Habitual chocolate consumption and the risk of incident heart failure among healthy men and women

Acknowledgments The authors would like to thank the participants of the EPIC-Norfolk cohort. We thank the nutritionist team and data management team of the EPIC-Norfolk cohort. The EPIC-Norfolk study was supported by grants from the Medical Research Council and Cancer Research UK. Funders had no role in study design or interpretation of the findings.

Habitual chocolate consumption and the risk of incident heart failure among healthy men and women

Acknowledgments The authors would like to thank the participants of the EPIC-Norfolk cohort. We thank the nutritionist team and data management team of the EPIC-Norfolk cohort. The EPIC-Norfolk study was supported by grants from the Medical Research Council and Cancer Research UK. Funders had no role in study design or interpretation of the findings.

From the Cover: Arsenite Uncouples Mitochondrial Respiration and Induces a Warburg-like Effect in Caenorhabditis elegans.

Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Angiotensin receptors and β-catenin regulate brain endothelial integrity in malaria

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum–infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter–endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin–induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC–induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised.

Adiposity, Sex Hormones, and Repetitive Element DNA Methylation in Healthy Postmenopausal Women

INTRODUCTION: Low levels of methylation within repetitive DNA elements, such as long interspersed nuclear element-1 (LINE-1) and Alu repeats, are believed to epigenetically predispose an individual to cancer and other diseases. The extent to which lifestyle factors affect the degree of DNA methylation within these genomic regions has yet to be fully understood. Adiposity and sex hormones are established risk factors for certain types of cancer and other illnesses, particularly amongst postmenopausal women. The aim of the current investigation is to assess the impact of adiposity and sex hormones on LINE-1 and Alu methylation in healthy postmenopausal women. METHODS: A cross-sectional study was conducted using baseline data from an ancillary study of the Alberta Physical Activity and Breast Cancer Prevention (ALPHA) Trial. Current adiposity was measured using a dual-energy x-ray absorptiometry (DXA) scan, computed tomography (CT) scan, and balance beam scale. Historical weights were self-reported in a questionnaire. Current endogenous sex hormone concentrations were measured in fasting blood serum. Estimated lifetime number of menstrual cycles was used as a proxy for cumulative exposure to ovarian sex hormones. Repetitive element methylation was quantified in white blood cells using a pyrosequencing assay. Linear regression was used to model the relations of interest while adjusting for important confounders. RESULTS: Adiposity and serum estrogen concentrations were positively related to LINE-1 methylation but were not associated with Alu methylation. Cumulative ovarian sex hormone exposure had a “U-shaped” relation with LINE-1 regardless of folate intake and a negative relation with Alu methylation amongst low folate consumers. Androgens were not associated with repetitive element DNA methylation in this population. CONCLUSION: Adiposity and estrogens appear to play a role in maintaining high levels of repetitive element DNA methylation in healthy postmenopausal women. LINE-1 methylation may be a mechanism whereby estrogen exposure protects against cardiovascular and neurodegenerative illnesses. These results add to the growing body of literature showing how the epigenome is shaped by our lifestyle choices. Future prospective studies assessing the relation between levels of repetitive element DNA methylation in healthy individuals and subsequent disease risk are needed to better understand the clinical significance of these results.

Synthetic Polymer and Microbubble Systems for Gene Delivery to the Brain

Thesis (Ph.D.)--University of Washington, 2016-08

Perturbations in Blood Phosphatidylcholine and Sphingomyelin Fatty Acid Composition in a Sample Population of Cigarette Smokers

