 at GIPR (n=146). We found that GRS was related to greater glycemic responses after consuming a high GI/high Carb diet, regardless of baseline glycemic status (Fig). When testing diet replacement effects, combined effects of high GI and high Carb (vs. low GI/low Carb) on glycemic responses were significantly modified by GRS (Pinteraction<0.05). In conclusion, T2D-risk-related GIPR variants modified the effects of dietary carbohydrates on glucose tolerance. GIPR variants may be related to deteriorating glucose tolerance after eating a high GI, high Carb diet. Disclosure Y.Heianza: None. X.Wang: None. Q.Xue: None. L.J.Appel: None. F.Sacks: None. L.Qi: None. Funding National Institutes of Health (2P20GM109036-06A1-7233, DK091718, DK100383, DK115679)

The central melanocortin system is essential for the regulation of food intake and body weight in humans and rodents. Agouti-related protein (AgRP) is the sole orexigenic component of the central melanocortin system and is conserved across mammalian species. AgRP is currently known to express exclusively in the mediobasal hypothalamus, and hypothalamic AgRP-expressing neurons are essential for feeding regulation. Here we describe a previously unknown population of AgRP cells in the area postrema (AP) and the adjacent commissural nucleus of the solitary tract (cNTS) of the causal brainstem. AgRP was expressed in the embryonic AP, and hindbrain AgRP expression in adult mice was low under ad libitum fed condition but increased with food deprivation. AgRP cells in the AP and cNTS consisted of locally projecting neurons as well as tanycyte-like cells. In mice that lack hypothalamic AgRP neurons, diphtheria-toxin mediated ablation of AgRP cells led to anorexia and weight loss, whereas chemogenetic activation of AgRP neurons resulted in hyperphagia and weight gain. Moreover, focal transcranial photo-stimulation of AgRP cells in the AP and cNTS with a step-function opsin with ultra-high light sensitivity (SOUL) stimulated feeding in mice with or without hypothalamic AgRP neurons, suggesting that the hyperphagic effects of hindbrain AgRP neurons are independent of hypothalamic AgRP neurons. Collectively, our study indicates that the hindbrain has a balanced melanocortin system, complete with both orexigenic and anorexigenic components, and that AgRP neurons in the hindbrain drive feeding in adult animals. Disclosure T.P.Bachor: None. K.Attal: None. E.Vagena: None. F.Mifsud: None. V.Pham: None. M.Valdearcos-contreras: None. C.Vaisse: None. A.Xu: Advisory Panel; Pennington Biomedical Research Center, Research Support; Eli Lilly and Company. Funding National Institute of Diabetes and Digestive and Kidney Diseases (1P30DK098722-01A1, P30DK63720-06A1)

Retrograde transport of viral vectors following intramuscular injection allows for targeted gene delivery to motoneuron pools. Gene delivery to the hypoglossal (XII) motoneurons is of potential interest in disorders involving impaired tongue muscle control such as dysphagia, dysarthria, and possibly obstructive sleep apnea. Using a murine model of Pompe disease, our group has shown that a single intramuscular injection of 1.00e11 vector genomes (vg) of recombinant adeno‐associated virus serotype 9 (AAV9) into the base of the tongue produces sustained transgene expression in a subset of XII motoneurons. Here we investigated whether distributing the virus over a broader portion of the tongue via two near‐simultaneous injections would increase XII motoneuron transduction as compared to a single injection. Four week old 129SVE mice were anesthetized and administered either a unilateral, single 20μL injection (n=2) or bilateral, 10μL injections (n=2) to the anterior base of the tongue targeting the insertion point for the genioglossus muscle. In both cases, a total of 1.00e11 vg of AAV9 encoding green fluorescent protein (GFP) with expression driven by the chicken β‐actin promotor (AAV9‐CBA‐GFP) was given. The medulla was histologically evaluated 6 weeks after tongue injection; GFP was detected using immunohistochemistry. Robust XII motoneuron GFP expression was observed in the caudal XII nucleus after either single or dual AAV9 injection; initial observations suggest no differences in XII motoneuron transduction between the two delivery methods. With both approaches, GFP expression was primarily restricted to XII motoneurons, but was also sporadically present in neurons throughout the medulla. The data confirm that tongue injection of AAV9 drives robust transgene expression in the XII motoneuron pool, and suggest that expression is similar whether using the single or dual injection approach.Support or Funding Information2R01HD052682‐06A1

