👤 Shivangi Patel

Bardet-Biedl syndrome (BBS) is a human genetic disorder associated with several phenotypes including hypertension. Here we used the hypertensive Bbs4 knockout mouse model (Bbs4-/-) to test the hypothesis that areas of the brain involved in cardiovascular regulation (CVR) exhibit abnormalities in primary neuronal cilia (PNC) structure and density. We utilized immunocytochemical localization of adenylyl cyclase-III (ACIII), a specific marker for PNC, to identify the changes in PNC length and density in commissural nucleus of solitary tract (cNTS), area postrema (AP), rostroventrolateral medulla (RVLM) and subfornical organ (SFO). A quantitative analysis of the morphology and distribution of ACIII-immunoreactive PNC revealed dramatic alterations in the length and number of cilia in SFO of Bbs4-/- mice compared to wild type (WT) littermates. The significant reduction in the PNC length but not in the number was observed in cNTS and RVLM. Surprisingly, no significant changes in length and distribution of PNC were documented in the AP. We found that in all investigated areas of the brain the number of neurons did not display significant changes in Bbs4-/- when compared to the corresponding areas of WT mice. This data suggests that loss of the Bbs4 gene differentially affects the PNC in the brain areas involved in CVR; and the pathology of PNC in selected regions of CVR can cause a failure in signal transduction and may contribute to the hypertension associated with Bbs4-/- mouse model. Show less

Microtubule actin crosslinking factor 1 (MACF1) plays a role in the coordination of microtubules and actin in multiple cellular processes. Here, we show that MACF1 is also critical for ciliogenesis in multiple cell types. Ablation of Macf1 in the developing retina abolishes ciliogenesis, and basal bodies fail to dock to ciliary vesicles or migrate apically. Photoreceptor polarity is randomized, while inner retinal cells laminate correctly, suggesting that photoreceptor maturation is guided by polarity cues provided by cilia. Deletion of MACF1 in adult photoreceptors causes reversal of basal body docking and loss of outer segments, reflecting a continuous requirement for MACF1 function. MACF1 also interacts with the ciliary proteins MKKS and TALPID3. We propose that a disruption of trafficking across microtubles to actin filaments underlies the ciliogenesis defect in cells lacking MACF1 and that MKKS and TALPID3 are involved in the coordination of microtubule and actin interactions. Show less

Sequence variants affecting blood lipids and coronary artery disease (CAD) may enhance understanding of the atherogenicity of lipid fractions. Using a large resource of whole-genome sequence data, we examined rare and low-frequency variants for association with non-HDL cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides in up to 119,146 Icelanders. We discovered 13 variants with large effects (within ANGPTL3, APOB, ABCA1, NR1H3, APOA1, LIPC, CETP, LDLR, and APOC1) and replicated 14 variants. Five variants within PCSK9, APOA1, ANGPTL4, and LDLR associate with CAD (33,090 cases and 236,254 controls). We used genetic risk scores for the lipid fractions to examine their causal relationship with CAD. The non-HDL cholesterol genetic risk score associates most strongly with CAD (P = 2.7 × 10(-28)), and no other genetic risk score associates with CAD after accounting for non-HDL cholesterol. The genetic risk score for non-HDL cholesterol confers CAD risk beyond that of LDL cholesterol (P = 5.5 × 10(-8)), suggesting that targeting atherogenic remnant cholesterol may reduce cardiovascular risk. Show less