Docosahexaenoic (DHA) and arachidonic acids (AA) are polyunsaturated fatty acids (PUFAs), major components of brain tissue and neural systems, and the precursors of a number of biologically active metabolites with functions in inflammation resolution, neuroprotection and other actions. As PUFAs are highly susceptible to peroxidation, we hypothesised whether cigarette smokers would present altered PUFAs levels in plasma and erythrocyte phospholipids. Adult males from Indian, Sri-Lankan or Bangladeshi genetic backgrounds who reported smoking between 20 and 60 cigarettes per week were recruited. The control group consisted of matched non-smokers. A blood sample was taken, plasma and erythrocyte total lipids were extracted, phospholipids were separated by thin layer chromatography, and the fatty acid content analysed by gas chromatography. In smokers, dihomo-gamma-linolenic acid, the AA precursor, was significantly reduced in plasma and erythrocyte phosphatidylcholine. AA and DHA were significantly reduced in erythrocyte sphingomyelin. Relatively short term smoking has affected the fatty acid composition of plasma and erythrocyte phospholipids with functions in neural tissue composition, cell signalling, cell growth, intracellular trafficking, neuroprotection and inflammation, in a relatively young population. As lipid peroxidation is pivotal in the pathogenesis of atherosclerosis and neurodegenerative diseases such as Alzheimer disease, early effects of smoking may be relevant for the development of such conditions.

Immune function, gene expression, blood indices and performance in transition dairy cows affected by diet and inflammation

The transition period is associated with the peak incidence of production problems, metabolic disorders and infectious diseases in dairy cows (Drackley, 1999). During this time the cow’s immune system seems to be weakened; it is apparent that metabolic challenges associated with the onset of lactation are factors capable of affecting immune function. However, the reasons for this state are not entirely clear (Goff, 2006). The negative energy balance associated with parturition leads to extensive mobilization of fatty acids stored in adipose tissue, thus, causing marked elevations in blood non-esterified fatty acids (NEFA) and B-hydroxybutyrate (BHBA) concentrations (Drackley et al., 2001). Prepartal level of dietary energy can potentially affect adipose tissue deposition and, thus, the amount of NEFA released into blood and available for metabolism in liver (Drackley et al., 2005). The current feeding practices for pregnant non-lactating cows has been called into question because increasing amounts of moderate-to-high energy diets (i.e. those more similar to lactation diets in the content of energy) during the last 3 wk postpartum have largely failed to overcome peripartal health problems, excessive body condition loss after calving, or declining fertility (Beever, 2006). Current prepartal feeding practices can lead to elevated intakes of energy, which can increase fat deposition in the viscera and upon parturition lead to compromised liver metabolism (Beever, 2006, Drackley et al., 2005). Our general hypothesis was that overfeeding dietary energy during the dry period, accompanied by the metabolic challenges associated with the onset of lactation would render the cow’s immune function less responsive early postpartum. The chapters in this dissertation evaluated neutrophil function, metabolic and inflammation indices and gene expression affected by the plane of dietary energy prepartum and an early post-partum inflammatory challenge in dairy cows. The diet effect in this experiment was transcendental during the transition period and potentially during the entire lactation. Changes in energy balance were observed and provided a good model to study the challenges associated with the onset of lactation. Overall the LPS model provided a consistent response representing an inflammation incident; however the changes in metabolic indices were sudden and hard to detect in most of the cases during the days following the challenge. In general overfeeding dietary energy during the dry period resulted in a less responsive immune function during the early postpartum. In other words, controlling the dietary energy prepartum has more benefits for the dairy cow during transition.

A systems approach to cardiovascular health and disease with a focus on aortic wave dynamics

Cardiovascular diseases (CVDs) have reached an epidemic proportion in the US and worldwide with serious consequences in terms of human suffering and economic impact. More than one third of American adults are suffering from CVDs. The total direct and indirect costs of CVDs are more than $500 billion per year. Therefore, there is an urgent need to develop noninvasive diagnostics methods, to design minimally invasive assist devices, and to develop economical and easy-to-use monitoring systems for cardiovascular diseases. In order to achieve these goals, it is necessary to gain a better understanding of the subsystems that constitute the cardiovascular system. The aorta is one of these subsystems whose role in cardiovascular functioning has been underestimated. Traditionally, the aorta and its branches have been viewed as resistive conduits connected to an active pump (left ventricle of the heart). However, this perception fails to explain many observed physiological results. My goal in this thesis is to demonstrate the subtle but important role of the aorta as a system, with focus on the wave dynamics in the aorta.