Evidence on the benefits of non-nutritive sweeteners for weight management and improving insulin sensitivity and glucose metabolism is inconsistent. Recently, high levels of circulating erythritol, a sugar alcohol used as one of the non-nutritive sweeteners, have been linked to increased risks of type 2 diabetes and its vascular complications. Whether changes in circulating erythritol levels are related to insulin sensitivity and glucose metabolism remains unknown. Here, we investigated whether changes in plasma erythritol induced by weight-loss diet interventions were related to improved insulin sensitivity and glucose metabolism. This study included overweight or obese participants without diabetes or unstable cardiovascular disease from the POUNDS Lost trial with available data on circulating erythritol at baseline (n=805) and its changes from baseline to 6 months (n=670) and 2 years (n=533) during weight-loss diet interventions. At baseline, higher plasma levels of erythritol were related to hyperinsulinemia and greater degrees of insulin resistance assessed by HOMA-IR, even after adjusting for covariates, including BMI (P <0.05 for all). In response to weight-loss dietary interventions, a greater decrease in plasma erythritol was significantly associated with larger reductions of fasting insulin (p= 0.0001) and HOMA-IR (p=0.039) at 6 months, regardless of the initial levels of BMI, erythritol, and the respective outcome. Similarly, we found a significant association between long-term changes in erythritol and a 2-year reduction of fasting insulin (p=0.0027). In conclusion, circulating plasma erythritol, a marker of non-nutritive sweetener consumption, was significantly associated with hyperinsulinemia and insulin sensitivity among adults with overweight or obesity. Weight-loss diet-induced decreases in plasma erythritol were related to improved insulin sensitivity. Disclosure Y. Heianza: None. Q. Xue: None. J. Rood: None. G. Bray: Advisory Panel; Medifast, Inc., Herbalife International of America, Inc. F. Sacks: None. L. Qi: None. Funding National Institutes of Health (DK091718, DK100383, DK115679, 2P20GM109036-06A1-7233)

Of the 37 million Americans with type II diabetes, a substantial proportion have a normal body mass index (BMI). These populations suffer a disproportionately high burden of mortality; the development of diabetic cardiomyopathy (DCM) is likely a contributor. Myocardial steatosis, a known consequence of diabetes, is a significant part of DCM's pathogenesis; unfortunately, there is no feasible way to screen patients for steatosis. Exosomal microRNAs (miRNAs) are a promising biomarker for this purpose. However, no study has evaluated miRNA signatures in normal-weight patients with diabetes. Serum samples of obese/overweight and normal-weight diabetic subjects were obtained via IMPACT and STRONG-D clinical trial protocols respectively. Exosomes were extracted using the ExoTIC (exosome total isolation chip) platform. MiRNA-1 levels from extracellular vesicles were quantified using RT-qPCR and compared via unpaired student's two-tailed t-test. A total of 8 samples (4 normal-weight and 4 overweight/obese) were analyzed. Overall, the obese cohort had a mean age of 63.7 vs. 57.2 for the normal-weight cohort. The obese cohort had a mean BMI of 29.7 vs. 23.1 for the normal weight cohort. Both cohorts were composed of 75% men and 25% women. The obese cohort had a mean hemoglobin A1c of 7.2% vs. 8.0% in the normal-weight cohort. Serum miRNA-1 levels were significantly higher in normal-weight diabetic subjects (p = 0.0004). In this pilot study, patients with diabetes of a normal weight had significantly higher levels of serum miRNA-1, implying that they may have more myocardial steatosis and possibly, more cardiac dysfunction. This pathophysiologic difference could help explain the significant morbidity and mortality that diabetic patients of a normal weight suffer. Further directions for our study include the complete quantification of both miRNA-1 and miRNA-133a among our entire cohort, as well as assessment of echocardiographic parameters. Disclosure S.Palanisamy: None. U.Parlatan: None. M.Ozen: None. A.H.Karim: None. D.Akin: None. U.Demirci: None. L.Palaniappan: None. Funding National Institutes of Health (1R18DK096394-01A1, 2R01DK081371-06A1)

