Large-scale analysis to identify risk factors for ovarian cancer.

OBJECTIVE: Ovarian cancer is characterized by late-stage diagnoses and poor prognosis. We aimed to identify factors that can inform prevention and early detection of ovarian cancer. METHODS: We used a data-driven machine learning approach to identify predictors of epithelial ovarian cancer from 2920 input features measured 12.6 years (IQR 11.9 to 13.3 years) before diagnoses. Analyses included 221 732 female participants in the UK Biobank without a history of cancer. During the follow-up 1441 women developed ovarian cancer. For factors that contributed to model prediction, we used multivariate logistic regression to evaluate the association with ovarian cancer, with evidence for causality tested by Mendelian randomization (MR) analyses in the Ovarian Cancer Genetics Consortium (25 509 cases). RESULTS: Greater parity and ever-use of oral contraception were associated with lower ovarian cancer risk (ever vs never OR 0.74, 95% CI 0.66 to 0.84). After adjustment for established risk factors, greater height, weight, and greater red blood cell distribution width were associated with increased ovarian cancer risk, while higher aspartate aminotransferase levels and mean corpuscular volume were associated with lower risk. MR analyses confirmed observational associations with anthropometric/adiposity traits (eg, body fat percentage per standard deviation (SD); OR inverse-variance weighted (ORIVW) 1.28, 95% CI 1.13 to 1.46) and aspartate aminotransferase (ORIVW 0.87, 95% CI 0.78 to 0.98). MR also provided genetic evidence for a protective association of higher total serum protein on ovarian cancer, higher lymphocyte count on serous and endometrioid ovarian cancer, and greater forced expiratory volume in 1 s on serous ovarian cancer among other findings. CONCLUSIONS: This study shows that certain risk factors for ovarian cancer are modifiable, suggesting that weight reduction and interventions to reduce the number of ovulations may provide potential for future prevention. We also identified blood biomarkers associated with ovarian cancer years before diagnoses, warranting further investigation.

Hypothesis-free discovery of novel cancer predictors using machine learning.

BACKGROUND: Cancer is a leading cause of morbidity and mortality worldwide, and better understanding of the risk factors could enhance prevention. METHODS: We conducted a hypothesis-free analysis combining machine learning and statistical approaches to identify cancer risk factors from 2828 potential predictors captured at baseline. There were 459,169 UK Biobank participants free from cancer at baseline and 48,671 new cancer cases during the 10-year follow-up. Logistic regression models adjusted for age, sex, ethnicity, education, material deprivation, smoking, alcohol intake, body mass index and skin colour (as a proxy for sun sensitivity) were used for obtaining adjusted odds ratios, with continuous predictors presented using quintiles (Q). RESULTS: In addition to smoking, older age and male sex, positively associating features included several anthropometric characteristics, whole body water mass, pulse, hypertension and biomarkers such as urinary microalbumin (Q5 vs. Q1 OR 1.16, 95% CI = 1.13-1.19), C-reactive protein (Q5 vs. Q1 OR 1.20, 95% CI = 1.16-1.24) and red blood cell distribution width (Q5 vs. Q1 OR 1.18, 95% CI = 1.14-1.21), among others. High-density lipoprotein cholesterol (Q5 vs. Q1 OR 0.84, 95% CI = 0.81-0.87) and albumin (Q5 vs. Q1 OR 0.84, 95% CI = 0.81-0.87) were inversely associated with cancer. In sex-stratified analyses, higher testosterone increased the risk in females but not in males (Q5 vs. Q1 ORfemales 1.23, 95% CI = 1.17-1.30). Phosphate was associated with a lower risk in females but a higher risk in males (Q5 vs. Q1 ORfemales 0.94, 95% CI = 0.90-0.99 vs. ORmales 1.09, 95% CI 1.04-1.15). CONCLUSIONS: This hypothesis-free analysis suggests personal characteristics, metabolic biomarkers, physical measures and smoking as important predictors of cancer risk, with further studies needed to confirm causality and clinical relevance.

Metabolic profile-based subgroups can identify differences in brain volumes and brain iron deposition.

