Associations Between Genetically Predicted Iron Status and Cardiovascular Disease Risk: A Mendelian Randomization Study.

BACKGROUND: Mendelian randomization (MR) studies suggest a causal effect of iron status on cardiovascular disease (CVD) risk, but it is unknown if these associations are confounded by pleiotropic effects of the instrumental variables on CVD risk factors. We aimed to investigate the effect of iron status on CVD risk controlling for CVD risk factors. METHODS AND RESULTS: Iron biomarker instrumental variables (total iron-binding capacity [n=208 422], transferrin saturation [n=198 516], serum iron [n=236 612], ferritin [n=257 953]) were selected from a European genome-wide association study meta-analysis. We performed 2-sample univariate MR of each iron trait on CVD outcomes (all-cause ischemic stroke, cardioembolic ischemic stroke, large-artery ischemic stroke, small-vessel ischemic stroke, and coronary heart disease) from MEGASTROKE (n=440 328) and CARDIoGRAMplusC4D (Coronary Artery Disease Genome Wide Replication and Meta-Analysis Plus the Coronary Artery Disease Genetics) (n=183 305). We then implemented multivariate MR conditioning on 7 CVD risk factors from independent European samples to evaluate their potential confounding or mediating effects on the observed iron-CVD associations. With univariate MR analyses, we found higher genetically predicted iron status to be associated with a greater risk of cardioembolic ischemic stroke (transferrin saturation: odds ratio, 1.17 [95% CI, 1.03-1.33]; serum iron: odds ratio, 1.21 [95% CI, 1.02-1.44]; total iron-binding capacity: odds ratio, 0.81 [95% CI, 0.69-0.94]). The detrimental effects of iron status on cardioembolic ischemic stroke risk remained unaffected when adjusting for CVD risk factors (all P<0.05). Additionally, we found diastolic blood pressure to mediate between 7.1 and 8.8% of the total effect of iron status on cardioembolic ischemic stroke incidence. Univariate MR initially suggested a protective effect of iron status on large-artery stroke and coronary heart disease, but controlling for CVD factors using multivariate MR substantially diminished these associations (all P>0.05). CONCLUSIONS: Higher iron status was associated with a greater risk of cardioembolic ischemic stroke independent of CVD risk factors, and this effect was partly mediated by diastolic blood pressure. These findings support a role of iron status as a modifiable risk factor for cardioembolic ischemic stroke.

Associations between genetically predicted iron status and cardiovascular disease risk: A Mendelian randomization study.

Background: Mendelian randomization (MR) studies suggest a causal effect of iron (Fe) status on cardiovascular disease (CVD) risk, but it is unknown if these associations are confounded by pleiotropic effects of the instrumental variables (IV) on CVD risk factors. We aimed to investigate the effect of Fe status on CVD risk controlling for CVD risk factors. Methods: Fe biomarker IVs (total Fe binding capacity (TIBC, n=208,422), transferrin saturation (TSAT, n=198,516), serum Fe (SI, n=236,612), ferritin (n=257,953)) were selected from a European GWAS meta-analysis. We performed two-sample univariate (UV) MR of each Fe trait on CVD outcomes (all-cause ischemic stroke (IS), cardioembolic IS (CES), large artery IS (LAS), small vessel IS (SVS), and coronary heart disease (CHD)) from MEGASTROKE (n=440,328) and CARDIoGRAMplusC4D (n=183,305). We then implemented multivariate (MV) MR conditioning on six CVD risk factors from independent European samples to evaluate their potential confounding and/or mediating effects on the observed Fe-CVD associations. Results: With UVMR analyses, we found higher genetically predicted Fe status to be associated with a greater risk of CES (TSAT: OR 1.17 [95%CI 1.03, 1.33], SI: OR 1.21 [ 95%CI 1.02, 1.44]; TIBC: OR 0.81 [95%CI 0.69, 0.94]). The detrimental effects of Fe status on CES risk remained unaffected when adjusting for CVD risk factors (all P<0.05). Additionally, we found diastolic blood pressure (DBP) to mediate between 7.1-8.8% of the total effect of Fe status on CES incidence. While UVMR initially suggested a protective effect of Fe status on LAS and CHD, MVMR analyses factoring CVD risk factors revealed a complete annulment of this perceived protective effect (all P>0.05). Discussion: Higher Fe status was associated with a greater risk of CES independent of CVD risk factors, and this effect was partly mediated by DBP. These findings support a role of Fe status as a modifiable risk factor for CES.

