👤 W Schlegel

Cholesteryl ester transfer protein (CETP) inhibitor is a target for both lowering low-density lipoproteins and raising high-density lipoproteins. Anacetrapib was the lead compound in our cholesteryl ester transfer protein inhibitor program. Preclinical studies were initiated to support the safety of anacetrapib deposition in adipose tissue, followed by a clinical trial to evaluate the effects of anacetrapib in people with vascular disease. An ultra-high performance liquid chromatography/tandem mass spectrometry method was developed to determine tissue anacetrapib concentrations in the adipose of three animal species and humans. The assays were validated in the concentration ranges of 5-5000 ng/ml and 0.1-100 μg/ml. The anacetrapib concentrations in adipose tissue from preclinical and clinical studies were determined. Show less

Pre-pregnancy overweight and obesity promote deleterious health impacts on both mothers during pregnancy and the offspring. Significant changes in the maternal peripheral blood mononuclear cells (PBMCs) gene expression due to obesity are well-known. However, the impact of pre-pregnancy overweight on immune cell gene expression during pregnancy and its association with maternal and infant outcomes is not well explored. Blood samples were collected from healthy normal weight (NW, pre-pregnancy BMI 18.5-24.9) or overweight (OW, pre-pregnancy BMI 25-29.9) 2nd parity pregnant women at 12, 24 and 36 weeks of pregnancy. PBMCs were isolated from the blood and subjected to mRNA sequencing. Maternal and infant microbiota were analyzed by 16S rRNA gene sequencing. Integrative multi-omics data analysis was performed to evaluate the association of gene expression with maternal diet, gut microbiota, milk composition, and infant gut microbiota. Gene expression analysis revealed that 453 genes were differentially expressed in the OW women compared to NW women at 12 weeks of pregnancy, out of which 354 were upregulated and 99 were downregulated. Several up-regulated genes in the OW group were enriched in inflammatory, chemokine-mediated signaling and regulation of interleukin-8 production-related pathways. At 36 weeks of pregnancy healthy eating index score was positively associated with several genes that include, DTD1, ELOC, GALNT8, ITGA6-AS1, KRT17P2, NPW, POT1-AS1 and RPL26. In addition, at 36 weeks of pregnancy, genes involved in adipocyte functions, such as NG2 and SMTNL1, were negatively correlated to human milk 2'FL and total fucosylated oligosaccharides content collected at 1 month postnatally. Furthermore, infant Akkermansia was positively associated with maternal PBMC anti-inflammatory genes that include CPS1 and RAB7B, at 12 and 36 weeks of pregnancy. These findings suggest that prepregnancy overweight impacts the immune cell gene expression profile, particularly at 12 weeks of pregnancy. Furthermore, deciphering the complex association of PBMC's gene expression levels with maternal gut microbiome and milk composition and infant gut microbiome may aid in developing strategies to mitigate obesity-mediated effects. Show less

Hypothermic-oxygenated-machine-perfusion (HOPE) allows assessment/reconditioning of livers procured from high-risk donors before transplantation. Graft referral to HOPE mostly depends on surgeons' subjective judgment, as objective criteria are still insufficient. We investigated whether analysis of effluent fluids collected upon organ flush during static-cold-storage can improve selection criteria for HOPE utilization. Effluents were analyzed to determine cytolysis enzymes, metabolites, inflammation-related mediators, and damage-associated-molecular-patterns. Molecular profiles were assessed by unsupervised cluster analysis. Differences between "machine perfusion (MP)-yes" vs. "MP-no"; "brain-death (DBD) vs. donation-after-circulatory-death (DCD)"; "early-allograft-dysfunction (EAD)-yes" vs. "EAD-no" groups, as well as correlation between effluent variables and transplantation outcome, were investigated. Livers assigned to HOPE ( Show less

Post-prandial hyperlipidemia has emerged as a cardiovascular risk factor with limited therapeutic options. The Liver X receptors (Lxrs) are nuclear hormone receptors that regulate cholesterol elimination. Knowledge of their role in regulating the absorption and handling of dietary fats is incomplete. The purpose of this study was to determine the role of intestinal Lxrα in post-prandial intestinal lipid transport. Using Lxrα knockout ( Show less

The conventional ECG is commonly used to screen for hypertrophic cardiomyopathy (HCM), but up to 25% of adults and possibly larger percentages of children with HCM have no distinctive abnormalities on the conventional ECG, whereas 5 to 15% of healthy young athletes do. Recently, a 5-min resting advanced 12-lead ECG test ("A-ECG score") showed superiority to pooled criteria from the strictly conventional ECG in correctly identifying adult HCM. The purpose of this study was to evaluate whether in children and young adults, A-ECG scoring could detect echocardiographic HCM associated with the MYBPC3 genetic mutation with greater sensitivity than conventional ECG criteria and distinguish healthy young controls and athletes from persons with MYBPC3 HCM with greater specificity. Five-minute 12-lead ECGs were obtained from 15 young patients (mean age 13.2years, range 0-30years) with MYBPC3 mutation and phenotypic HCM. The conventional and A-ECG results of these patients were compared to those of 198 healthy children and young adults (mean age 13.2, range 1month-30years) with unremarkable echocardiograms, and to those of 36 young endurance-trained athletes, 20 of whom had athletic (physiologic) left ventricular hypertrophy. Compared with commonly used, age-specific pooled criteria from the conventional ECG, a retrospectively generated A-ECG score incorporating results from just 2 derived vectorcardiographic parameters (spatial QRS-T angle and the change in the vectorcardiographic QRS azimuth angle from the second to the third eighth of the QRS interval) increased the sensitivity of ECG for identifying MYBPC3 HCM from 46% to 87% (p<0.05). Use of the same score also demonstrated superior specificity in a set of 198 healthy controls (94% vs. 87% for conventional ECG criteria; p<0.01) including in a subset of 36 healthy, young endurance-trained athletes (100% vs. 69% for conventional ECG criteria, p<0.001). In children and young adults, a 2-parameter 12-lead A-ECG score is retrospectively significantly more sensitive and specific than pooled, age-specific conventional ECG criteria for detecting MYBPC3-HCM and in distinguishing such patients from healthy controls, including endurance-trained athletes. Show less

Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists' induction of hepatic lipogenesis and hypertriglyceridemia. Elucidating the function of Lxrs in regulating enterocyte lipid handling might identify novel aspects of lipid metabolism that are pharmacologically amenable. We took a genetic approach centered on the single Lxr gene nr1h3 in zebrafish to study the role of Lxr in enterocyte lipid metabolism. Loss of nr1h3 function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal nr1h3 activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets. This delay in transport of ingested neutral lipids protects animals from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of acsl3a, which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. Forced overexpression of acls3a in enterocytes delays, in part, the appearance of neutral lipids in the vasculature of zebrafish larvae. Activation of Lxr in the intestine cell-autonomously regulates the rate of delivery of absorbed lipids by inducting a temporary lipid intestinal droplet storage depot. Show less

Activation of mitogen-activated protein kinases (MAPKs), their upstream activators MAPK kinases (MAPKKs or MEKs) and induction of MKP-1 (CL100/3CH134) and MKP-3 (Pyst1/rVH6) dual-specificity MAPK phosphatases (MKPs) were studied in the mouse embryonic stem cell line P19 during the 7 day induction of neuronal differentiation triggered by aggregation and retinoic acid. ERK (extracellular signal-regulated kinase), but not JNK (c-Jun N-terminal kinase), was found activated with biphasic kinetics: a first transient phase on days 1 and 2, followed by a second activation that was sustained until the appearance of a neuronal phenotype. MEK activation appeared coincident with ERK activation. Cytosolic MKP-3 was induced in parallel to ERK activation, the induction being dependent on ERK activation, as was shown using the MEK-1 inhibitor PD98059. In contrast, nuclear MKP-1 was transiently elevated at 48 h, coincident with ERK inactivation and independently of ERK activity. As shown by cell fractionation, activated ERK is translocated to the nucleus. The complementary induction of ERK-specific phosphatases MKP-1 and MKP-3 permits precise and independent control of cytoplasmic and nuclear ERK activity, most probably required to properly induce a complex cellular programme of differentiation. Show less

MKP-1 (also known as CL100, 3CH134, Erp, and hVH-1) exemplifies a class of dual-specificity phosphatase able to reverse the activation of mitogen-activated protein (MAP) kinase family members by dephosphorylating critical tyrosine and threonine residues. We now report the cloning of MKP-3, a novel protein phosphatase that also suppresses MAP kinase activation state. The deduced amino acid sequence of MKP-3 is 36% identical to MKP-1 and contains the characteristic extended active-site sequence motif VXVHCXXGXSRSXTXXXAYLM (where X is any amino acid) as well as two N-terminal CH2 domains displaying homology to the cell cycle regulator Cdc25 phosphatase. When expressed in COS-7 cells, MKP-3 blocks both the phosphorylation and enzymatic activation of ERK2 by mitogens. Northern analysis reveals a single mRNA species of 2.7 kilobases with an expression pattern distinct from other dual-specificity phosphatases. MKP-3 is expressed in lung, heart, brain, and kidney, but not significantly in skeletal muscle or testis. In situ hybridization studies of MKP-3 in brain reveal enrichment within the CA1, CA3, and CA4 layers of the hippocampus. Metrazole-stimulated seizure activity triggers rapid (<1 h) but transient up-regulation of MKP-3 mRNA in the cortex, piriform cortex, and some amygdala nuclei. Metrazole stimulated similar regional up-regulation of MKP-1, although this was additionally induced within the thalamus. MKP-3 mRNA also undergoes powerful induction in PC12 cells after 3 h of nerve growth factor treatment. This response appears specific insofar as epidermal growth factor and dibutyryl cyclic AMP fail to induce significant MKP-3 expression. Subcellular localization of epitope-tagged MKP-3 in sympathetic neurons reveals expression in the cytosol with exclusion from the nucleus. Together, these observations indicate that MKP-3 is a novel dual-specificity phosphatase that displays a distinct tissue distribution, subcellular localization, and regulated expression, suggesting a unique function in controlling MAP kinase family members. Identification of a second partial cDNA clone (MKP-X) encoding the C-terminal 280 amino acids of an additional phosphatase that is 76% identical to MKP-3 suggests the existence of a distinct structurally homologous subfamily of MAP kinase phosphatases. Show less