The operation of a healthy heart is based on an optimized balance between its pumping characteristics and the hemodynamics of the aorta and vascular branches. The delicate balance between the aorta and heart can be impaired due to aging, smoking, or disease. The heart generates pulsatile flow that produces pressure and flow waves as it enters into the compliant aorta. These aortic waves propagate and reflect from reflection sites (bifurcations and tapering). They can act constructively and assist the blood circulation. However, they may act destructively, promoting diseases or initiating sudden cardiac death. These waves also carry information about the diseases of the heart, vascular disease, and coupling of heart and aorta. In order to elucidate the role of the aorta as a dynamic system, the interplay between the dominant wave dynamic parameters is investigated in this study. These parameters are heart rate, aortic compliance (wave speed), and locations of reflection sites. Both computational and experimental approaches have been used in this research. In some cases, the results are further explained using theoretical models.

The main findings of this study are as follows: (i) developing a physiologically realistic outflow boundary condition for blood flow modeling in a compliant vasculature; (ii) demonstrating that pulse pressure as a single index cannot predict the true level of pulsatile workload on the left ventricle; (iii) proving that there is an optimum heart rate in which the pulsatile workload of the heart is minimized and that the optimum heart rate shifts to a higher value as aortic rigidity increases; (iv) introducing a simple bio-inspired device for correction and optimization of aortic wave reflection that reduces the workload on the heart; (v) deriving a non-dimensional number that can predict the optimum wave dynamic state in a mammalian cardiovascular system; (vi) demonstrating that waves can create a pumping effect in the aorta; (vii) introducing a system parameter and a new medical index, Intrinsic Frequency, that can be used for noninvasive diagnosis of heart and vascular diseases; and (viii) proposing a new medical hypothesis for sudden cardiac death in young athletes.

Evaluation of influenza vaccination services and users’ perception about its utility: a study in a community pharmacy in Lisbon, Portugal

Poster presented at the 25th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID). Copenhagen, Denmark, 25–28 April 2015

Correlates of Vitamin D Status in Healthy Older Adults Living in Miami-Dade and its Association with Non-Skeletal Outcomes: A Cross-Sectional Study

Examining factors that affect vitamin D status in the fast-growing elderly population of Miami-Dade, Florida, is needed. Vitamin D deficiency in older adults has been linked to correlates of disability, including falls and fractures, and cardiovascular disease. The purpose of this study was to determine the proportion of vitamin D insufficient individuals and their relationship with vitamin D insufficiency in older adults (n=97) living in Miami-Dade. We evaluated the association between vitamin D status and 1) dual task physical performance to understand the link between vitamin D and cognition in the context of mobility; and 2) cardiometabolic risk, measured by galvanic skin response, pulse oximetry, and blood pressure to create a composite score based on autonomic nervous system and endothelial function. Participants completed baseline assessments that included serum levels of vitamin D, anthropometrics, body composition, dual task physical performance and cardiometabolic risk. Surveys to evaluate vitamin D intake, sun exposure, physical activity, and depressive symptoms were completed. Spearman’s correlations, independent t-tests, paired t-tests, repeated measures ANOVAs, and multiple logistic and linear regressions were used to examine the relationship of vitamin D insufficiency (25(OH)D /ml) and sufficiency (25(OH)D ≥30 ng/ml) with determinants of vitamin D status, dual task physical performance variables and cardiometabolic risk scores. Although the proportion of vitamin D insufficient individuals was lower when compared to the prevalance of the general United States elderly population, it was still common in healthy community-dwelling older adults living in Miami-Dade County, especially among Hispanics. Factors that affected skin synthesis (ethnicity, and sun exposure), and bioavailability/metabolism (obesity) were significant predictors of vitamin D status. Vitamin D insufficiency was not significantly correlated with worse dual task physical performance; however, cognitive performance was worse in the vitamin D insufficient group. Our results suggest a relationship of vitamin D insufficiency with executive dysfunction, and support an association with cardiometabolic risk using an innovative electro-sensor complex, possibly by modulating autonomic nervous system activity and vascular function, thus affecting cardiac performance.

