Understanding cell–nanoparticle interactions is critical to developing effective nanosized drug delivery systems. Nanoparticles have already advanced the treatment of several challenging conditions including cancer and human immunodeficiency virus (HIV), yet still hold the potential to improve drug delivery to elusive target sites. Even though most nanoparticles will encounter blood at a certain stage of their transport through the body, the interactions between nanoparticles and blood cells is still poorly understood and the importance of evaluating nanoparticle hemocompatibility is vastly understated. In contrast to most review articles that look at the interference of nanoparticles with the intricate coagulation cascade, this review will explore nanoparticle hemocompatibility from a cellular angle. The most important functions of the three cellular components of blood, namely erythrocytes, platelets and leukocytes, in hemostasis are highlighted. The potential deleterious effects that nanoparticles can have on these cells are discussed and insight is provided into some of the complex mechanisms involved in nanoparticle–blood cell interactions. Throughout the review, emphasis is placed on the importance of undertaking thorough, all-inclusive hemocompatibility studies on newly engineered nanoparticles to facilitate their translation into clinical application. Blood is not only the first contact for nanoparticles (NPs) administered intravenously, but also the gateway for all NPs, administered via other routes, to reach their target tissues or organs. The size of NPs allows them to easily distribute throughout the body, traverse biological barriers and enter the systemic circulation where they can readily penetrate cells . The sizThe moment NPs reach the blood system they come into direct contact with blood cells, endothelial cells and plasma proteins, where they can affect the intricate structure and critical functions of these blood components. Plasma proteins instantly adsorb to the surface of NPs to form a protein corona that significantly influences their interaction with blood components and may even lead to increased cellular activation . RecentlThe most common NPs encountered are carbon-based NPs (fullerenes and carbon nanotubes), metal NPs, ceramic NPs, semiconductors (quantum dots), polymeric NPs and lipid-based NPs . Each coHemocompatibility testing refers to the evaluation of critical interactions between foreign materials and the different components of blood to determine if any adverse effects may arise from the exposure of these foreign materials to blood . The maiHistorically, the role of erythrocytes in hemostasis was neglected and pushed aside as unimportant by researchers. However, clinical evidence argues differently and indicates that erythrocytes have a critical role in normal vascular functioning. For example, a large study with a population of more than 8000 subjects found the incidence of cardiovascular disease (CVD) to be double in the high-hematocrit group compared to the low-hematocrit group, clearly demonstrating the importance of erythrocyte count in normal hemostasis . Such obDue to their large size and abundance in the blood, erythrocytes mainly contribute to hemostasis and thrombosis through their rheological properties . HemorheThere are three main factors that can influence blood viscosity, namely hematocrit, erythrocyte aggregation and erythrocyte deformation . All thrHematocrit refers to the percentage erythrocytes as a function of blood volume and causes an exponential increase in blood viscosity in large blood vessels compared to a linear increase in small vessels where erythrocytes can only pass through in single-file . HematocIn addition to altering the hematocrit and hemorheology, a few other mechanisms have been suggested whereby hemolysis can contribute to thrombosis, including the release of erythrocyte-derived macrovesicles, activation of the complement cascade and release of free hemoglobin and heme into circulation which sequesters nitric oxide . HemolysThe method whereby hemolysis yields adverse effects on hemostasis is prompt and clearly defined, however the methods whereby NPs interact with erythrocytes to cause hemolysis has not been fully elucidated. Different mechanisms have been suggested, including interactions with the erythrocyte membrane, cellular uptake, internalization and oxidative stress. The most prominent mechanism is the direct interaction of NPs with the erythrocyte membrane which can lead to injury, detrimental morphological changes and cytoskeletal distortions . In the 2), and hydroxyapatite NPs, to be more hemolytic than larger particles [The hemolytic activity of most NPs is concentration-, structure-, size- and shape-dependent. Silica NP toxicity, for example, is directly proportional to the amount of reactive silanol groups exposed on the NP surface which depends on both the size and geometry of the NP . Accordiarticles . Indeed,Erythrocyte aggregation is largely attributed to the discord morphology and electrostatic interactions between erythrocytes. At low shear rates, erythrocytes tend to form linear face-to-face cell stacks or arrays referred to as rouleaux . Under pRecent studies have shown that cell–NP interactions can cause significant erythrocyte aggregation with damaging consequences, despite the NPs having little hemolytic activity. More and more studies are including erythrocyte aggregation, in addition to cell viability and hemolysis assays, to demonstrate the hemocompatibility of NPs. A recent study by Avsievich et al. showed that erythrocytes formed dissimilar aggregates with irregular shapes and sizes when incubated with different inorganic and polymeric NPs . Among tTwo models of aggregation, namely the ‘bridging’ and the ‘depletion’ model, are commonly used to explain the interactions between macromolecules, such as fibrinogen, and erythrocytes. In the bridging model, macromolecules adsorb onto erythrocyte membranes and ensue attractive bridging forces. Once these attractive forces overcome the normal