Mechanosensory feedback loops during chronic inflammation.

Epithelial tissues are crucial to maintaining healthy organization and compartmentalization in various organs and act as a first line of defense against infection in barrier organs such as the skin, lungs and intestine. Disruption or injury to these barriers can lead to infiltration of resident or foreign microbes, initiating local inflammation. One often overlooked aspect of this response is local changes in tissue mechanics during inflammation. In this mini-review, we summarize known molecular mechanisms linking disruption of epithelial barrier function to mechanical changes in epithelial tissues. We consider direct mechanisms, such as changes in the secretion of extracellular matrix (ECM)-modulating enzymes by immune cells as well as indirect mechanisms including local activation of fibroblasts. We discuss how these mechanical changes can modulate local immune cell activity and inflammation and perturb epithelial homeostasis, further dysregulating epithelial barrier function. We propose that this two-way relationship between loss of barrier function and altered tissue mechanics can lead to a positive feedback loop that further perpetuates inflammation. We discuss this cycle in the context of several chronic inflammatory diseases, including inflammatory bowel disease (IBD), liver disease and cancer, and we present the modulation of tissue mechanics as a new framework for combating chronic inflammation.

Time series transcriptome analysis implicates the circadian clock in the Drosophila melanogaster female's response to sex peptide.

Sex peptide (SP), a seminal fluid protein of Drosophila melanogaster males, has been described as driving a virgin-to-mated switch in females, through eliciting an array of responses including increased egg laying, activity, and food intake and a decreased remating rate. While it is known that SP achieves this, at least in part, by altering neuronal signaling in females, the genetic architecture and temporal dynamics of the female's response to SP remain elusive. We used a high-resolution time series RNA-sequencing dataset of female heads at 10 time points within the first 24 h after mating to learn about the genetic architecture, at the gene and exon levels, of the female's response to SP. We find that SP is not essential to trigger early aspects of a virgin-to-mated transcriptional switch, which includes changes in a metabolic gene regulatory network. However, SP is needed to maintain and diversify metabolic changes and to trigger changes in a neuronal gene regulatory network. We further find that SP alters rhythmic gene expression in females and suggests that SP's disruption of the female's circadian rhythm might be key to its widespread effects.

Analysis of Collective Migration Patterns Within Tumors.

Metastasis is a hallmark of cancer and the leading cause of mortality among cancer patients. Cancer, in its most deadly form, is thus not only a disease of uncontrolled cell growth but also a disease of uncontrolled cell migration. The study of tumor cell migration requires both experimental systems that are representative of the complex tumor environment as well as quantitative tools to analyze migration patterns. In this chapter, we focus on experimental and analytical methods to capture and analyze cell migration in live explants from mouse intestinal tumors. We first describe a protocol to extract and perform ex vivo live imaging on intestinal tumors in mice. We then provide a step-by-step image analysis workflow using freely available software and custom analysis scripts for extracting several parameters related to collective cell migration and cell and tissue organization.

Collective Cell Migration on Collagen-I Networks: The Impact of Matrix Viscoelasticity.

Collective cell migration on extracellular matrix (ECM) networks is a key biological process involved in development, tissue homeostasis and diseases such as metastatic cancer. During invasion of epithelial cancers, cell clusters migrate through the surrounding stroma, which is comprised primarily of networks of collagen-I fibers. There is growing evidence that the rheological and topological properties of collagen networks can impact cell behavior and cell migration dynamics. During migration, cells exert mechanical forces on their substrate, resulting in an active remodeling of ECM networks that depends not only on the forces produced, but also on the molecular mechanisms that dictate network rheology. One aspect of collagen network rheology whose role is emerging as a crucial parameter in dictating cell behavior is network viscoelasticity. Dynamic reorganization of ECM networks can induce local changes in network organization and mechanics, which can further feed back on cell migration dynamics and cell-cell rearrangement. A number of studies, including many recent publications, have investigated the mechanisms underlying structural changes to collagen networks in response to mechanical force as well as the role of collagen rheology and topology in regulating cell behavior. In this mini-review, we explore the cause-consequence relationship between collagen network viscoelasticity and cell rearrangements at various spatiotemporal scales. We focus on structural alterations of collagen-I networks during collective cell migration and discuss the main rheological parameters, and in particular the role of viscoelasticity, which can contribute to local matrix stiffening during cell movement and can elicit changes in cell dynamics.