Bionano Electronics: Magneto-Electric Nanoparticles for Drug Delivery, Brain Stimulation and Imaging Applications

Nanoparticles are often considered as efficient drug delivery vehicles for precisely dispensing the therapeutic payloads specifically to the diseased sites in the patient’s body, thereby minimizing the toxic side effects of the payloads on the healthy tissue. However, the fundamental physics that underlies the nanoparticles’ intrinsic interaction with the surrounding cells is inadequately elucidated. The ability of the nanoparticles to precisely control the release of its payloads externally (on-demand) without depending on the physiological conditions of the target sites has the potential to enable patient- and disease-specific nanomedicine, also known as Personalized NanoMedicine (PNM). In this dissertation, magneto-electric nanoparticles (MENs) were utilized for the first time to enable important functions, such as (i) field-controlled high-efficacy dissipation-free targeted drug delivery system and on-demand release at the sub-cellular level, (ii) non-invasive energy-efficient stimulation of deep brain tissue at body temperature, and (iii) a high-sensitivity contrasting agent to map the neuronal activity in the brain non-invasively. First, this dissertation specifically focuses on using MENs as energy-efficient and dissipation-free field-controlled nano-vehicle for targeted delivery and on-demand release of a anti-cancer Paclitaxel (Taxol) drug and a anti-HIV AZT 5’-triphosphate (AZTTP) drug from 30-nm MENs (CoFe2O4-BaTiO3) by applying low-energy DC and low-frequency (below 1000 Hz) AC fields to separate the functions of delivery and release, respectively. Second, this dissertation focuses on the use of MENs to non-invasively stimulate the deep brain neuronal activity via application of a low energy and low frequency external magnetic field to activate intrinsic electric dipoles at the cellular level through numerical simulations. Third, this dissertation describes the use of MENs to track the neuronal activities in the brain (non-invasively) using a magnetic resonance and a magnetic nanoparticle imaging by monitoring the changes in the magnetization of the MENs surrounding the neuronal tissue under different states. The potential therapeutic and diagnostic impact of this innovative and novel study is highly significant not only in HIV-AIDS, Cancer, Parkinson’s and Alzheimer’s disease but also in many CNS and other diseases, where the ability to remotely control targeted drug delivery/release, and diagnostics is the key.

Enterococcus faecalis prophage dynamics and contributions to pathogenic traits

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.

Public health implications of dietary fiber intake to reduce the cost of chronic diseases

Objective: To assess the epidemiological evidence on dietary fiber intake and chronic diseases and make public health recommendations for the population in Romania based on their consumption. Populations that consume more dietary fiber from cereals, fruits and vegetables have less chronic disease. Dietary Reference Intakes recommend consumption of 14 g dietary fiber per 1,000 kcal, or 25 g for adult women and 38 g for adult men, based on epidemiologic studies showing protection against cardiovascular disease, stroke, hypertension, diabetes, obesity, metabolic syndrome, gastrointestinal disorders, colorectal -, breast -, gastric -, endometrial -, ovarian - and prostate cancer. Furthermore, increased consumption of dietary fiber improves serum lipid concentrations, lowers blood pressure, blood glucose leads to low glycemic index, aids in weight loss, improve immune function, reduce inflammatory marker levels, reduce indicators of inflammation. Dietary fibers contain an unique blend of bioactive components including resistant starches, vitamins, minerals, phytochemicals and antioxidants. Dietary fiber components have important physiological effects on glucose, lipid, protein metabolism and mineral bioavailability needed to prevent chronic diseases. Materials and methods: Data regarding diet was collected based on questionnaires. We used mathematical formulas to calculate the mean dietary fiber intake of Romanian adult population and compared the results with international public health recommendations. Results: Based on the intakes of vegetables, fruits and whole cereals we calculated the Mean Dietary Fiber Intake/day/person (MDFI). Our research shows that the national average MDFI was 9.8 g fiber/day/person, meaning 38% of Dietary Requirements, and the rest of 62% representing a “fiber gap” that we have to take into account. This deficiency predisposes to chronic diseases. Conclusions and recommendations:The poor control of relationship between dietary fiber intake and chronic diseases is a major issue that can result in adverse clinical and economic outcomes. The population in Romania is at risk to develop such diseases due to the deficient fiber consumption. A model of chronic diseases costs is needed to aid attempts to reduce them while permitting optimal management of the chronic diseases. This paper presents a discussion of the burden of chronical disease and its socio-economic implications and proposes a model to predict the costs reduction by adequate intake of dietary fiber.