repulsive forces between membrane surfaces, erythrocyte aggregation occurs. This model assumes that macromolecules form ‘cross-bridges’ that facilitate interactions between adjacent erythrocytes . The depDeformability refers to the ability of erythrocytes to change their geometry in response to changes in the blood flow and shear rates . This alDifferent NPs, all promising candidates in nanomedicine, have been found to interact with erythrocyte membranes in such a way that the cell’s intrinsic function in oxygen transport is threatened and its commended deformability is altered . Hemorhe3O4 magnetic NPs on PS expression in erythrocytes to evaluate their nanotoxicity. Their research showed that these extensively explored NPs cause PS externalization on the erythrocyte surface through the induction of oxidative stress. This led to unavoidable eryptosis and subsequent changes in hemorheology, erythrocyte rigidity and aggregation which can alter the thrombotic potential of blood and again highlight the great importance of ensuring NP hemocompatibility [Besides hemorheology, erythrocytes also contribute towards coagulation and blood clotting through exposure of phosphatidylserine (PS) on their membrane surfaces . In normtibility . A more tibility .Platelets are well renowned for their pivotal role in preventing blood loss by forming a hemostatic plug following vascular injury. However, the role of platelets in normal hemostasis goes beyond simple clot formation during hemorrhaging and is in fact a continuous physiological process that keeps the vascular system intact . EverydaAdhesion is a rapid process that takes place within seconds of vascular injury and entails the deceleration of rapidly moving platelets to allow adherence to the vascular wall . Owing tSilica NPs have been intensely studied for their use in nanomedicine fields including drug delivery systems, bioimaging and diagnosis. However, an increasing amount of studies have found that engineered silica NPs may hold adverse implications for vascular hemostasis that will limit their biomedical application . Saikia Platelets can be activated by numerous stimuli from various origins, including the platelets themselves that secrete activating agonists like adenosine diphosphate (ADP) and thromboxane A2, after adhesion to the endothelium. Other common endogenous stimuli include collagen, thrombin, epinephrine and complement C5b-9 . ActivatOnce platelets become activated, conformational changes allow the integrin GPIIb/IIIa to bind to various ligands  and catch passing platelets to build the platelet plug further. Interaction between this glycoprotein receptor and circulating proteins is essential to the final stage of platelet aggregation and helps stabilize the formed platelet aggregate . InhibitOne of the key determinants of NP interactions with platelets is the surface chemistry of the particles. Ionizable sialic groups on the cellular membrane of platelets confer a net negative charge to the platelet surface which can be neutralized by positive charges on the surface of NPs through cross-bridge formation. Cationic NPs can therefore facilitate platelet–platelet interactions and enhance platelet aggregation . Indeed,However, interaction through electrostatic surface charges is not the only mechanism whereby NPs lead to platelet aggregation, and each nanosystem must be investigated as an independent case . AmongstHowever, the potential in vivo toxicities that can arise from NP interactions with platelets are perhaps the most significant encouragement to carefully evaluate any possible interactions of engineered NPs with blood platelets . These pAnother cellular constituent of blood, which is often overlooked in terms of contribution to hemostasis, is represented by leukocytes. These cells are subdivided into monocytes, granulocytes and lymphocytes  and playActivated platelets regulate the proinflammatory activity of leukocytes through two mechanisms, namely degranulation, which exposes leukocytes to activating factors such as chemokines, and P-selectin exposure, which is a surface receptor that interacts with the leukocyte surface receptor PSGL-1 . This inIndeed, quiescent leukocytes play a vital role in maintaining blood fluidity by expressing the anticoagulant factors, endothelial protein C receptor (EPCR), thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI), under normal physiological conditions . HoweverThis strong interrelation between the immune and coagulation systems makes leukocyte activation an important parameter during the hemocompatibility evaluation of nanomaterials, in addition to its normal inclusion in immunotoxicity studies . FurtherOne reason for the high accumulation of NPs within leukocytes is plasma proteins that adsorb to the surface of NPs, marking them for clearance by leukocytes . This adThe proteins that preferentially bind to polymeric NPs, liposomes, carbon nanotubes and metal oxide particles are albumin, fibrinogen, apolipoprotein, complement and immunoglobulins . Each ofTo conduct a comprehensive study of the potential adverse effects of NPs on leukocytes, their influence in the characteristic functions of leukocytes must be considered. These include chemotaxis, cytokine secretion, oxidative burst and phagocytosis, which should be studied in conjunction with basic changes in leukocyte morphology and apoptosis caused by NPs . Figure Chemotaxis entails the recruitment or attraction of leukocytes to an environment with a higher concentration of toxic, sensory chemicals thereby allowing them to move towards intruding substances and phagocytose them . KojouriOne method whereby leukocytes react to foreign irritants is degranulation and the subsequent release of digesting enzymes  and other factors  that aid the removal of irritants. Recent studies also show that certain cytokines released by neutrophils have a distinct role in the resolution of the inflammatory process . NPs thaOver the years, numerous studies have proved the stimulating effect of chitosan NPs on leukocyte secretion of different cytokines