Dysmorphology syndromes are among the most common referrals to clinical genetics specialists. Inability to match the dysmorphology pattern to a known syndrome can pose a major diagnostic challenge. With an aim to accelerate the establishment of new syndromes and their genetic etiology, we describe our experience with multiplex consanguineous families that appeared to represent novel autosomal recessive dysmorphology syndromes at the time of evaluation. Combined autozygome/exome analysis of multiplex consanguineous families with apparently novel dysmorphology syndromes. Consistent with the apparent novelty of the phenotypes, our analysis revealed a strong candidate variant in genes that were novel at the time of the analysis in the majority of cases, and 10 of these genes are published here for the first time as novel candidates (CDK9, NEK9, ZNF668, TTC28, MBL2, CADPS, CACNA1H, HYAL2, CTU2, and C3ORF17). A significant minority of the phenotypes (6/31, 19%), however, were caused by genes known to cause Mendelian phenotypes, thus expanding the phenotypic spectrum of the diseases linked to these genes. The conspicuous inheritance pattern and the highly specific phenotypes appear to have contributed to the high yield (90%) of plausible molecular diagnoses in our study cohort. Reporting detailed clinical and genomic analysis of a large series of apparently novel dysmorphology syndromes will likely lead to a trend to accelerate the establishment of novel syndromes and their underlying genes through open exchange of data for the benefit of patients, their families, health-care providers, and the research community.Genet Med 18 7, 686-695. Show less

Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element) is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes. Show less

Multiple autoimmune syndrome (MAS), an extreme phenotype of autoimmune disorders, is a very well suited trait to tackle genomic variants of these conditions. Whole exome sequencing (WES) is a widely used strategy for detection of protein coding and splicing variants associated with inherited diseases. The DNA of eight patients affected by MAS [all of whom presenting with Sjögren's syndrome (SS)], four patients affected by SS alone and 38 unaffected individuals, were subject to WES. Filters to identify novel and rare functional (pathogenic-deleterious) homozygous and/or compound heterozygous variants in these patients and controls were applied. Bioinformatics tools such as the Human gene connectome as well as pathway and network analysis were applied to test overrepresentation of genes harbouring these variants in critical pathways and networks involved in autoimmunity. Eleven novel and rare functional variants were identified in cases but not in controls, harboured in: MACF1, KIAA0754, DUSP12, ICA1, CELA1, LRP1/STAT6, GRIN3B, ANKLE1, TMEM161A, and FKRP. These were subsequently subject to network analysis and their functional relatedness to genes already associated with autoimmunity was evaluated. Notably, the LRP1/STAT6 novel mutation was homozygous in one MAS affected patient and heterozygous in another. LRP1/STAT6 disclosed the strongest plausibility for autoimmunity. LRP1/STAT6 are involved in extracellular and intracellular anti-inflammatory pathways that play key roles in maintaining the homeostasis of the immune system. Further; networks, pathways, and interaction analyses showed that LRP1 is functionally related to the HLA-B and IL10 genes and it has a substantial impact within immunological pathways and/or reaction to bacterial and other foreign proteins (phagocytosis, regulation of phospholipase A2 activity, negative regulation of apoptosis and response to lipopolysaccharides). Further, ICA1 and STAT6 were also closely related to AIRE and IRF5, two very well known autoimmunity genes. Novel and rare exonic mutations that may account for autoimmunity were identified. Among those, the LRP1/STAT6 novel mutation has the strongest case for being categorised as potentially causative of MAS given the presence of intriguing patterns of functional interaction with other major genes shaping autoimmunity. Show less

AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism by which AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L. Show less

Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone secreted from gastrointestinal K cells in response to food intake, has an important role in the control of whole-body metabolism. GIP signals through activation of the GIP receptor (GIPR), a G-protein-coupled receptor (GPCR). Dysregulation of this pathway has been implicated in the development of metabolic disease. Here we demonstrate that GIPR is constitutively trafficked between the plasma membrane and intracellular compartments of both GIP-stimulated and unstimulated adipocytes. GIP induces a downregulation of plasma membrane GIPR by slowing GIPR recycling without affecting internalization kinetics. This transient reduction in the expression of GIPR in the plasma membrane correlates with desensitization to the effects of GIP. A naturally occurring variant of GIPR (E354Q) associated with an increased incidence of insulin resistance, type 2 diabetes, and cardiovascular disease in humans responds to GIP stimulation with an exaggerated downregulation from the plasma membrane and a delayed recovery of GIP sensitivity following cessation of GIP stimulation. This perturbation in the desensitization-resensitization cycle of the GIPR variant, revealed in studies of cultured adipocytes, may contribute to the link of the E354Q variant to metabolic disease. Show less