Pompe disease, caused by deficiency of acid alpha‐glucosidase (GAA), leads to glycogen accumulation, disruption of cellular architecture and neuromuscular impairments. Respiratory and lingual motor systems often decline first, and respiratory motoneurons typically show profound histopathology and glycogen accumulation at an early age. Although neuropathology is now firmly established in Pompe tissues from both humans and animal models, the associated mechanisms have not been evaluated. Therefore, we studied the cervical spinal cord of adult Pompe (Gaa−/−) and wild‐type (129SVE) mice using histological methods and mRNA analyses with the Affymetrix Mouse Gene Array 2.0ST. Pathway analyses of the mRNA data were done using the Broad Institute's Molecular Signature Database. In separate animals, cervical tissues were stained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and ionized calcium binding adaptor molecule 1 (IBA‐1) to assess DNA fragmentation and microglial morphology, respectively. From these analyses, several themes emerged (FDR q values <0.01). First, neuronal loss is an important feature of Pompe disease, and occurs in motoneurons before the onset of overt respiratory symptoms. Putative phrenic motoneurons were TUNEL‐positive, and pathway analyses showed significant upregulation of apoptotic processes in the Gaa−/− cervical spinal cord. Second, pro‐inflammatory mRNA signaling is significantly upregulated in the Gaa−/− cervical cord, and this is associated with greater neuronal co‐localization of microglia. Third, multiple signal transduction pathways are altered in the Gaa−/− cervical spinal cord. Glutamatergic signaling pathways were downregulated, as were well‐known “synaptic plasticity pathways” including genes related to long‐term potentiation. Finally, there was a robust change in cellular metabolism pathways in the Gaa−/− spinal cord that included upregulation of macromolecule, lipid, carbohydrate, and protein metabolic pathways. These data confirm that absence of functional GAA protein triggers a complex neurodegenerative process, and suggest that alterations in synaptic transmission and neuroplasticity processes may contribute to motor phenotypes in Pompe disease. Most importantly, the results underscore the importance of therapeutically targeting neuropathology in Pompe disease.Support or Funding InformationCTSA U54‐TR001012; Craig H. Neilsen Foundation 313369 (SMFT); 2R01HD052682‐06A1 (DDF, BJB)

Cluster‐of‐differentiation gene 44, in particular CD44 variant isoforms (CD44v), emerges during regeneration of the gastric epithelium in response to injury. In particular, CD44v9 is expressed within Spasmolytic Polypeptide/TFF2‐Expressing Metaplasia (SPEM) glands during gastric repair, but the function is unknown. Here we tested the hypothesis that CD44v9 marks a regenerative cell lineage that plays a functional role in gastric repair. Acetic acid injury was induced in CD44‐deficient (CD44KO) and C57BL/6 (BL6) mice. Mouse‐derived gastric organoids expressing CD44v9 were transplanted at the ulcer site of CD44KO mice. Ulcers, expression of CD44v9, proliferation and histology were measured at days 1, 3, 5 and 7 post‐injury. Human‐derived gastric organoids from elderly patients (>55 years) or young patients (14–20 years) were generated and transplanted into the stomachs of NOD scid gamma (NSG) mice post‐injury. Ulcers were induced in NRGS mice expressing human‐derived immune cells (huNRGS) and the immune phenotype analyzed by CyTOF. CD44v9 expression emerged at the ulcer margin during gastric ulcer repair. Compared to BL6 mice that healed within 7 days post‐injury, CD44KO mice exhibited loss of ulcer repair and gastric epithelial regeneration. Transplantation of CD44v9‐expressing gastric organoids into the stomachs of CD44KO mice promoted ulcer repair. Compared to NSG mice exhibiting loss of CD44v9 expression and gastric repair in response to injury, transplantation of human‐derived gastric organoids from young stomachs promoted repair. Transplantation of organoids derived from aged stomachs did not promote repair. Compared to NRGS mice, huNRGS animals exhibited smaller ulcer sizes and an infiltration of human CD163+ macrophages that correlated with an emergence of CD44v9 expression. Thus, CD44v9 marks a regenerative cell lineage. Macrophages infiltrating the injured gastric mucosa may induce the emergence of CD44v9 during regeneration of the epithelium.Support or Funding InformationThis work was supported by NIH 2 R01 DK083402‐06A1 grant, College of Medicine Bridge Funding Program (Zavros), and the University of Cincinnati Graduate School Dean's Fellowship, Albert J. Ryan Fellowship and 2T32GM105526‐04 (Bertaux‐Skeirik).