including IL-18, IL-1β and granulocyte-colony stimulating factor (G-CSF) which Nolte et al. found to be upregulated by more than 40% ,93,94,95A common technique to determine leukocyte activation and degranulation is by measuring the release of elastase from neutrophils. Fang et al. compared the influence of cationic lipid and polymeric (PLGA) NPs on leukocyte elastase release and found both to induce a threefold increase in elastase, even though the lipid NPs generally had a greater effect on the neutrophils . The resAnother interesting study looked at the direct effects of drug-free poly(ε-caprolactone) lipid-core nanocapsules (LNCs) on leukocytes and found that these particles inhibit the secretory abilities of leukocytes in both basal and stimulated conditions. This suggest that even carrier particles can have distinct effects on leukocyte function and must be carefully evaluated and then used to guide their therapeutic applications to avoid discrepancies between the immunological effects of the carrier and the drug itself .2•−) and hydrogen peroxide (H2O2). These substances can react with hypochlorous acid within the cell to produce free radical species that help in the degradation of NPs and other exogenic substances. However, stressful conditions such as infection, inflammation or exposure to engineered or environmental NPs may significantly increase ROS production in leukocytes that may disturb homeostasis and cause cellular damage and tissue injury [In combination with cytokine release, oxidative burst is another method employed by leukocytes to eliminate unwanted or exogenic substances like microbes and NPs . Reactive injury .A recent study showed that quantum dots thrust neutrophils into a hyperactive state with an overproduction of ROS and a more pronounced respiratory burst compared to the control . AnotherA fascinating study done by Pujari-Palmer et al. evaluated the influence of NP morphology on the acute inflammatory response and oxidative burst. The fiber morphology caused the highest ROS production in both granulocytes and monocytes with 45% and 40% increases, respectively. This excessive free radical production proved toxic to both the phagocyte and the surrounding cells, with blebbing occurring within 1 hour of contact with the fibers. It was concluded that fibers, when compared to sheets and dots, cause the highest acute inflammation and that NP morphology, independent of charge and chemistry, has an important influence on NP–cell interactions and the subsequent induced inflammatory response .Another important observation is that a positive charge on the surface of NPs can, in addition to warranting effective cellular drug delivery, also ensue immunotoxicity through the stimulation of inflammatory immune responses. Several researchers have found that cationic liposomes can stimulate neutrophils and induce oxidative bursts in these cells ,105,106.Before either cytokine release or oxidative burst can occur, interaction between NPs and leukocytes, with subsequent activation of the immune cells, is necessary. This takes place through the initial phagocytic uptake of NPs by leukocytes, which is one of the greatest barriers to effective, targeted delivery of NPs in vivo . PhagocyIn a recent study, Ho et al. looked at the number of bacteria ingested by polymorphonuclear (PMN) cells after administration of sub-lethal doses of citrated Ag-NPs to determine the influence of these NPs on the phagocytic abilities of PMN cells. They found that Ag-NPs can significantly suppress the phagocytic ability of PMN cells and suggest that agglomeration of NPs in intracytoplasmic vacuoles restricted the expansion capability of PMN cells which might explain the suppressive phagocytic effects of Ag-NPs . HoweverOn the other hand, Babin et al. found that several NPs can, in addition to being taken up by phagocytosis themselves, directly enhance the ability of neutrophils to phagocytose other particles, including opsonized erythrocytes and fluorescent latex beads. The results revealed that NPs can act in the same way as granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance phagocytosis by a Syk-dependent mechanism. According to the authors of this study, the influence of naked NPs on the biology and functioning of neutrophils  has been greatly neglected in the past and must be carefully evaluated during future studies involving NP interactions with immune cells . 2+ ions damaged the cellular membrane in a concentration- and time-dependent manner that corresponded with the cell viability assay results. They also demonstrated a statistically significant, concentration-dependent increase in DNA damage induced by Co-NPs at both 3 and 6 μM. Lastly, they found that imbalance of the oxidative/antioxidative system may play an important role as these NPs markedly interfered with the activity of several anti-oxidant enzymes in the lysosomes [In addition to the above-mentioned studies where the effect of NPs on leukocyte functions are scrutinized, basic cytotoxic effects such as induced apoptosis, necrosis and morphological changes in the immune cells must not be overlooked. Different mechanisms have been proposed to explain the cytotoxic effect of NPs on leukocytes. These include cellular membrane damage as indicated by lactate dehydrogenase (LDH) release, imbalances between oxidant and anti-oxidant processes that can lead to ROS generation and DNA damage as well as disruption of the autophagy and endo-lysosomal pathways ,114. Colysosomes . Autophagy and lysosomal dysfunction are considered underlying mechanisms of NP cytotoxicity. NP endocytosis typically results in concentration of NPs inside lysosomes, making the lysosomal compartment the most common intracellular site of NP sequestration and degradation . DysfuncIn terms of NP hemocompatibility and NP–blood cell interactions, NP surface chemistry plays an irrefutable role . Not onlIt is widely accepted that cationic NPs will favorably interact with the negatively charged cell membrane surface, allowing these NPs to easily translocate across the cell