Cerebral vasospasm is a major contributor to delayed morbidity following aneurysmal subarachnoid hemorrhage. We sought to evaluate differential plasma protein levels across time in patients with aneurysmal subarachnoid hemorrhage to identify potential biomarkers and to better understand the pathogenesis of cerebral vasospasm. Nine female patients with aneurysmal subarachnoid hemorrhage underwent serial analysis of 239 different serum protein levels using quantitative, multiplexed immunoassays (DiscoveryMAP 250+ v2.0, Myriad RBM, Austin, TX, USA) on post-hemorrhage days 0 and 5. A repeated measures analysis of variance determined that mean protein concentration decreased significantly in patients who developed vasospasm versus those who did not for alpha-2-macroglobulin (F [1.00,7.00]=16.33, p=0.005), angiogenin (F [1.00,7.00]=7.65, p=0.028), apolipoprotein A-IV (F [1.00,7.00]=6.308, p=0.040), granulocyte colony-stimulating factor (F [1.00,7.00]=9.08, p=0.020), macrophage-stimulating protein (F [1.00,7.00]=24.21, p=0.002), tetranectin (F [1.00,7.00]=5.46, p<0.039), vascular endothelial growth factor receptor 3 (F [1.00,7.00]=6.94, p=0.034), and significantly increased for vitronectin (F [1.00,7.00]=5.79, p=0.047). These biomarkers may be of value in detecting cerebral vasospasm, possibly aiding in the identification of patients at high-risk prior to neurological deterioration. Show less

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous inherited human disorder displaying a pleotropic phenotype. Many of the symptoms characterized in the human disease have been reproduced in animal models carrying deletions or knock-in mutations of genes causal for the disorder. Thinning of the cerebral cortex, enlargement of the lateral and third ventricles, and structural changes in cilia are among the pathologies documented in these animal models. Ciliopathy is of particular interest in light of recent studies that have implicated primary neuronal cilia (PNC) in neuronal signal transduction. In the present investigation, we tested the hypothesis that areas of the brain responsible for learning and memory formation would differentially exhibit PNC abnormalities in animals carrying a deletion of the Bbs4 gene (Bbs4-/-). Immunohistochemical localization of adenylyl cyclase-III (ACIII), a marker restricted to PNC, revealed dramatic alterations in PNC morphology and a statistically significant reduction in number of immunopositive cilia in the hippocampus and amygdala of Bbs4-/- mice compared to wild type (WT) littermates. Western blot analysis confirmed the decrease of ACIII levels in the hippocampus and amygdala of Bbs4-/- mice, and electron microscopy demonstrated pathological alterations of PNC in the hippocampus and amygdala. Importantly, no neuronal loss was found within the subregions of amygdala and hippocampus sampled in Bbs4-/- mice and there were no statistically significant alterations of ACIII immunopositive cilia in other areas of the brain not known to contribute to the BBS phenotype. Considered with data documenting a role of cilia in signal transduction these findings support the conclusion that alterations in cilia structure or neurochemical phenotypes may contribute to the cognitive deficits observed in the Bbs4-/- mouse mode. Show less

During animal cell division, an actin-based ring cleaves the cell into two. Problems with this process can cause chromosome missegregation and defects in cytoplasmic inheritance and the partitioning of organelles, which in turn are associated with human diseases. Although much is known about how chromosome segregation is coupled to cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging small interfering RNA screen, we identify Myosin-XIX (Myo19) as a novel regulator of cell division. Previously, this actin-based motor was shown to control the interphase movement of mitochondria. Our analysis shows that Myo19 is indeed localized to mitochondria and that its silencing leads to defects in the distribution of mitochondria within cells and in mitochondrial partitioning at division. Furthermore, many Myo19 RNAi cells undergo stochastic division failure--a phenotype that can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion, implying that mitochondria can physically interfere with cytokinesis. Strikingly, using live imaging we also observe the inappropriate movement of mitochondria to the poles of spindles in cells depleted for Myo19 as they enter anaphase. Since this phenocopies the results of an acute loss of actin filaments in anaphase, these data support a model whereby the Myo19 actin-based motor helps to control mitochondrial movement to ensure their faithful segregation during division. The presence of DNA within mitochondria makes their inheritance an especially important aspect of symmetrical cell division. Show less