AIMS: To evaluate associations of metabolic profiles and biomarkers with brain atrophy, lesions, and iron deposition to understand the early risk factors associated with dementia. MATERIALS AND METHODS: Using data from 26 239 UK Biobank participants free from dementia and stroke, we assessed the associations of metabolic subgroups, derived using an artificial neural network approach (self-organizing map), and 39 individual biomarkers with brain MRI measures: total brain volume (TBV), grey matter volume (GMV), white matter volume (WMV), hippocampal volume (HV), white matter hyperintensity (WMH) volume, and caudate iron deposition. RESULTS: In metabolic subgroup analyses, participants characterized by high triglycerides and liver enzymes showed the most adverse brain outcomes compared to the healthy reference subgroup with high-density lipoprotein cholesterol and low body mass index (BMI) including associations with GMV (ßstandardized -0.20, 95% confidence interval [CI] -0.24 to -0.16), HV (ßstandardized -0.09, 95% CI -0.13 to -0.04), WMH volume (ßstandardized 0.22, 95% CI 0.18 to 0.26), and caudate iron deposition (ßstandardized 0.30, 95% CI 0.25 to 0.34), with similar adverse associations for the subgroup with high BMI, C-reactive protein and cystatin C, and the subgroup with high blood pressure (BP) and apolipoprotein B. Among the biomarkers, striking associations were seen between basal metabolic rate (BMR) and caudate iron deposition (ßstandardized 0.23, 95% CI 0.22 to 0.24 per 1 SD increase), GMV (ßstandardized -0.15, 95% CI -0.16 to -0.14) and HV (ßstandardized -0.11, 95% CI -0.12 to -0.10), and between BP and WMH volume (ßstandardized 0.13, 95% CI 0.12 to 0.14 for diastolic BP). CONCLUSIONS: Metabolic profiles were associated differentially with brain neuroimaging characteristics. Associations of BMR, BP and other individual biomarkers may provide insights into actionable mechanisms driving these brain associations.

A Gut-Intrinsic Melanocortin Signaling Complex Augments L-Cell Secretion in Humans.

OBJECTIVE: Hypothalamic melanocortin 4 receptors (MC4R) are a key regulator of energy homeostasis. Brain-penetrant MC4R agonists have failed, as concentrations required to suppress food intake also increase blood pressure. However, peripherally located MC4R may also mediate metabolic benefits of MC4R activation. Mc4r transcript is enriched in mouse enteroendocrine L cells and peripheral administration of the endogenous MC4R agonist, α-melanocyte stimulating hormone (α-MSH), triggers the release of the anorectic hormones Glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in mice. This study aimed to determine whether pathways linking MC4R and L-cell secretion exist in humans. DESIGN: GLP-1 and PYY levels were assessed in body mass index-matched individuals with or without loss-of-function MC4R mutations following an oral glucose tolerance test. Immunohistochemistry was performed on human intestinal sections to characterize the mucosal MC4R system. Static incubations with MC4R agonists were carried out on human intestinal epithelia, GLP-1 and PYY contents of secretion supernatants were assayed. RESULTS: Fasting PYY levels and oral glucose-induced GLP-1 secretion were reduced in humans carrying a total loss-of-function MC4R mutation. MC4R was localized to L cells and regulates GLP-1 and PYY secretion from ex vivo human intestine. α-MSH immunoreactivity in the human intestinal epithelia was predominantly localized to L cells. Glucose-sensitive mucosal pro-opiomelanocortin cells provide a local source of α-MSH that is essential for glucose-induced GLP-1 secretion in small intestine. CONCLUSION: Our findings describe a previously unidentified signaling nexus in the human gastrointestinal tract involving α-MSH release and MC4R activation on L cells in an autocrine and paracrine fashion. Outcomes from this study have direct implications for targeting mucosal MC4R to treat human metabolic disorders.

Augmented capacity for peripheral serotonin release in human obesity.