membrane with subsequent high levels of cellular internalization . Anionic20 and negatively charged Au-NP70 to be proapoptotic towards leukocytes [Like most cell membranes, the erythrocyte surface is negatively charged and can readily interact with cationic NPs. Indeed, Han et al. found that HAP-NPs exhibited surface charge dependent erythrocyte aggregation due to electrostatic interactions between NPs and the erythrocyte membrane which resulted in unstructured agglutination. Surface functionalization of the HAP-NPs with negatively charged groups annulated the erythrocyte aggregating effects of these NPs . Plateleukocytes . SurfaceThe importance of NP geometry or shape is often overlooked in terms of nanotoxicity but can easily be exploited to improve the hemocompatibility of NPs. NP geometry determines the effective surface area of the NP, the tendency of NPs to agglomerate, the interactions of NPs with plasma proteins, the mechanism and extend of cellular uptake and therefore also the potential toxicity of NPs .However, the importance of NP geometry in terms of hemocompatibility is clearly seen in the case of amorphous silica NPs where the extent of hemolysis is concentration- and geometry-dependent. With increasing concentration, the impact of NP geometry becomes more pronounced and geometries with a low aspect ratio, such as spherical NPs, induce significant hemolysis while geometries with a high aspect ratio is associated with negligible hemolytic activity . The geoAnother critical effect of NP geometry is its influence on particle margination. As noted earlier, NP- and platelet margination is central to normal hemostasis, NP hemocompatibility and the therapeutic efficacy of NPs. NPs with complex geometries and high aspect ratios demonstrate greater margination, bringing them into close contact with the endothelial wall and platelet-dense region of capillaries . DecuzziNP geometry also defines the cellular uptake efficacy, with rod-shaped NPs internalized most readily, followed by spheres and cylinders, while cubes are not easily internalized . BartnecAccording to Park et al., NP porosity and crystallinity is a pertinent driving force behind the hemotoxic effects of certain NPs and must be carefully considered when designing new nanosized biomaterials . The difThe hemotoxicity of silica NPs is undeniable, with adverse effects witnessed towards erythrocytes, platelets and leukocytes. Yu et al. studied the impact or NP porosity on human erythrocytes and found that nonporous Stöber NPs caused an immediate onset of hemolysis while no hemolytic activity was observed for mesoporous silica NPs . On the One of the main strategies to improve the hemocompatibility of NPs is surface modification which alters chemistry and properties of the NP surface that comes into direct contact with blood. This can often mask the inherent toxicity of the NPs, extend their circulation half-life and drastically alter their interactions with blood cells . HerrmanHowever, an intriguing study done by He et al. found an unexpected negative effect of PEGylated Au-NPs on erythrocyte function. Although these NPs did not induce hemolysis, they did alter erythrocyte deformability and oxygen delivering ability through interaction with the erythrocyte membrane . The autKim et al. studied the influence of three different surface functionalized  graphene quantum dots (GQDs) on the hemorheological properties of erythrocytes. They found that carboxylated GQDs had more substantial hemolytic activity and caused more significant changes in erythrocyte deformability and aggregation than non-functionalized or hydroxylated GQDs . No clarFrom the above-mentioned studies it is apparent that surface chemistry plays an important role NP–blood cell interactions and can have a significant influence on the overall hemocompatibility of NPs. However, it must be considered that even though certain trends, such as greater toxicity of cationic NPs compared to anionic NPs, can be identified in the discussed studies, this should by no means be inferred upon all NPs. Each newly engineered NP must be reviewed as an independent, solitary case that has the potential to interact with blood cells in a previously unseen manner.The blood is a unique biological fluid with every component playing a distinct role in the effective functioning of the intricate system. This biological fluid is responsible for the transport of all NPs to their target tissues, and it is important that researchers confirm the compatibility of engineered NPs with all three cellular constituents of blood, namely erythrocytes, platelets and leukocytes. This will not only give a more accurate and complete picture of the engineered NP’s hemocompatibility but will also help ensure its optimum safety and enhance its effective translation into clinical application.Erythrocytes mainly contribute to hemostasis through hemorheological mechanisms such as pseudoplastic flow and axial margination. When NPs interact with erythrocytes they can induce hemolysis, increase erythrocyte aggregation or decrease erythrocyte deformability that will prevent erythrocytes from performing their basic functions in hemostasis. NPs can also induce phosphatidylserine exposure on erythrocyte membranes that will create a pro-thrombotic surface for platelets to adhere to and a subsequent increased risk of thrombosis . HemolysPlatelets are well-known for their critical role in hemostasis through hemostatic plug formation. Platelets, however, are also responsible for the overall regulation of hemostasis and play a vital role in immunological and inflammatory responses . NPs canLeukocytes also play a distinct role in hemostasis through the secretion of procoagulant and anticoagulant factors that link the coagulation and immune systems in an inseparable fashion . LeukocyThe surface chemistry of NPs has a pertinent influence on their hemocompatibility and interactions with blood cells. Surface charge, geometry, porosity and surface functionalization with specific polymers or functional groups are the most important surface properties of NPs in terms of their hemocompatibility and must be carefully considered during the assessment of NP hemocompatibility .Within the scope of research and development, it is critical to view each newly engineered NP as an individual case without conferring any characteristics of similar particles or the bulk material to the engineered NP. Even slight changes to an existing and well-studied particle can result in an entirely new toxicological profile that must be evaluated from scratch . ShvedovYet the value of understanding NP–blood cell interactions goes beyond improving NP hemocompatibility. This knowledge is central to new and upcoming research on synthetic blood components, artificial blood production and modified blood cell components for novel oxygen delivery to tissues affected by ischemia and trauma . All the