The nuclear receptor liver X receptor [LXR] is activated by endogenous oxidized derivatives of cholesterol. It constitutes a critical receptor in the regulation of various physiological functions related to the development of metabolic and cardiovascular diseases, such as atherosclerosis and diabetes, as well as various other disorders. Both isoforms of LXR, LXRα [NR1H3] and LXRβ [NR1H2], form heterodimers with the isoforms of the retinoid X receptor [RXR], which then regulate the gene expression by binding to DNA sequences associated with target genes. LXR acts as a cholesterol sensor in response to an increased concentration of cholesterol in cells and induces the transcription of genes that protect cells from cholesterol overload. LXRs play numerous roles in controlling cholesterol homeostasis via their actions on bile acid synthesis and metabolism/excretion, reverse cholesterol transport and cholesterol absorption/excretion in the intestines. Therefore, these receptors show great potential as pharmacological targets for anti-atherosclerotic activities. Recent discoveries have also emphasized the important involvement of LXRs in the pathogenesis of diabetes, Alzheimer's disease, inflammation, adrenal steroid synthesis, skin aging and male fertility. However, LXR activation has also been shown to stimulate lipogenesis via sterol regulatory element binding protein-1c, leading to liver steatosis and hypertriglyceridemia. This review summarizes recent scientific discoveries and the biological actions of LXR with a special focus on the involvement of this type of receptor in important diseases and conditions. Show less

Liver X receptors (LXRs) α and β are nuclear hormone receptors that are widely expressed in the kidney. They promote cholesterol efflux from cells and inhibit inflammatory responses by regulating gene transcription. Here, we hypothesised (1) that LXR deficiency would promote renal decline in a mouse model of diabetes by accelerating intraglomerular cholesterol accumulation and, conversely, (2) that LXR agonism would attenuate renal decline in diabetes. Diabetes was induced with streptozotocin (STZ) and maintained for 14 weeks in Lxrα/β (+/+) (Lxrα, also known as Nr1h3; Lxrβ, also known as Nr1h2) and Lxrα/β (-/-) mice. In addition, STZ-injected DBA/2J mice were treated with vehicle or the LXR agonist N,N-dimethyl-hydroxycholenamide (DMHCA) (80 mg/kg daily) for 10 weeks. To determine the role of cholesterol in diabetic nephropathy (DN), mice were placed on a Western diet after hyperglycaemia developed. Even in the absence of diabetes, Lxrα/β (-/-) mice exhibited a tenfold increase in the albumin:creatinine ratio and a 40-fold increase in glomerular lipid accumulation compared with Lxrα/β (+/+) mice. When challenged with diabetes, Lxrα/β (-/-) mice showed accelerated mesangial matrix expansion and glomerular lipid accumulation, with upregulation of inflammatory and oxidative stress markers. In the DN-sensitive STZ DBA/2J mouse model, DMHCA treatment significantly decreased albumin and nephrin excretion (by 50% each), glomerular lipids and plasma triacylglycerol (by 70%) and cholesterol (by 48%); it also decreased kidney inflammatory and oxidative stress markers compared with vehicle-treated mice. These data support the idea that LXR plays an important role in the normal and diabetic kidney, while showing that LXR, through its inhibitory effect on inflammation and cholesterol accumulation in glomeruli, could also be a novel therapeutic target for DN. Show less