Abstract Objectives The potential impact of seafood consumption and long chain n-3 polyunsaturated fatty acids (LCn-3PUFA) supplement use on the development of insulin resistance and type 2 diabetes mellitus (T2DM) is not yet fully clarified. The aim of this large cohort study was to investigate the associations between prenatal intake of total seafood, lean fish, fatty fish and LCn-3PUFA supplement use and the risk of T2DM in women after pregnancy. Methods The study subjects (n = 60 831, median age 31 years) participates in the ongoing population-based Norwegian Mother and Child Cohort study (MoBa) initiated and maintained by the Norwegian Institute of Public Health. Recruitment lasted from 1999 through 2009. The MoBa database is linked to the Medical Birth Registry of Norway. For the current study we also obtained permission to link the data to the Norwegian Prescription Database for ascertainment of medications dispensed for diabetes (ATC code A10) 90 days or more after delivery. Dietary intake was obtained by a validated 255-item semi-quantitative food frequency questionnaire (FFQ) and assessed habitual diet during the first four to five months of pregnancy. Results During a median (IQR) follow up time of 7.5 (6.5, 8.5) years, T2DM was identified for 711 (1.2%) of the participants. Adjusted linear Cox regression analyses estimated a decreased risk of T2DM with increased lean fish intake as g/1000 kcal intake (HR 0.98, 95% CI 0.97, 0.99, P = 0.010). Modeling intake as quintiles, a decreased risk of T2DM was seen among those in quintiles two to five compared to the first quintile of energy adjusted lean fish intake (P for trend across quintiles = 0.002). No associations between total seafood, fatty fish, or LCn-3PUFA supplement use and pharmacologically treated T2DM were identified in adjusted models. Conclusions Intake of lean fish was associated with decreased risk of T2DM. Funding Sources The Norwegian Mother and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NINDS (grant no.1 UO1 NS 047537–01 and grant no.2 UO1 NS 047537-06A1).

Pompe disease is a neuromuscular disorder characterized by a systemic lack of the glycogen metabolizing enzyme acid‐alpha glucosidase. The result is extensive intracellular lysosomal glycogen accumulation and disruption of cellular architecture and function. The hypoglossal (XII) motor system is particularly susceptible to pathology in Pompe disease and both tongue and XII motoneurons show substantial pathology early in the disease progression. Patients often present with macroglossia and tongue weakness, and animal models show pathology in tongue muscles and XII motoneurons. Initial clinical testing indicates that intramuscular gene therapy with adeno‐associated virus (AAV) is safe in Pompe patients and recent work in animal models demonstrates that AAV serotype 9 (AAV9) can produce persistent transgene expression in both tongue muscle and XII motoneurons following a single tongue injection. In ongoing studies, we are using Pompe mice (Gaa−/−) to evaluate the impact of a modified GAA protein with expression being driven by the Desmin promoter. The GAA protein has been modified to allow for IGFII receptor mediated uptake (IGFIIcoGAA) to improve cellular and lysosomal uptake. Initial tests in Gaa−/− mice indicate increased GAA activity without a concomitant increase in antibody response following AAV9‐IGFIIcoGAA delivery to the tibialis anterior muscle. To determine if the new vector offers improved efficacy in the tongue motor system, adult Gaa−/− mice were injected into the base of the tongue with 7μL (1e11 vg/μL) of AAV9‐DES‐coGAA or AAV9‐DES‐IGFIIcoGAA, diluted to 20 μL in lactated ringers (LR), or an equivalent sham injection (20 μL LR). Tongue, medulla and XII nerve tissues were harvested 3‐mo post‐injection. Tissues were paraffin embedded, cut at 7μm and stained with Periodic acid‐Schiff (PAS) to visualize glycogen. Initial qualitative evaluation of tongue histology confirms that both vectors achieved significant clearance of glycogen from tongue myofibers. This was illustrated by the absence of PAS staining at the site of injection and the absence of the prototypical Gaa−/− muscle histopathology characterized by vacuolization and disrupted cellular architecture. Ongoing work is focused on evaluating XII nerve and motoneurons for quantitative comparison of tissues from the three treatment groups.Support or Funding Information2R01HD052682‐06A1 (DDF and BJB)