Combining these and other innovative imaging protocols with more sophisticated techniques to analyse ever-increasing datasets holds the exciting prospect of transforming understanding of the biological processes and temporal evolution of the ALS syndrome, and can only benefit from multicentre collaboration across the entire ALS research community.Objective markers of disease sensitive to the clinical activity, symptomatic progression, and underlying substrates of neurodegeneration are highly coveted in amyotrophic lateral sclerosis in order to more eloquently stratify the highly heterogeneous phenotype and facilitate the discovery of effective disease modifying treatments for patients. Magnetic resonance imaging (MRI) is a promising, non-invasive biomarker candidate whose acquisition techniques and analysis methods are undergoing constant evolution in the pursuit of parameters which more closely represent biologically-applicable tissue changes. Neurite Orientation Dispersion and Density Imaging , and quantitative Magnetization Transfer Imaging (qMTi) are two such emerging modalities which have each broadened the understanding of other neurological disorders and have the potential to provide new insights into structural alterations initiated by the disease process in ALS. Furthermore, novel neuroimaging data analysis approaches such as Event-Based Modeling (EBM) may be able to circumvent the requirement for longitudinal scanning as a means to comprehend the dynamic stages of neurodegeneration  Standard clinical assessment is usually adequate for diagnosis, however there is a pressing need for non-invasive neuroimaging biomarkers that may differentiate between the various phenotypes within the ALS syndrome, provide more accurate prognostic information, and monitor responses to therapeutic interventions. There is also a need for neuroimaging techniques which have the potential to interrogate the specific mechanisms of neurodegeneration, given that conventional MRI primarily aims to exclude alternative diagnoses  are required as objective biological markers of disease activity iagnoses . As suchiagnoses –15 and biagnoses , 16, 17.iagnoses , 19 whiciagnoses . Additioiagnoses , 21, 22,iagnoses , 23 haveiagnoses –26. Neve3 . For this reason, any interpretation of the signal and its origin requires some degree of “modeling.” More than one model has been proposed and each typically incorporates slightly differing mathematical assumptions to interpret and model the signal, thus providing only indirect inferences on anatomical configurations. For instance, DTI assumes that water movement will obey Gaussian properties and is widely accepted to lose consistency when neuronal fibers bend or fan out within a voxel, or where otherwise aligned fiber tracts are crossing each other , and extra-neurite water , respectively , hindered anisotropic and restricted anisotropic components. These are known as Vectively –31. The ectively   within the semisolid tissue. The latter protons do not directly contribute to the MRI signal and are “silent” in diffusion sequences  , but canf], and transverse relaxation time of the free pool [T2F]. Although qMTi is yet to be explored in patients with ALS, studies in multiple sclerosis (MS) have demonstrated reductions in f and kf, and increased T2F in acute inflammatory lesions with a subsequent return to baseline over several months , including the macromolecular pool fraction , forward exchange rate of magnetization transfer . CurrenUltimately, all modeling is an attempt to separate meaningful information from randomness. MRI techniques differentially model the signal to derive parameters that plausibly relate to tissue microstructure properties; these parameters can then be modeled further using the EBM to reveal patterns that exist within the data, but which still require human assessment and interpretation . Although the innovative imaging and data analysis techniques presented here constitute a selection of available methods or protocols, their use singly and in combination has the potential to transform our understanding of the biological processes and temporal evolution of ALS, which is likely to benefit further from multicenter collaboration across the entire ALS research community.AWB and MCG performed a review of the literature and drafted the paper. MC and PNL provided specialized expertise and critical appraisal of the article for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA structure often plays a key role in determining the function of non-coding and coding transcripts. Whereas transcript abundance can help to identify transcripts of interest in or under some conditions, the RNA structure can help to explain how the transcript might function. Hence, identifying RNA structures encoded in genomic sequence is of great interest. Since RNA molecules are often folded intensively into structures, the functional important RNA structure is typically conserved and can be more reliably detected by considering the conservation of base pairs rather than the folding of a single sequence . AlthougThe detection of RNA secondary structures throughout multiple organisms can be achieved through different strategies: 1) excising the structure