Collective effects of human genomic variation on microbiome function.

Studies of the impact of host genetics on gut microbiome composition have mainly focused on the impact of individual single nucleotide polymorphisms (SNPs) on gut microbiome composition, without considering their collective impact or the specific functions of the microbiome. To assess the aggregate role of human genetics on the gut microbiome composition and function, we apply sparse canonical correlation analysis (sCCA), a flexible, multivariate data integration method. A critical attribute of metagenome data is its sparsity, and here we propose application of a Tweedie distribution to accommodate this. We use the TwinsUK cohort to analyze the gut microbiomes and human variants of 250 individuals. Sparse CCA, or sCCA, identified SNPs in microbiome-associated metabolic traits (BMI, blood pressure) and microbiome-associated disorders (type 2 diabetes, some neurological disorders) and certain cancers. Both common and rare microbial functions such as secretion system proteins or antibiotic resistance were found to be associated with host genetics. sCCA applied to microbial species abundances found known associations such as Bifidobacteria species, as well as novel associations. Despite our small sample size, our method can identify not only previously known associations, but novel ones as well. Overall, we present a new and flexible framework for examining host-microbiome genetic interactions, and we provide a new dimension to the current debate around the role of human genetics on the gut microbiome.

Extent of myosin penetration within the actin cortex regulates cell surface mechanics.

In animal cells, shape is mostly determined by the actomyosin cortex, a thin cytoskeletal network underlying the plasma membrane. Myosin motors generate tension in the cortex, and tension gradients result in cellular deformations. As such, many cell morphogenesis studies have focused on the mechanisms controlling myosin activity and recruitment to the cortex. Here, we demonstrate using super-resolution microscopy that myosin does not always overlap with actin at the cortex, but remains restricted towards the cytoplasm in cells with low cortex tension. We propose that this restricted penetration results from steric hindrance, as myosin minifilaments are considerably larger than the cortical actin meshsize. We identify myosin activity and actin network architecture as key regulators of myosin penetration into the cortex, and show that increasing myosin penetration increases cortical tension. Our study reveals that the spatial coordination of myosin and actin at the cortex regulates cell surface mechanics, and unveils an important mechanism whereby myosin size controls its action by limiting minifilament penetration into the cortical actin network. More generally, our findings suggest that protein size could regulate function in dense cytoskeletal structures.

A polygenic-score-based approach for identification of gene-drug interactions stratifying breast cancer risk.

An individual's genetics can dramatically influence breast cancer (BC) risk. Although clinical measures for prevention do exist, non-invasive personalized measures for reducing BC risk are limited. Commonly used medications are a promising set of modifiable factors, but no previous study has explored whether a range of widely taken approved drugs modulate BC genetics. In this study, we describe a quantitative framework for exploring the interaction between the genetic susceptibility of BC and medication usage among UK Biobank women. We computed BC polygenic scores (PGSs) that summarize BC genetic risk and find that the PGS explains nearly three-times greater variation in disease risk within corticosteroid users compared to non-users. We map 35 genes significantly interacting with corticosteroid use (FDR < 0.1), highlighting the transcription factor NRF2 as a common regulator of gene-corticosteroid interactions in BC. Finally, we discover a regulatory variant strongly stratifying BC risk according to corticosteroid use. Within risk allele carriers, 18.2% of women taking corticosteroids developed BC, compared to 5.1% of the non-users (with an HR = 3.41 per-allele within corticosteroid users). In comparison, there are no differences in BC risk within the reference allele homozygotes. Overall, this work highlights the clinical relevance of gene-drug interactions in disease risk and provides a roadmap for repurposing biobanks in drug repositioning and precision medicine.

Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration.

Intestinal organoids capture essential features of the intestinal epithelium such as crypt folding, cellular compartmentalization and collective movements. Each of these processes and their coordination require patterned forces that are at present unknown. Here we map three-dimensional cellular forces in mouse intestinal organoids grown on soft hydrogels. We show that these organoids exhibit a non-monotonic stress distribution that defines mechanical and functional compartments. The stem cell compartment pushes the extracellular matrix and folds through apical constriction, whereas the transit amplifying zone pulls the extracellular matrix and elongates through basal constriction. The size of the stem cell compartment depends on the extracellular-matrix stiffness and endogenous cellular forces. Computational modelling reveals that crypt shape and force distribution rely on cell surface tensions following cortical actomyosin density. Finally, cells are pulled out of the crypt along a gradient of increasing tension. Our study unveils how patterned forces enable compartmentalization, folding and collective migration in the intestinal epithelium.