Toxoplasma gondii is an obligate intracellular eukaryotic parasite able to infect mammals including humans. After initial infection, people can remain latently infected for many years, however, parasite reactivation in people who have AIDS or are otherwise immunocompromised, and during pregnancy, has dramatic deleterious consequences. Both host and parasite factors are responsible for disease progression, and in recent years it has become evident that the host‐parasite interplay is complex with significant amount of crosstalk. As an obligate intracellular parasite that is dependent on the host for biomaterials, T. gondii has to acquire nutrients yet protect itself from the host response. In order to successfully survive and replicate within its host, parasites must subvert host machinery to their benefit, simultaneously avoiding host defenses and early host demise.Recent studies have demonstrated that T. gondii infection induces significant changes to the host transcriptome, and some of these changes occur in genes involved in the nutrient pathways of glucose and lipid metabolism. Furthermore, it has been shown that one of the main pathways altered during parasite infection includes Hypoxia‐inducible Factor (HIF) ‐ a central regulator of host metabolism also targeted during other parasitic infection, suggesting that parasites intentionally subvert host metabolism. Metabolic landscape restructuring has been shown to occur and be essential in a wide range of processes including immune cell activation and in cancer. Oncogenic transformation causes cellular metabolic shift away from oxidative phosphorylation toward glycolysis under aerobic conditions – known as the “Warburg effect”. Recent studies revealed that metabolic shifts also occur during infection by some pathogens including viruses, bacteria and other parasites (Mycobacteria, Theileria and Leishmania). To date, the exact changes that take place in the host metabolism during infection with T. gondii have not been described.Objectivecharacterize metabolic shifts in the host cell during infection with T. gondii parasites.MethodsSeahorse extracellular flux analysis allows sensitive evaluation of cell's metabolism including both glycolytic and oxidative phosphorylation components. We utilize Seahorse flux analyzer to study glycolysis and mitochondrial respiration in fibroblast cells infected with T. gondii parasites.ResultsModulation of HIF, together with computational analyses, suggest that T. gondii parasites induce metabolic dysregulation in the host cell. Our results demonstrate that similar to other pathogens, infection with T. gondii induces metabolic alteration in infected cells. However, unlike classical Warburg effect of cancer cells, T. gondii do not cause a shift away from mitochondrial metabolism but instead lead to increase in both glycolysis and mitochondrial respiration.ConclusionsWe have developed framework utilizing Seahorse bioanalyzer to study metabolic states of cells during parasite infection, and have demonstrated that metabolic alterations during infection with T. gondii parasites are distinct from changes observed in cancer and immune cell activation. Further studies will focus on elucidating mechanism of this metabolic interplay between the parasite and its host.Support or Funding Information2T32AI070117‐06A1 Geographic Medicine and Emerging Infections

Arterial stiffness and echocardiograms were assessed in participants with youth-onset DM (N=399, 22.8 ± 5.1 years, duration 10.8 ± 3.2 years, 63% female, 45% non-Hispanic White; 33% non-Hispanic Black, 13.5% Hispanic, 8% Other, 44% type 2 DM (T2D)) . Cardiovascular risk factors (CVRF) , arterial stiffness and cardiac measures were compared between those with or without elevated arterial stiffness (stiff = Pulse Wave Velocity [PWV] ≥ 90th% for lean controls) . Association between PWV and cardiac parameters after adjusting for CVRF was assessed. Participants with high PWV were 4.6 yrs older, had 0.6 yrs longer diabetes duration, were more likely to be female (71.5 vs. 54.9%) , non-Hispanic White (68.8 v 42.3) , have T2D (72.0 v 20.2%) , higher BMI (35.6 v 26.3 kg/m2) , BP (122/79 v 109/72 mmHg) , LDL-C (113 v 100 mg/dl) , CRP (0.6 v 0.1mg/dL) , and HbA1c (9.6 v 8.9%; all p≤0.04) . Those with high PWV had higher left ventricular mass index (LVMI) and lower systolic and diastolic function. PWV remained a significant predictor of LVMI (R2 0.37) , systolic (EF, R2 0.235) and diastolic (e’/a’, R2 0.461) function after adjustment for CVRF, all p≤0.0002. Higher arterial stiffness is associated with adverse changes in cardiac structure and function and was more prevalent in persons with T2D compared to type 1 DM. Disclosure