Polynucleotides containing consecutive tracts of guanines can adopt an intramolecular G-quadruplex structure where multiple planar tetrads of hydrogen-bound guanines stack on top of each other. Remodeling of G-quadruplexes impacts numerous aspects of nucleotide biology including transcriptional and translational control. RNA helicase associated with AU-rich element (RHAU), a member of the ATP-dependent DEX(H/D) family of RNA helicases, has been established as a major cellular quadruplex resolvase. RHAU contains a core helicase domain responsible for ATP binding/hydrolysis/helicase activity and is flanked on either side by N- and C-terminal extensions. The N-terminal extension is required for quadruplex recognition, and we have previously demonstrated complex formation between this domain and a quadruplex from human telomerase RNA. Here we used an integrated approach that includes small angle x-ray scattering, nuclear magnetic resonance spectroscopy, circular dichroism, and dynamic light scattering methods to demonstrate the recognition of G-quadruplexes by the N-terminal domain of RHAU. Based on our results, we conclude that (i) quadruplex from the human telomerase RNA and its DNA analog both adopt a disc shape in solution, (ii) RHAU53-105 adopts a defined and extended conformation in solution, and (iii) the N-terminal domain mediates an interaction with a guanine tetrad face of quadruplexes. Together, these data form the foundation for understanding the recognition of quadruplexes by the N-terminal domain of RHAU. Show less

Macrophages are central to the inflammatory processes driving rheumatoid arthritis (RA) synovitis. The molecular pathways that are induced in synovial macrophages and thereby promote RA disease pathology remain poorly understood. We used microarray to characterise the transcriptome of synovial fluid (SF) macrophages compared with matched peripheral blood monocytes from patients with RA (n=8). Using in silico pathway mapping, we found that pathways downstream of the cholesterol activated liver X receptors (LXRs) and those associated with Toll-like receptor (TLR) signalling were upregulated in SF macrophages. Macrophage differentiation and tumour necrosis factor α promoted the expression of LXRα. Furthermore, in functional studies we demonstrated that activation of LXRs significantly augmented TLR-driven cytokine and chemokine secretion. The LXR pathway is the most upregulated pathway in RA synovial macrophages and activation of LXRs by ligands present within SF augments TLR-driven cytokine secretion. Since the natural agonists of LXRs arise from cholesterol metabolism, this provides a novel mechanism that can promote RA synovitis. Show less

Hyperglycaemia disproportionately affects African-Americans (AfAs). We tested the transferability of 18 single-nucleotide polymorphisms (SNPs) associated with glycaemic traits identified in European ancestry (EuA) populations in 5,984 non-diabetic AfAs. We meta-analysed SNP associations with fasting glucose (FG) or insulin (FI) in AfAs from five cohorts in the Candidate Gene Association Resource. We: (1) calculated allele frequency differences, variations in linkage disequilibrium (LD), fixation indices (F(st)s) and integrated haplotype scores (iHSs); (2) tested EuA SNPs in AfAs; and (3) interrogated within ± 250 kb around each EuA SNP in AfAs. Allele frequency differences ranged from 0.6% to 54%. F(st) exceeded 0.15 at 6/16 loci, indicating modest population differentiation. All iHSs were <2, suggesting no recent positive selection. For 18 SNPs, all directions of effect were the same and 95% CIs of association overlapped when comparing EuA with AfA. For 17 of 18 loci, at least one SNP was nominally associated with FG in AfAs. Four loci were significantly associated with FG (GCK, p = 5.8 × 10(-8); MTNR1B, p = 8.5 × 10(-9); and FADS1, p = 2.2 × 10(-4)) or FI (GCKR, p = 5.9 × 10(-4)). At GCK and MTNR1B the EuA and AfA SNPs represented the same signal, while at FADS1, and GCKR, the EuA and best AfA SNPs were weakly correlated (r(2) <0.2), suggesting allelic heterogeneity for association with FG at these loci. Few glycaemic SNPs showed strict evidence of transferability from EuA to AfAs. Four loci were significantly associated in both AfAs and those with EuA after accounting for varying LD across ancestral groups, with new signals emerging to aid fine-mapping. Show less