from sequence-based alignments, 2) aligning the predicted structure of individual sequences, or 3) simultaneously optimizing structure and alignment. This is reviewed in reference [In reference , Ivanov Complementing the simultaneous fold and align strategy, which has a relatively high computational cost, using sequence-based alignments combined with a sliding window approach is faster. This strategy is used by Thiel et al. in  who, in Another key challenge in detecting RNA structures is searching for those who change very dramatically in size and thereby structure over evolutionary time. A prime example is telomerase RNA, which is difficult to detect through both sequence and structure homology due to its variation. Waldi et al.  address High-throughput methods for RNA structure determination  provide Structural motifs are key components in the underlying architecture of the RNA structure . In partMany RNA molecules function by binding to proteins. The interactions between the RNA and protein give rise to the specific formation of molecular structures. Even though experimental data can provide the most reliable insight, computational analyses can be sufficient to point out relevant directions and to form hypotheses. In reference , Nithin Finally, some transcripts function through their ability to form circles and tens of thousands of circular RNAs have already been reported . Detecti


The emergence and spread of infections can contribute to the decline and extinction of populations, particularly in conjunction with anthropogenic environmental change. The importance of heterogeneity in processes of transmission, resistance and tolerance is increasingly well understood in theory, but empirical studies that consider both the demographic and behavioural implications of infection are scarce. Non-random mixing of host individuals can impact the demographic thresholds that determine the amplification or attenuation of disease prevalence. Risk assessment and management of disease in threatened wildlife populations must therefore consider not just host density, but also the social structure of host populations. Here we integrate the most recent developments in epidemiological research from a demographic and social network perspective, and synthesize the latest developments in social network modelling for wildlife disease, to explore their applications to disease management in populations in decline and at risk of extinction. We use simulated examples to support our key points and reveal how disease-management strategies can and should exploit both behavioural and demographic information to prevent or control the spread of disease. Our synthesis highlights the importance of considering the combined impacts of demographic and behavioural processes in epidemics to successful disease management in a conservation context.This article is part of the theme issue ‘Linking behaviour to dynamics of populations and communities: application of novel approaches in behavioural ecology to conservation’. For e in 2008 ,6.The impact of infectious diseases on small or declining populations can arise from spillover and emergence of novel pathogens in a species (or population), or a sudden change in the epidemiology of an existing pathogen, caused by environmental change or demographic shifts ,7. RegarNetwork approaches allow the inclusion of host population structure into transmission models –15. ReceOne potential problem with using a simple network modelling approach is that it may underestimate the influence of demographic changes on the spread of infection, and therefore the consequences of diseases at different stages of population decline. For example, if host contact networks were to become more clustered and modular as population density decreased then disease might be contained within a subset of these modules and therefore be more likely to die out at lower population densities (see ). HoweveHere we synthesize some of the most recent developments in the application of social network approaches to studying wildlife disease dynamics, focusing on directly transmitted infections. We highlight the value of combining such approaches with demographic modelling to describe the temporal dynamics of infection in small or declining populations, and to inform the design of effective disease management interventions in threatened wildlife populations.2.The most effective approach to forecasting how pathogens might spread through and impact wildlife populations is to use simulation models of infection across empirically-derived networks ,14. WhenWe provide a simple example of the use of an SIR model to understand the risks posed by disease transmission in a small and fragmented population  results 3.Many network models of infection have considered pathogen transmission on a static contact network. Such an approach can provide an accurate model of real-world disease spread in some contexts, such as with fast spreading epidemics (e.g. see ), and haTheoretical models that incorporate network dynamics alongside transmission have provided insights into how changes in behaviour  can alter disease dynamics ,37. InfeUnderstanding how changes in behaviour and social networks over time influence the spread of infection will also be critical to informing disease management interventions ,34. For 4.Many threatened populations will occupy fragmented landscapes and occur in discontinuous sub-populations. Therefore, understanding the potential impact of infectious disease in these contexts may rely on integrating ideas from metapopulation dynamics, habitat connectivity and movement ecology into social network approaches. For example, low population connectivity can result in reduced epidemic coupling that promotes the global persistence of infection ,48. If ta); (ii) interconnected networks would make it possible to consider how the transmission of pathogens between multiple hosts could exacerbate the disease threat posed to endangered populations (figure 2b), or an interconnected network framework can also be used to incorporate indirect