E.M. Urbina: Advisory Panel; Astellas Pharma Inc. D. Dabelea: None. R. Dagostino Jr: None. S.R. Daniels: None. L.M. Dolan: None. G. Imperatore: None. E. Lustigova: None. S.M. Marcovina: None. A.K. Mottl: Advisory Panel; Bayer AG. Board Member; Bayer AG. Research Support; Alexion Pharmaceuticals, Inc., Aurinia, Bayer AG, Boehringer Ingelheim International GmbH, Pfizer Inc. C. Pihoker: None. A.S. Shah: None. Funding Grant Support (SEARCH 4) : The SEARCH for Diabetes in Youth Cohort Study (1R01DK127208-01, 1UC4DK108173) is funded by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases and supported by the Centers for Disease Control and Prevention. The Population Based Registry of Diabetes in Youth Study (1U18DP006131, U18DP006133, U18DP006134, U18DP006136, U18DP006138, and U18DP006139) is funded by the Centers for Disease Control and Prevention (DP-15-002) and supported by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases. Grant Support (SEARCH 1, 2, 3) : SEARCH for Diabetes in Youth is funded by the Centers for Disease Control and Prevention (PA numbers 00097, DP-05-069, and DP-10-001) and supported by the National Institute of Diabetes and Digestive and Kidney Diseases. Kaiser Permanente Southern California (U48/CCU919219, UDP000246, and U18DP002714) , University of Colorado Denver (U48/CCU819241-3, UDP000247, and U18DP000247-06A1) , Cincinnati's Children's Hospital Medical Center (U48/CCU519239, UDP000248, and 1U18DP002709) , University of North Carolina at Chapel Hill (U48/CCU419249, UDP000254, and U18DP002708) , Seattle Children's Hospital (U58/CCU019235-4, UDP000244, and U18DP002710-01] and Wake Forest University School of Medicine (U48/CCU919219, UDP000250, and 200-2010-35171) .

We previously reported that neurogenic hypertension is associated with a reduction of Angiotensin Converting Enzyme 2 (ACE2) activity and an increase in A Disintegrin And Metalloprotease 17 (ADAM17) activity in the hypothalamus. In addition, we showed that silencing ADAM17 or blocking Angiotensin (Ang)‐II type 1 (AT1) receptors in the central nervous system (CNS) prevented DOCA‐salt hypertension, confirming the pivotal role of AT1R and ADAM17 in neurogenic hypertension. However, the interaction between AT1 receptors, ADAM17 and ACE2 is still unclear. Since ADAM17 is known to be expressed in multiple cell types and can be activated by various receptors, we tested the hypothesis that neuronal AT1a receptors (AT1aR) are necessary for ADAM17‐mediated ACE2 shedding in neurogenic hypertension. Male neuronal AT1aR knock‐down (AT1aR floxed crossed with Nefh‐cre recombinase mice, 12–16 week‐old, n=10) and control littermates (n=10) were implanted with telemetry probes for continuous recording of blood pressure (BP) and heart rate (HR). Following DOCA‐salt treatment (DOCA 1mg/g body weight sc + 1% saline po for 3 weeks), both strains showed increased BP, however mean arterial pressure and HR were significantly lower in neuronal AT1aR knock‐down mice after 19 days of treatment, compared to the controls. Meanwhile, autonomic function and urinary norepinephrine levels were assessed in conscious mice before and after 19 days of treatment. At baseline level, autonomic function and urinary norepinephrine levels were identical between AT1aR knock‐down and their wild type (WT) counterparts. However, the deleterious changes caused by DOCA‐salt administration on sympathetic drive and vagal tone were partially reverted by neuronal AT1aR knock‐down (P<0.05), whereas DOCA‐salt‐induced cardiac and renal hypertrophy were not corrected. Furthermore, enzyme activity assays from the hypothalamus of these DOCA‐salt‐treated mice revealed that ADAM17 activity was significantly decreased (−37.4 ±10.5%, P<0.05) in neuronal AT1aR knock‐down animals. Unlike WT group, there was no marked difference in ADAM17 activity between DOCA‐salt treated and sham AT1aR knock‐down mice, which remained at baseline levels. Concomitant to the down‐regulation of ADAM17, both the expression and activity of ACE2 were found to be significantly (P<0.05) up‐regulated in the hypothalamus of neuronal AT1aR knock‐down mice, by +20.7 ±8.5% and +32.3 ±10.1%, respectively. Preliminary results show an increasing tendency for ADAM17 inhibitory protein, TIMP‐3, in the hypothalamus of AT1aR knock‐down mice. To further confirm the pivotal role of neuronal AT1R, hypothalamic primary neurons isolated from neonates were exposed to Ang‐II (300 nM, 24 h) after 14 days of culture. Western blotting reveals that Ang‐II significantly increased the expression of mature ADAM17 and this effect could be blocked by pre‐treatment with the AT1R antagonist, Losartan (10 μM, 30 min prior). Together our data provide strong evidence that activation of neuronal AT1aR is responsible for ADAM17‐mediated ACE2 shedding and the maintenance of neurogenic hypertension.Support or Funding Information American Heart Association Postdoctoral Fellowship (15POST25000010) NIH/NHLBI 2R01HL093178‐06A1 NIH COBRE P30GM106392