BACKGROUND/OBJECTIVES: Evidence from animal studies highlights an important role for serotonin (5-HT), derived from gut enterochromaffin (EC) cells, in regulating hepatic glucose production, lipolysis and thermogenesis, and promoting obesity and dysglycemia. Evidence in humans is limited, although elevated plasma 5-HT concentrations are linked to obesity. SUBJECTS/METHODS: We assessed (i) plasma 5-HT concentrations before and during intraduodenal glucose infusion (4 kcal/min for 30 min) in non-diabetic obese (BMI 44 ± 4 kg/m2, N = 14) and control (BMI 24 ± 1 kg/m2, N = 10) subjects, (ii) functional activation of duodenal EC cells (immunodetection of phospho-extracellular related-kinase, pERK) in response to glucose, and in separate subjects, (iii) expression of tryptophan hydroxylase-1 (TPH1) in duodenum and colon (N = 39), and (iv) 5-HT content in primary EC cells from these regions (N = 85). RESULTS: Plasma 5-HT was twofold higher in obese than control responders prior to (P = 0.025), and during (iAUC, P = 0.009), intraduodenal glucose infusion, and related positively to BMI (R2 = 0.334, P = 0.003) and HbA1c (R2 = 0.508, P = 0.009). The density of EC cells in the duodenum was twofold higher at baseline in obese subjects than controls (P = 0.023), with twofold more EC cells activated by glucose infusion in the obese (EC cells co-expressing 5-HT and pERK, P = 0.001), while the 5-HT content of EC cells in duodenum and colon was similar; TPH1 expression was 1.4-fold higher in the duodenum of obese subjects (P = 0.044), and related positively to BMI (R2 = 0.310, P = 0.031). CONCLUSIONS: Human obesity is characterized by an increased capacity to produce and release 5-HT from the proximal small intestine, which is strongly linked to higher body mass, and glycemic control. Gut-derived 5-HT is likely to be an important driver of pathogenesis in human obesity and dysglycemia.

Huntingtin-associated protein 1: Eutherian adaptation from a TRAK-like protein, conserved gene promoter elements, and localization in the human intestine.

BACKGROUND: Huntingtin-associated Protein 1 (HAP1) is expressed in neurons and endocrine cells, and is critical for postnatal survival in mice. HAP1 shares a conserved "HAP1_N" domain with TRAfficking Kinesin proteins TRAK1 and TRAK2 (vertebrate), Milton (Drosophila) and T27A3.1 (C. elegans). HAP1, TRAK1 and TRAK2 have a degree of common function, particularly regarding intracellular receptor trafficking. However, TRAK1, TRAK2 and Milton (which have a "Milt/TRAK" domain that is absent in human and rodent HAP1) differ in function to HAP1 in that they are mitochondrial transport proteins, while HAP1 has emerging roles in starvation response. We have investigated HAP1 function by examining its evolution, and upstream gene promoter sequences. We performed phylogenetic analyses of the HAP1_N domain family of proteins, incorporating HAP1 orthologues (identified by genomic synteny) from 5 vertebrate classes, and also searched the Dictyostelium proteome for a common ancestor. Computational analyses of mammalian HAP1 gene promoters were performed to identify phylogenetically conserved regulatory motifs. RESULTS: We found that as recently as marsupials, HAP1 contained a Milt/TRAK domain and was more similar to TRAK1 and TRAK2 than to eutherian HAP1. The Milt/TRAK domain likely arose post multicellularity, as it was absent in the Dictyostelium proteome. It was lost from HAP1 in the eutherian lineage, and also from T27A3.1 in C. elegans. The HAP1 promoter from human, mouse, rat, rabbit, horse, dog, Tasmanian devil and opossum contained common sites for transcription factors involved in cell cycle, growth, differentiation, and stress response. A conserved arrangement of regulatory elements was identified, including sites for caudal-related homeobox transcription factors (CDX1 and CDX2), and myc-associated factor X (MAX) in the region of the TATA box. CDX1 and CDX2 are intestine-enriched factors, prompting investigation of HAP1 protein expression in the human duodenum. HAP1 was localized to singly dispersed mucosal cells, including a subset of serotonin-positive enterochromaffin cells. CONCLUSION: We have identified eutherian HAP1 as an evolutionarily recent adaptation of a vertebrate TRAK protein-like ancestor, and found conserved CDX1/CDX2 and MAX transcription factor binding sites near the TATA box in mammalian HAP1 gene promoters. We also demonstrated that HAP1 is expressed in endocrine cells of the human gut.

Fusion Pore Size Limits 5-HT Release From Single Enterochromaffin Cell Vesicles.

Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.

Huntingtin-associated protein-1 is a synapsin I-binding protein regulating synaptic vesicle exocytosis and synapsin I trafficking.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1(-/-) cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1(-/-) brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling.

Deficiency of huntingtin has pleiotropic effects in the social amoeba Dictyostelium discoideum.

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell-cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca(2+) or Mg(2+) but not pulses of cAMP. Although hd(-) cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd(-) cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein.