transmission via the environment . It is clear, therefore, that the multilayer network approach has considerable value for the study of wildlife disease and could be directly applicable in a conservation context through better integrating the role of alternative hosts and impact of interactions between multiple pathogens and/or parasites.Perhaps an even more powerful approach will be to integrate networks of different types of interactions across spatial scales. The advent of multilayer network analysis has resulted in a toolkit that can analyse complex systems containing multiple network types , with pofigure 2gure 2c; ); and  transmission can prevent host extinctions being caused by disease alone. As a population declines, transmission rates also decline and may reach a point where the pathogen is unlikely to be maintained within the population unless it has alternative host species, is able to persist in the environment, persists in a covert/latent state or is able to re-infect hosts  . CombineWe provide an example of a combined network-demographic model of infection . These sBox 1.Model outline. An initial population consisting of 10 groups of 10 individuals with a modular social network structure was seeded with three initially infected individuals. In a given time-step: (i) infected individuals transmit stochastically according to their contact structure and a fixed transmission probability, (ii) mortality is simulated stochastically with separate probabilities controlling the baseline (susceptible) mortality and additional , and (iii) recruitment is simulated stochastically using a fixed birth rate and the probability of being recruited into a group is inversely proportional to group size (encoding density-dependent recruitment). Once recruited, individuals' social connections were fixed, meaning that network dynamics were caused only by demographic processes, and meant that disease transmission was more density-dependent than frequency-dependent.Endemic phase. The model was allowed to run for 800 time-steps with the original conditions to ensure that disease was endemic within the population.Change phase. After 800 time-steps we set up four sets of prospective future conditions: (i) a control condition (no change to any parameters), (ii) increased pathogen virulence  mortality), (iii) increased connectivity , and (iv) increased pathogen virulence and increased connectivity. The two ‘treatments’ were designed to reflect potentially realistic future changes to the system; increased disease-induced mortality caused by increased pathogen virulence or reduced host tolerance, and increased connectivity caused by social perturbation in response to disturbance. After the change in parameters the simulation was run for 400 additional time-steps (50 repeat runs) and the results are shown in 6.In many cases, intervention may be required to prevent the spread of infection in an endangered wildlife population. Management interventions can be categorized as approaches targeted at the host, the pathogen or the environment . Using aIn many small populations, culling of hosts is often not appropriate for control of disease, especially if it is non-selective, as it may be necessary to remove too many individuals and therefore contribute to the population becoming unviable . For exaBox 2.Model outline. The initial population and model structure were the same as that described in Epidemic phase. Three individuals were initially infected, and infection was allowed to spread for five time-steps prior to detection (designed to replicate the likely lag between disease emergence and detection).Vaccination phase. At the sixth time-step individuals were vaccinated according to four different programmes: (i) no vaccination (control), (ii) 20% of individuals vaccinated at random, (iii) 20% of individuals with the highest degree vaccinated, and (iv) 20% of individuals with the highest betweenness centrality vaccinated. Vaccine efficacy was assumed to be 100%, and vaccination was assumed to prevent infection if an individual selected was susceptible.Post-vaccination phase. Subsequent to vaccination the model was run for an additional 79 time-steps . The vaccination and post-vaccination phases were repeated 50 times. The results, which reveal that targeted vaccinations are slightly more effective than random vaccination with a less variable outcome, are presented in Networks may provide critical insights into how best to target vaccination programmes in wildlife populations if individuals behave consistently over time. For example, in highly modular static networks, targeting vaccination at ‘bridge’ individuals  can greatly reduce the level of vaccination required to eradicate a disease or prevent it from spreading . SimilarIt is unlikely to always be feasible to target vaccinations based on known network positions. Quantifying empirical social networks is time-consuming and expensive , and mayMycoplasma gallisepticum in house finches Haemorhous mexicanus [Network approaches can also be used to guide management interventions aimed at modifying the environment. Supplemental feeding of wildlife, for instance, can alter transmission dynamics by altering social network structure and/or providing well-connected hubs of indirect environmental transmission . Therefoexicanus . This po7.Disease can cause or contribute to the decline and extinction of threatened species, and the threat posed may be exacerbated by anthropogenically-driven environmental change resulting in the emergence of novel pathogens or changes in the epidemiology of existing diseases. Developing an understanding of how population dynamics, population connectivity and social behaviour interact to determine the vulnerability of small and declining populations to new and existing pathogens will be crucial to recognizing and developing effective means of managing such threats. New developments in epidemiological modelling that combine social networks and demographic parameters offer a predictive framework that may be instrumental in achieving this goal.