Endothelial progenitor cells (EPCs), critical for mediating vascular repair, are dysfunctional in a hyperglycemic and/or hypercholesterolemic environment. Their dysfunction contributes to the progression of diabetic macro- and microvascular complications. Activation of "cholesterol-sensing" nuclear receptors, the liver X receptors (LXRα/LXRβ), protects against atherosclerosis by transcriptional regulation of genes important in promoting cholesterol efflux and inhibiting inflammation. We hypothesized that LXR activation with a synthetic ligand would correct diabetes-induced EPC dysfunction and improve diabetic retinopathy. Studies were performed in streptozotocin (STZ)-injected DBA/2J mice fed a high-fat Western diet (DBA/STZ/WD) and treated with the LXR agonist GW3965 and in LXRα(-/-), LXRβ(-/-), and LXRα/β(-/-) mice. Retinas were evaluated for number of acellular capillaries and glial fibrillary acidic protein (GFAP) immunoreactivity. Bone marrow EPCs were analyzed for migratory function and gene expression. Compared with vehicle-treated DBA/STZ/WD mice, GW3965 treated mice showed fewer acellular capillaries and reduced GFAP expression. These mice also exhibited enhanced EPC migration and restoration of inflammatory and oxidative stress genes toward nondiabetic levels. LXRα(-/-), LXRβ(-/-), and LXRα/β(-/-) mice developed acellular capillaries and EPC dysfunction similar to the DBA/STZ/WD mice. These studies support a key role for LXR in retinal and bone marrow progenitor dysfunction associated with type 1 diabetes. LXR agonists may represent promising pharmacologic targets for correcting retinopathy and EPC dysfunction. Show less

The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer. Show less

Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor-γ (PPARγ). Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2-3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = -0.41, p<0.001; rho = -0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = -0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression. ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia-related, persistent disruption of a key component of RCT. Show less

Although widely prescribed for their potent antiinflammatory actions, glucocorticoid drugs (e.g., dexamethasone) cause undesirable side effects that are features of the metabolic syndrome, including hyperglycemia, fatty liver, insulin resistance, and type II diabetes. Liver x receptors (LXRs) are nuclear receptors that respond to cholesterol metabolites and regulate the expression of a subset of glucocorticoid target genes. Here, we show LXRβ is required to mediate many of the negative side effects of glucocorticoids. Mice lacking LXRβ (but not LXRα) were resistant to dexamethasone-induced hyperglycemia, hyperinsulinemia, and hepatic steatosis, but remained sensitive to dexamethasone-dependent repression of the immune system. In vivo, LXRα/β knockout mice demonstrated reduced dexamethasone-induced expression of the key hepatic gluconeogenic gene, phosphoenolpyruvate carboxykinase (PEPCK). In perfused liver and primary mouse hepatocytes, LXRβ was required for glucocorticoid-induced recruitment of the glucocorticoid receptor to the PEPCK promoter. These findings suggest a new avenue for the design of safer glucocorticoid drugs through a mechanism of selective glucocorticoid receptor transactivation. Show less

The National Heart, Lung, and Blood Institute's Candidate Gene Association Resource (CARe), a planned cross-cohort analysis of genetic variation in cardiovascular, pulmonary, hematologic, and sleep-related traits, comprises >40,000 participants representing 4 ethnic groups in 9 community-based cohorts. The goals of CARe include the discovery of new variants associated with traits using a candidate gene approach and the discovery of new variants using the genome-wide association mapping approach specifically in African Americans. CARe has assembled DNA samples for >40,000 individuals self-identified as European American, African American, Hispanic, or Chinese American, with accompanying data on hundreds of phenotypes that have been standardized and deposited in the CARe Phenotype Database. All participants were genotyped for 7 single-nucleotide polymorphisms (SNPs) selected based on prior association evidence. We performed association analyses relating each of these SNPs to lipid traits, stratified by sex and ethnicity, and adjusted for age and age squared. In at least 2 of the ethnic groups, SNPs near CETP, LIPC, and LPL strongly replicated for association with high-density lipoprotein cholesterol concentrations, PCSK9 with low-density lipoprotein cholesterol levels, and LPL and APOA5 with serum triglycerides. Notably, some SNPs showed varying effect sizes and significance of association in different ethnic groups. The CARe Pilot Study validates the operational framework for phenotype collection, SNP genotyping, and analytic pipeline of the CARe project and validates the planned candidate gene study of approximately 2000 biological candidate loci in all participants and genome-wide association study in approximately 8000 African American participants. CARe will serve as a valuable resource for the scientific community. Show less

Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. The current investigation was undertaken to examine the role of Wnt/beta-catenin pathway in regulating the growth and maintenance of colonospheres. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant) were used. Colonospheres formed in vitro exhibited higher expression of colon CSCs markers LGR5, CD44, CD166 and Musashi-1 along with putative CSC marker EpCAM, compared to the corresponding parental cancer cells and also exhibit the ability to form spheroids under extreme limiting dilution, indicating the predominance of CSCs in colonospheres. Colonospheres formed by HCT-116 cells show over 80% of the cells to be CD44 positive, compared to Show less

The tricho-rhino-phalangeal syndrome type II (TRPS II) is characterized by sparse scalp hair, a long nose with a bulbous tip, a long flat philtrum, cone-shaped epiphyses of the phalanges, retarded bone age in infancy and multiple cartilaginous exostoses. All patients have a hemizygous deletion on chromosome 8q23.3-24.11 which spans at least the 2.8 Mb-region from TRPS1 through EXT1. Only patients with deletions that extend beyond this interval tend to have mental retardation. Here we describe a 14.5-year-old girl with mental retardation and TRPS II. Her facial features are only mild, but she has the typical skeletal features including cone-shaped epiphyses at the phalanges, retarded bone age, multiple exostoses and short stature. She is the first patient with TRPS II and a molecularly proven mosaic interstitial deletion in 8q22.3-q24.13. The deletion is one of the largest ever found in TRPS II, and spans 19.79 Mb and 50 genes or loci including TRPS1 and EXT1. The degree of mosaicism is 7% in lymphocytes from peripheral blood and 97% in skin fibroblasts. Show less

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity. Show less

The mRNA expression of lipogenic genes Scd-1 and Fas is regulated partly by the insulin-sensitive transcription factor SREBP-1c and liver X receptor alpha (LXRalpha). Compared with normal mice, the increase in the mRNA expression of hepatic Scd-1, Fas, and Srebp-1c was severely attenuated in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice during the transition from the starved to the re-fed states. The concentration of the membrane-bound form of SREBP-1c was also lower in the livers of the PPARalpha-deficient mice during re-feeding but there was little difference in the concentration of the active, nuclear form, or in the abundance of Insig-2a mRNA. The response of plasma insulin to starvation and re-feeding was normal in the PPARalpha-deficient mice. Rat hepatocytes transfected with an adenovirus encoding a dominant negative form of PPARalpha were resistant to the stimulatory effects of insulin on Fas and Scd-1 mRNA expression in vitro. When LXRalpha was activated in vivo by inclusion of a non-steroidal ligand in the diet, the expression of the mRNA for hepatic Srebp-1c, Fas, and Scd-1 was increased severalfold in mice of both genotypes and resistance associated with PPARalpha deficiency was abolished during re-feeding. However, although re-feeding the LXRalpha ligand induced the immature form of SREBP-1c equally in the livers of both genotypes, the concentration of the nuclear form remained relatively low in the livers of the PPARalpha-deficient mice. We conclude that intact PPARalpha is required to mediate the response of Scd-1 and Fas gene expression to insulin and that this is normally achieved directly by activation of LXRalpha. Show less