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1ß). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1ß (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Phenome-wide association study of ovarian cancer identifies common comorbidities and reveals shared genetics with complex diseases and biomarkers.

BACKGROUND: Ovarian cancer (OC) is commonly diagnosed among older women who have comorbidities. This hypothesis-free phenome-wide association study (PheWAS) aimed to identify comorbidities associated with OC, as well as traits that share a genetic architecture with OC. METHODS: We used data from 181,203 white British female UK Biobank participants and analysed OC and OC subtype-specific genetic risk scores (OC-GRS) for an association with 889 diseases and 43 other traits. We conducted PheWAS and colocalization analyses for individual variants to identify evidence for shared genetic architecture. RESULTS: The OC-GRS was associated with 10 diseases, and the clear cell OC-GRS was associated with five diseases at the FDR threshold (p = 5.6 × 10-4 ). Mendelian randomizaiton analysis (MR) provided robust evidence for the association of OC with higher risk of "secondary malignant neoplasm of digestive systems" (OR 1.64, 95% CI 1.33, 2.02), "ascites" (1.48, 95% CI 1.17, 1.86), "chronic airway obstruction" (1.17, 95% CI 1.07, 1.29), and "abnormal findings on examination of the lung" (1.51, 95% CI 1.22, 1.87). Analyses of lung spirometry measures provided further support for compromised respiratory function. PheWAS on individual OC variants identified five genetic variants associated with other diseases, and seven variants associated with biomarkers (all, p ≤ 4.5 × 10-8 ). Colocalization analysis identified rs4449583 (from TERT locus) as the shared causal variant for OC and seborrheic keratosis. CONCLUSIONS: OC is associated with digestive and respiratory comorbidities. Several variants affecting OC risk were associated with other diseases and biomarkers, with this study identifying a novel genetic locus shared between OC and skin conditions.

Milk consumption and risk of twelve cancers: A large-scale observational and Mendelian randomisation study.

BACKGROUND & AIMS: Milk consumption is a modifiable lifestyle factor that has been associated with several cancer types in observational studies. Limited evidence exists regarding the causality of these relationships. Using a genetic variant (rs4988235) near the lactase gene (LCT) locus that proxies milk consumption, we conducted a comprehensive survey to assess potential causal relationships between milk consumption and 12 types of cancer. METHODS: Our analyses were conducted using white British participants of the UK Biobank (n = up to 255,196), the FinnGen cohort (up to 260,405), and available cancer consortia. We included cancers with previous evidence of an association with milk consumption in observational studies, as well as cancers common in both UK Biobank and FinnGen populations (>1000 cases). We evaluated phenotypic associations of milk intake and cancer incidence in the UK Biobank, and then used a Mendelian randomisation (MR) approach to assess causality in the UK Biobank, FinnGen consortium, and combined analyses incorporating additional consortia data for five cancers. In MR meta-analyses, case numbers for cancers of breast, ovary, uterus, cervix, prostate, bladder and urinary tract, colorectum, and lung ranged between 6000 and 148,000 cases, and between 780 and 1342 cases for cancers of the liver, mouth, stomach and diffuse large B-cell lymphoma. RESULTS: In observational analyses, milk consumption was associated with higher risk of bladder and urinary tract cancer (OR 1.23, 95% CI 1.03-1.47), but not with any other cancer. This association was not confirmed in the MR analysis, and genetically predicted milk consumption showed a significant association only with lower risk of colorectal cancer (0.89, 0.81-0.98 per additional 50 g/day). In the MR analyses conducted among individual cohorts, genetically predicted milk consumption provided evidence for an association with lower colorectal cancer in the FinnGen cohort (0.85, 0.74-0.97), and in the UK Biobank greater risk of female breast cancer (1.12, 1.03-1.23), and uterine cancer in pre-menopausal females (3.98, 1.48-10.7). CONCLUSION: In a comprehensive survey of milk-cancer associations, we confirm of a protective role of milk consumption for colorectal cancer. Our analyses also provide some suggestion for higher risks of breast cancer and premenopausal uterine cancer, warranting further investigation.

Alterations in GLP-1 and PYY release with aging and body mass in the human gut.