INTRODUCTIONBurn and blast injuries present with serious risk for complications including fibrosis and calcification of the soft tissues. Plasmin, the active form of plasminogen, is the key serine protease responsible for the degradation of fibrin (fibrinolysis). Our lab has previously shown that plasmin is essential for the prevention of muscle fibrosis and calcification following injury. We hypothesized that 1) plasminogen is consumed in response to severe burn, and 2) this transient plasminogen deficiency impairs proper muscle regeneration.METHODSTo test our first hypothesis in humans, we collected blood from 10 control subjects at one time‐point and 20 burn patients with burns ranging from 25–75% total body surface area (TBSA) at 0, 3, 7, and 14 days post‐admission. Plasma plasminogen levels were assessed by human plasminogen ELISA. Methods including human subjects were pre‐approved by Vanderbilt's IRB.Because our lab has demonstrated that plasmin activity prevents muscle calcification, we used the formation of dystrophic calcification and as an output measure for impaired muscle regeneration. To assess muscle regeneration following severe burn in mice, we induced bilateral muscle injuries in the mice by injecting cardiotoxin (CTX) into the calf muscles and then we administered a 30% TBSA burn to the dorsum by scalding in 100°C water. Control groups received either the burn alone or the CTX injection alone. Another group of mice received the same injury and burn concurrent with a subcutaneous injection of either an antisense oligonucleotide (ASO) against plasmin's inhibitor, α2‐antiplasmin, or a control ASO, beginning 2 weeks prior to injury. Following the burn injury, weekly radiographs of the lower extremities were taken up to 28 days post‐injury (DPI) to assess dystrophic calcification. Following sacrifice at 28 DPI, ex vivo micro‐computed tomography (μCT) was performed on the lower extremities to further assess muscle calcification.To assess plasminogen consumption following severe burn in mice, we collected blood from animals with either CTX‐induced injury, injury with burn (as described above) or no injury following sacrifice at 3 DPI. Plasma plasminogen levels were assessed by mouse plasminogen ELISA. Vanderbilt IACUC pre‐approved all animal experiments.RESULTSBurn patients exhibited a significant plasminogen deficit compared to control subjects at 0 and 3 days post‐admission, followed by a return to control plasminogen levels between 7 and 14 days (Figure 1).Consistent with the human subjects, mice exhibited a significant deficit in circulating plasminogen following burn when compared to both uninjured mice and mice given CTX injection alone.In the animal models assessing muscle regeneration, following the CTX‐induced injury alone, mice did not form significant dystrophic calcification in the injured muscles, but the mice that received the CTX injection and the burn developed dystrophic calcification in the injured muscle at 7 DPI. This phenotype in the burn mice was rescued by a systemic increase in plasmin activity through weekly administration of α2AP ASO (Figure 2).DISCUSSIONWhile many response mechanisms following severe burn have been implicated in impaired healing, the loss of plasminogen and the consequences of this have not been well characterized. Conclusively, this study suggests that severe burn increases the risk of the formation of calcification in injured muscle due to consumption of plasminogen.Support or Funding InformationNIH T32 AR059039‐06A1 Rheumatology Training Grant, Vanderbilt Department of Orthopaedics and Rehabilitation, Caitlin Lovejoy Fund