Despite its widespread use to assess fibrosis, liver biopsy has several important drawbacks, including that is it semi-quantitative, invasive, and limited by sampling and observer variability. Non-invasive serum biomarkers may more accurately reflect the fibrogenetic process. To identify potential biomarkers of fibrosis, we compared serum protein expression profiles in patients with chronic hepatitis C (CHC) virus infection and fibrosis. Twenty-one patients with no or mild fibrosis (METAVIR stage F0, F1) and 23 with advanced fibrosis (F3, F4) were retrospectively identified from a pedigreed database of 1600 CHC patients. All samples were carefully phenotyped and matched for age, gender, race, body mass index, genotype, duration of infection, alcohol use, and viral load. Expression profiling was performed in a blinded fashion using a 2D polyacrylamide gel electrophoresis/LC-MS/MS platform. Partial least squares discriminant analysis and likelihood ratio statistics were used to rank individual differences in protein expression between the 2 groups. Seven individual protein spots were identified as either significantly increased (alpha2-macroglobulin, haptoglobin, albumin) or decreased (complement C-4, serum retinol binding protein, apolipoprotein A-1, and two isoforms of apolipoprotein A-IV) with advanced fibrosis. Three individual proteins, haptoglobin, apolipoprotein A-1, and alpha2-macroglobulin, are included in existing non-invasive serum marker panels. Biomarkers identified through expression profiling may facilitate the development of more accurate marker algorithms to better quantitate hepatic fibrosis and monitor disease progression. Show less

Combined hyperlipidemia is a common disorder, characterized by a highly atherogenic lipoprotein profile and a substantially increased risk of coronary heart disease. The purpose of this study was to establish whether variations of apolipoprotein A5 (APOA5), a newly discovered gene of lipid metabolism located 30 kbp downstream of the APOA1/C3/A4 gene cluster, contributes to the transmission of familial combined hyperlipidemia (FCHL). We performed linkage and association tests on 128 families. Two independent alleles, APOA5c.56G and APOC3c.386G, of the APOA1/C3/A4/A5 gene cluster were overtransmitted in FCHL (P=0.004 and 0.007, respectively). This was paired with reduced transmission of the common APOA1/C3/A4/A5 haplotype (frequency 0.4461) to affected subjects (P=0.012). The APOA5c.56G genotype accounted for 7.3% to 13.8% of the variance in plasma triglyceride levels in probands (P<0.004). The APOC3c.386G genotypes accounted for 4.4% to 5.1% of the variance in triglyceride levels in FCHL spouses (P<0.007), suggesting that this allele marks a FCHL quantitative trait as well as representing a susceptibility locus for the condition. A combined linkage and association analysis establishes that variation at the APOA1/C3/A4/A5 gene cluster contributes to FCHL transmission in a substantial proportion of northern European families. Show less

To investigate prospectively the relationship between Jewish ethnicity and prostate cancer mortality. Men were selected from white male participants in two large American Cancer Society cohorts: Cancer Prevention Studies I (CPS-I) (enrolled in 1959 and followed through 1972) and II (CPS-II) (enrolled in 1982 and followed through 1996). During the follow-up periods there were 1,751 prostate cancer deaths among 417,018 men in CPS-1 and 3594 deaths among 447,780 men in CPS-II. Cox proportional hazards modeling was used to compute rate ratios (RR) and to adjust for known and suspected risk factors for prostate cancer. Prostate cancer death rates were substantially lower among Jewish men than other white men in both cohorts (multivariate adjusted rate ratios (RR) = 0.54, 95% CI = 0.38-0.77 in CPS-I; RR = 0.72, 95% CI = 0.61-0.86 in CPS-II). Factors such as tobacco avoidance and measured dietary patterns did not account for this difference. Lower prostate cancer death rates were observed among Jewish men regardless of place of birth of the participants or their parents. Prostate cancer death rates are lower among Jewish men in the US and in Israel than among non-Jewish US white men. This may reflect persistent differences in unknown environmental risk factors or possible genetic susceptibility. Show less