The lining of our intestinal surface contains an array of hormone-producing cells that are collectively our bodies' largest endocrine cell reservoir. These "enteroendocrine" (EE) cells reside amongst the billions of absorptive epithelial and other cell types that line our gastrointestinal tract and can sense and respond to the ever-changing internal environment in our gut. EE cells release an array of important signalling molecules that can act as hormones, including glucagon-like peptide (GLP-1) and peptide YY (PYY) which are co-secreted from L cells. While much is known about the effects of these hormones on metabolism, insulin secretion and food intake, less is understood about their secretion from human intestinal tissue. In this study we assess whether GLP-1 and PYY release differs across human small and large intestinal tissue locations within the gastrointestinal tract, and/or by sex, body weight and the age of an individual. We identify that the release of both hormones is greater in more distal regions of the human colon, but is not different between sexes. We observe a negative correlation of GLP-1 and BMI in the small, but not large, intestine. Increased aging correlates with declining secretion of both GLP-1 and PYY in human large, but not small, intestine. When the data for large intestine is isolated by region, this relationship with age remains significant for GLP-1 in the ascending and descending colon and in the descending colon for PYY. This is the first demonstration that site-specific differences in GLP-1 and PYY release occur in human gut, as do site-specific relationships of L cell secretion with aging and body mass.

Healthy Lifestyle, Genetic Risk and Brain Health: A Gene-Environment Interaction Study in the UK Biobank.

Genetic susceptibility and lifestyle affect the risk of dementia but there is little direct evidence for their associations with preclinical changes in brain structure. We investigated the association of genetic dementia risk and healthy lifestyle with brain morphometry, and whether effects from elevated genetic risk are modified by lifestyle changes. We used prospective data from up to 25,894 UK Biobank participants (median follow-up of 8.8 years), and defined healthy lifestyle according to American Heart Association criteria as BMI < 30, no smoking, healthy diet and regular physical activity). Higher genetic risk was associated with lower hippocampal volume (beta −0.16 cm3, 95% CI −0.22, −0.11) and total brain volume (−4.34 cm3, 95% CI −7.68, −1.01) in participants aged ≥60 years but not <60 years. Healthy lifestyle was associated with higher total brain, grey matter and hippocampal volumes, and lower volume of white matter hyperintensities, with no effect modification by age or genetic risk. In conclusion, adverse effects of high genetic risk on brain health were only found in older participants, while adhering to healthy lifestyle recommendations is beneficial regardless of age or genetic risk.

Infection of Human Retinal Pigment Epithelial Cells with Dengue Virus Strains Isolated during Outbreaks in Singapore.

Prevalence of dengue retinopathy varies across epidemics, with the disease linked to circulation of dengue virus serotype 1 (DENV-1). The retinal pigment epithelium has been implicated in the pathology. We investigated infectivity, molecular response, and barrier function of epithelial cells inoculated with DENV strains from different outbreaks in Singapore. Monolayers of human retinal pigment epithelial cells (multiple primary cell isolates and the ARPE-19 cell line) were inoculated with six DENV strains, at multiplicity of infection of 10; uninfected and recombinant strain-infected controls were included where relevant. Infectivity and cell response were assessed primarily by RT-qPCR on total cellular RNA, and barrier function was evaluated as electrical resistance across monolayers. Higher viral RNA loads were measured in human retinal pigment epithelial cells infected with DENV-1 strains from the 2005 Singapore epidemic, when retinopathy was prevalent, versus DENV-1 strains from the 2007 Singapore epidemic, when retinopathy was not observed. Type I interferon (IFN) transcripts (IFN-ß and multiple IFN-stimulated genes) were up-regulated, and impact on barrier function was more pronounced, for cells infected with DENV-1 strains from the 2005 versus the 2007 Singapore epidemics. Aside from serotype, strain of DENV may determine the potential to induce retinal pathology. Identification of molecular markers of disease-associated DENV strains may provide insights into the pathogenesis of dengue retinopathy.

Selective neuronal requirement for huntingtin in the developing zebrafish.