Abstract STUDY QUESTION Is cord blood DNA methylation associated with having been conceived by medically assisted reproduction? SUMMARY ANSWER This study does not provide strong evidence of an association of conception by medically assisted reproduction with variation in infant blood cell DNA methylation. WHAT IS KNOWN ALREADY Medically assisted reproduction consists of procedures used to help infertile/subfertile couples conceive, including ART. Due to its importance in gene regulation during early development programming, DNA methylation and its perturbations associated with medically assisted reproduction could reveal new insights into the biological effects of assisted reproductive technologies and potential adverse offspring outcomes. STUDY DESIGN, SIZE, DURATION We investigated the association of DNA methylation and medically assisted reproduction using a case–control study design (N = 205 medically assisted reproduction cases and N = 2439 naturally conceived controls in discovery cohorts; N = 149 ART cases and N = 58 non-ART controls in replication cohort). PARTICIPANTS/MATERIALS, SETTINGS, METHODS We assessed the association between medically assisted reproduction and DNA methylation at birth in cord blood (205 medically assisted conceptions and 2439 naturally conceived controls) at &gt;450 000 CpG sites across the genome in two sub-samples of the UK Avon Longitudinal Study of Parents and Children (ALSPAC) and two sub-samples of the Norwegian Mother, Father and Child Cohort Study (MoBa) by meta-analysis. We explored replication of findings in the Australian Clinical review of the Health of adults conceived following Assisted Reproductive Technologies (CHART) study (N = 149 ART conceptions and N = 58 controls). MAIN RESULTS AND THE ROLE OF CHANCE The ALSPAC and MoBa meta-analysis revealed evidence of association between conception by medically assisted reproduction and DNA methylation (false-discovery-rate-corrected P-value &lt; 0.05) at five CpG sites which are annotated to two genes (percentage difference in methylation per CpG, cg24051276: Beta = 0.23 (95% CI 0.15,0.31); cg00012522: Beta = 0.47 (95% CI 0.31, 0.63); cg17855264: Beta = 0.31 (95% CI 0.20, 0.43); cg17132421: Beta = 0.30 (95% CI 0.18, 0.42); cg18529845: Beta = 0.41 (95% CI 0.25, 0.57)). Methylation at three of these sites has been previously linked to cancer, aging, HIV infection and neurological diseases. None of these associations replicated in the CHART cohort. There was evidence of a functional role of medically assisted reproduction-induced hypermethylation at CpG sites located within regulatory regions as shown by putative transcription factor binding and chromatin remodelling. LIMITATIONS, REASONS FOR CAUTIONS While insufficient power is likely, heterogeneity in types of medically assisted reproduction procedures and between populations may also contribute. Larger studies might identify replicable variation in DNA methylation at birth due to medically assisted reproduction. WIDER IMPLICATIONS OF THE FINDINGS Newborns conceived with medically assisted procedures present with divergent DNA methylation in cord blood white cells. If these associations are true and causal, they might have long-term consequences for offspring health. STUDY FUNDING/COMPETING INTERESTS(S) This study has been supported by the US National Institute of Health (R01 DK10324), the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement no. 669545, European Union’s Horizon 2020 research and innovation programme under Grant agreement no. 733206 (LifeCycle) and the NIHR Biomedical Centre at the University Hospitals Bristol NHS Foundation Trust and the University of Bristol. The UK Medical Research Council and Wellcome (Grant ref: 102215/2/13/2) and the University of Bristol provide core support for ALSPAC. Methylation data in the ALSPAC cohort were generated as part of the UK BBSRC funded (BB/I025751/1 and BB/I025263/1) Accessible Resource for Integrated Epigenomic Studies (ARIES, http://www.ariesepigenomics.org.uk). D.C., J.J., C.L.R. D.A.L and H.R.E. work in a Unit that is supported by the University of Bristol and the UK Medical Research Council (Grant nos. MC_UU_00011/1, MC_UU_00011/5 and MC_UU_00011/6). B.N. is supported by an NHMRC (Australia) Investigator Grant (1173314). ALSPAC GWAS data were generated by Sample Logistics and Genotyping Facilities at Wellcome Sanger Institute and LabCorp (Laboratory Corporation of America) using support from 23andMe. The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, NIH/NIEHS (Contract no. N01-ES-75558), NIH/NINDS (Grant nos. (i) UO1 NS 047537-01 and (ii) UO1 NS 047537-06A1). For this work, MoBa 1 and 2 were supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES-49019) and the Norwegian Research Council/BIOBANK (Grant no. 221097). This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, Project no. 262700. D.A.L. has received support from national and international government and charity funders, as well as from Roche Diagnostics and Medtronic for research unrelated to this study. The other authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER N/A.