Ethnic Differences in Iron Status.

Iron is unique among all minerals in that humans have no regulatable excretory pathway to eliminate excess iron after it is absorbed. Iron deficiency anemia occurs when absorbed iron is not sufficient to meet body iron demands, whereas iron overload and subsequent deposition of iron in key organs occur when absorbed iron exceeds body iron demands. Over time, iron accumulation in the body can increase risk of chronic diseases, including cirrhosis, diabetes, and heart failure. To date, only ∼30% of the interindividual variability in iron absorption can be captured by iron status biomarkers or iron regulatory hormones. Much of the regulation of iron absorption may be under genetic control, but these pathways have yet to be fully elucidated. Genome-wide and candidate gene association studies have identified several genetic variants that are associated with variations in iron status, but the majority of these data were generated in European populations. The purpose of this review is to summarize genetic variants that have been associated with alterations in iron status and to highlight the influence of ethnicity on the risk of iron deficiency or overload. Using extant data in the literature, linear mixed-effects models were constructed to explore ethnic differences in iron status biomarkers. This approach found that East Asians had significantly higher concentrations of iron status indicators (serum ferritin, transferrin saturation, and hemoglobin) than Europeans, African Americans, or South Asians. African Americans exhibited significantly lower hemoglobin concentrations compared with other ethnic groups. Further studies of the genetic basis for ethnic differences in iron metabolism and on how it affects disease susceptibility among different ethnic groups are needed to inform population-specific recommendations and personalized nutrition interventions for iron-related disorders.

Cancer cells in the tumor core exhibit spatially coordinated migration patterns.

In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local 'currents' and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis.This article has an associated First Person interview with the first author of the paper.

3D cell migration in the presence of chemical gradients using microfluidics.

Chemotaxis is an important biological process involved in the development of multicellular organisms, immune response and cancer metastasis. In order to better understand how cells follow chemical cues in their native environments, we recently developed a microfluidics-based chemotaxis device that allows for observation of cells or cell aggregates in 3D networks in response to tunable chemical gradients (Aizel et al., 2017). Here, we describe the methods required for fabrication of this device as well as its use for live imaging experiments and subsequent analysis of imaging data. This device can be adapted to study a number of different cell arrangements and chemical gradients, opening new avenues of research in 3D chemotaxis.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

A tuneable microfluidic system for long duration chemotaxis experiments in a 3D collagen matrix.

In many cell types, migration can be oriented towards a chemical stimulus. In mammals, for example, embryonic cells migrate to follow developmental cues, immune cells migrate toward sites of inflammation, and cancer cells migrate away from the primary tumour and toward blood vessels during metastasis. Understanding how cells migrate in 3D environments in response to chemical cues is thus crucial to understanding directed migration in normal and disease states. To date, chemotaxis in mammalian cells has been primarily studied using 2D migration models. However, it is becoming increasingly clear that the mechanisms by which cells migrate in 2D and 3D environments dramatically differ, and cells in their native environments are confronted with a complex chemical milieu. To address these issues, we developed a microfluidic device to monitor the behaviour of cells embedded in a 3D collagen matrix in the presence of complex concentration fields of chemoattractants. This tuneable microsystem enables the generation of (1) homogeneous, stationary gradients set by a purely diffusive mechanism, or (2) spatially evolving, stationary gradients, set by a convection-diffusion mechanism. The device allows for stable gradients over several days and is large enough to study the behaviour of large cell aggregates. We observe that primary mature dendritic cells respond uniformly to homogeneous diffusion gradients, while cell behaviour is highly position-dependent in spatially variable convection-diffusion gradients. In addition, we demonstrate a directed response of cancer cells migrating away from tumour-like aggregates in the presence of soluble chemokine gradients. Together, this microfluidic device is a powerful system to observe the response of different cells and aggregates to tuneable chemical gradients.

Cancer-associated fibroblasts lead tumor invasion through integrin-ß3-dependent fibronectin assembly.

Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well documented. However, it is not clear whether CAFs remodel the matrix by other means, such as degradation, matrix deposition, or stiffening. We now show that CAFs assemble fibronectin (FN) and trigger invasion mainly via integrin-αvß3. In the absence of FN, contractility of the matrix by CAFs is preserved, but their ability to induce invasion is abrogated. When degradation is impaired, CAFs retain the capacity to induce invasion in an FN-dependent manner. The level of expression of integrins αv and ß3 and the amount of assembled FN are directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin-αvß3 expression as new hallmarks of CAFs that promote tumor invasion.

Estimating mating rates in wild Drosophila melanogaster females by decay rates of male reproductive proteins in their reproductive tracts.

Female Drosophila melanogaster frequently mate with multiple males in nature as shown through parentage analysis. Although polyandry is well documented, we know little about the timing between mating events in wild Drosophila populations due to the challenge of following behaviours of individual females. In this study, we used the presence of a male reproductive protein that is transferred to the female during mating (Sex Peptide, SP) to determine whether she had recently mated. We sampled females throughout the day, conducted control matings to determine the decay rate of SP within the female reproductive tract and performed computer simulations to fit the observed proportion of mated females to a nonhomogenous Poisson process that defined the expected time between successive matings for a given female. In our control matings, 100% of mated females tested positive for SP 0.5 h after the start of mating (ASM), but only 24% tested positive 24 h ASM. Overall, 35% of wild-caught females tested positive for the presence of SP. Fitting our observed data to our simple nonhomogenous Poisson model provided the inference that females are mating, on average, approximately every 27 h (with 95% credibility interval 23-31 h). Thus, it appears that females are mating a bit less frequently that once per day in this natural population and that mating events tend to occur either early in the morning or late in the afternoon.

Integrative modelling of tumour DNA methylation quantifies the contribution of metabolism.

Altered DNA methylation is common in cancer and often considered an early event in tumorigenesis. However, the sources of heterogeneity of DNA methylation among tumours remain poorly defined. Here we capitalize on the availability of multi-platform data on thousands of human tumours to build integrative models of DNA methylation. We quantify the contribution of clinical and molecular factors in explaining intertumoral variability in DNA methylation. We show that the levels of a set of metabolic genes involved in the methionine cycle is predictive of several features of DNA methylation in tumours, including the methylation of cancer genes. Finally, we demonstrate that patients whose DNA methylation can be predicted from the methionine cycle exhibited improved survival over cases where this regulation is disrupted. This study represents a comprehensive analysis of the determinants of methylation and demonstrates the surprisingly large interaction between metabolism and DNA methylation variation. Together, our results quantify links between tumour metabolism and epigenetics and outline clinical implications.

Modes of cancer cell invasion and the role of the microenvironment.

Metastasis begins with the invasion of tumor cells into the stroma and migration toward the blood stream. Human pathology studies suggest that tumor cells invade collectively as strands, cords and clusters of cells into the stroma, which is dramatically reorganized during cancer progression. Cancer cells in intravital mouse models and in vitro display many 'modes' of migration, from single isolated cells with round or elongated phenotypes to loosely-/non-adherent 'streams' of cells or collective migration of cell strands and sheets. The tumor microenvironment, and in particular stroma organization, influences the mode and dynamics of invasion. Future studies will clarify how the combination of stromal network structure, tumor cell signaling and extracellular signaling cues influence cancer cell migration and metastasis.

Stresses at the cell surface during animal cell morphogenesis.

Cell shape is determined by cellular mechanics. Cell deformations in animal cells, such as those required for cell migration, division or epithelial morphogenesis, are largely controlled by changes in mechanical stress and tension at the cell surface. The plasma membrane and the actomyosin cortex control surface mechanics and determine cell surface tension. Tension in the actomyosin cortex primarily arises from myosin-generated stresses and depends strongly on the ultrastructural architecture of the network. Plasma membrane tension is controlled mainly by the surface area of the membrane relative to cell volume and can be modulated by changing membrane composition, shape and the organization of membrane-associated proteins. We review here our current understanding of the control of cortex and membrane tension by molecular processes. We particularly highlight the need for studies that bridge the scales between microscopic events and emergent properties at the cellular level. Finally, we discuss how the mechanical interplay between membrane dynamics and cortex contractility is key to understanding the biomechanical control of cell morphogenesis.