Huntington's disease shares a common molecular basis with eight other neurodegenerative diseases, expansion of an existing polyglutamine tract. In each case, this repeat tract occurs within otherwise unrelated proteins. These proteins show widespread and overlapping patterns of expression in the brain and yet the diseases are distinguished by neurodegeneration in a specific subset of neurons that are most sensitive to the mutation. It has therefore been proposed that expansion of the polyglutamine region in these genes may result in perturbation of the normal function of the respective proteins, and that this perturbation in some way contributes to the neuronal specificity of these diseases. The normal functions of these proteins have therefore become a focus for investigation as potential pathogenic pathways. We have used synthetic antisense morpholinos to inhibit the translation of huntingtin mRNA during early zebrafish development and have previously reported the effects of huntingtin reduction on iron transport and homeostasis. Here we report an analysis of the effects of huntingtin loss-of-function on the developing nervous system, observing distinct defects in morphology of neuromasts, olfactory placode and branchial arches. The potential common origins of these defects were explored, revealing impaired formation of the anterior-most region of the neural plate as indicated by reduced pre-placodal and telencephalic gene expression with no effect on mid- or hindbrain formation. These investigations demonstrate a specific 'rate-limiting' role for huntingtin in formation of the telencephalon and the pre-placodal region, and differing levels of requirement for huntingtin function in specific nerve cell types.

Apolipoprotein E (APOE) genotype-associated disease risks: a phenome-wide, registry-based, case-control study utilising the UK Biobank.

BACKGROUND: The three main alleles of the APOE gene (ε4, ε3 and ε2) carry differential risks for conditions including Alzheimer's disease (AD) and cardiovascular disease. Due to their clinical significance, we explored disease associations of the APOE genotypes using a hypothesis-free, data-driven, phenome-wide association study (PheWAS) approach. METHODS: We used data from the UK Biobank to screen for associations between APOE genotypes and over 950 disease outcomes using genotype ε3ε3 as a reference. Data was restricted to 337,484 white British participants (aged 37-73 years). FINDINGS: After correction for multiple testing, PheWAS analyses identified associations with 37 outcomes, representing 18 distinct diseases. As expected, ε3ε4 and ε4ε4 genotypes associated with increased odds of AD (p ≤ 7.6 × 10-46), hypercholesterolaemia (p ≤ 7.1 × 10-17) and ischaemic heart disease (p ≤ 2.3 × 10-4), while ε2ε3 provided protection for the latter two conditions (p ≤ 3.7 × 10-10) compared to ε3ε3. In contrast, ε4-associated disease protection was seen against obesity, chronic airway obstruction, type 2 diabetes, gallbladder disease, and liver disease (all p ≤ 5.2 × 10-4) while ε2ε2 homozygosity increased risks of peripheral vascular disease, thromboembolism, arterial aneurysm, peptic ulcer, cervical disorders, and hallux valgus (all p ≤ 6.1 × 10-4). Sensitivity analyses using brain neuroimaging, blood biochemistry, anthropometric, and spirometric biomarkers supported the PheWAS findings on APOE associations with respective disease outcomes. INTERPRETATION: PheWAS confirms strong associations between APOE and AD, hypercholesterolaemia, and ischaemic heart disease, and suggests potential ε4-associated disease protection and harmful effects of the ε2ε2 genotype, for several conditions. FUNDING: National Health and Medical Research Council of Australia.

Sugar Responses of Human Enterochromaffin Cells Depend on Gut Region, Sex, and Body Mass.

Gut-derived serotonin (5-HT) is released from enterochromaffin (EC) cells in response to nutrient cues, and acts to slow gastric emptying and modulate gastric motility. Rodent studies also evidence a role for gut-derived 5-HT in the control of hepatic glucose production, lipolysis and thermogenesis, and in mediating diet-induced obesity. EC cell number and 5-HT content is increased in the small intestine of obese rodents and human, however, it is unknown whether EC cells respond directly to glucose in humans, and whether their capacity to release 5-HT is perturbed in obesity. We therefore investigated 5-HT release from human duodenal and colonic EC cells in response to glucose, sucrose, fructose and α-glucoside (αMG) in relation to body mass index (BMI). EC cells released 5-HT only in response to 100 and 300 mM glucose (duodenum) and 300 mM glucose (colon), independently of osmolarity. Duodenal, but not colonic, EC cells also released 5-HT in response to sucrose and αMG, but did not respond to fructose. 5-HT content was similar in all EC cells in males, and colonic EC cells in females, but 3 to 4-fold higher in duodenal EC cells from overweight females (p < 0.05 compared to lean, obese). Glucose-evoked 5-HT release was 3-fold higher in the duodenum of overweight females (p < 0.05, compared to obese), but absent here in overweight males. Our data demonstrate that primary human EC cells respond directly to dietary glucose cues, with regional differences in selectivity for other sugars. Augmented glucose-evoked 5-HT release from duodenal EC is a feature of overweight females, and may be an early determinant of obesity.

Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.