The clinical diagnosis of amyotrophic lateral sclerosis (ALS) relies on determination of progressive dysfunction of both cortical as well as spinal and bulbar motor neurons. However, the variable mix of upper and lower motor neuron signs result in the clinical heterogeneity of patients with ALS, resulting frequently in delay of diagnosis as well as difficulty in monitoring disease progression and treatment outcomes particularly in a clinical trial setting. As such, the present review provides an overview of recently developed novel non-invasive electrophysiological techniques that may serve as biomarkers to assess UMN and LMN dysfunction in ALS patients. Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease that was first described in the 1869 by Jean-Martin Charcot  althoughThe present review will provide an overview of recently developed neurophysiological biomarkers, with emphases on novel non-invasive electrophysiological techniques used to assess UMN and LMN dysfunction in ALS patients.An important component in the diagnosis of ALS relies on clinical features of UMN involvement in the presence of progressive LMN weakness , but oftSince its original description more than 3 decades ago , TranscrA/B receptor action  is a reflection of the ease with which corticomotoneurons are excited, hence the corticomotoneuronal membrane excitability, as well as the density of UMN projections onto motor neurons . ThroughThe central motor conduction time (CMCT) time is defined by the time interval between stimulation of the motor cortex and arrival of the corticospinal volleys at the spinal motor neurons, and is inferred from the motor evoked potential (MEP) onset latency . ProlongThe cortical silent period (CSP) refers to the interruption of voluntary electromyography activity in a target muscle after motor cortex stimulation , and theIn the paired-pulse paradigm, a conditioning stimulus (CS) precedes and is utilized to modulate the effect of a second test stimulus (TS). By varying the time interval between the paired pulses  a number of parameters can be determined, using either a constant stimulus method [in which the CS and TS are kept at a constant level and MEP amplitude is evaluated ] or the Reduction or absence of SICI, which is a biomarker of cortical interneuronal inhibitory GABAergic function, has been established as an early feature of ALS Figures , correlaThe reduction in SICI has been a widely reported feature present in both familial and sporadic forms of ALS with the alterations observed as an early feature , 57–62. The use of threshold-tracking TMS may be able to uncover UMN involvement in ALS phenotypes without clinically evident UMN signs such as the flail limb variant of ALS or progressive muscular atrophy (PMA). Moreover, this technique was able to reliably distinguish between ALS and other neurological mimic conditions including multifocal motor neuropathy, spinal muscular atrophy, Kennedy's disease, peripheral nerve hyperexcitability disorders, Hirayama disease, CIDP, lead neuropathy, hereditary spastic paraparesis, as well as hereditary motor neuropathy with pyramidal features –68.SICI abnormalities using the threshold-tracking technique, appear to be the most robust diagnostic parameter that is indicative of UMN dysfunction in ALS patients , 29, 69.in vivo monitoring of cortical function and particularly, SICI may also be an effective biomarker used to monitor the effects of novel therapeutics in a clinical trial setting.More recent studies have also documented increasing cortical hyperexcitability with advancing disease indicating that intracortical inhibitory neurons become progressively dysfunctional in ALS Figure  70). Re. Re70). Objective assessment of LMN dysfunction, utilizing neurophysiological techniques, appear to be more sensitive than clinical assessments , 78. ConAs such, various methods to approximate the number of motor units innervating individual muscles, including motor unit number estimation (MUNE), and motor unit number index (MUNIX), may potentially represent valuable biomarkers of LMN degeneration. Since the development of the first MUNE technique in 1971 , there hThe motor unit index (MUNIX) technique is a method designed to express the number of functioning motor units within a muscle as an index, instead of providing a direct measure of their absolute numbers. It is based on patients performing a voluntary contraction at various intensity levels and surface interference patterns being captured and decomposed to obtain a normalized motor unit size, which is then in turn divided into the maximal CMAP value to obtain the MUNIX , 89, 90.Recent studies using different MUNE methods have demonstrated potential utility for assessing disease progression in ALS patients as reflected by a progressive linear decline in MUNE counts , 91–94. The neurophysiological index (NI) is a potential electrophysiological biomarker in assessing lower motor neuron loss in ALS . Using aThe split-hand sign is documented as an early and specific clinical feature in patients with ALS that is not characteristic in other commonly encountered clinical mimics , 100. ItElectrical impedance myography (EIM) is a novel non-invasive form of testing to provide quantitative information on neuromuscular disorders that may be useful and reliable in assessing longitudinally the severity of a disease process –107. EIMAmyotrophic lateral sclerosis remains a devastating neurodegenerative disorder with a poor prognosis, much of which is attributable to frequent delays in diagnosis, an incomplete understanding of the underlying pathophysiological mechanisms, and the current lack of effective disease-modifying treatment available. As such, there is a critical need to devise accurate and reliable biomarkers to address the above shortfalls in current ALS management. The current review has presented recent developments in novel neurophysiological biomarkers that are able to effectively interrogate upper and lower motor neuron dysfunction and characterize their change over time with disease progression, thereby exhibiting the potential to improve diagnosis, as well as facilitating in the prognosis and monitoring of the effects of future therapeutic agents in a clinical trial setting.WH drafted manuscript and made all necessary edits and revisions as well as obtained required copyright permissions for figures. TD, SV, and MK revised and edited manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Children exposed to moderate levels of alcohol during pregnancy show growth deficits and intellectual and behavioral problems similar to, although less severe than, those found in children with fetal alcohol syndrome. Research has begun to examine the extent to which these problems affect the child’s ability to function on a day-to-day basis at school and with peers. Findings indicate that “ moderate” drinking has much more impact on child development when the mother consumes several drinks in a single day than when she drinks the same quantity in doses of one to two drinks per day over several days. Moderate drinkingSeveral studies have found that children exposed to alcohol during pregnancy at lower levels than FAS children experience moderate intellectual and behavioral deficits that resemble those of FAS children but on a less severe level . Most ofThis article summarizes the effects of moderate prenatal alcohol exposure on children’s growth, intellectual competence, and behavior as well as discusses research findings regarding the impact of these effects on children’s general ability to function. The article also investigates the doses and patterns of moderate drinking during pregnancy that have been linked to developmental problems in offspring.Children whose mothers drink moderately during pregnancy exhibit growth deficits as well as intellectual and behavioral impairment.a), and several studies have reported an association between prenatal alcohol exposure and slower-than-normal growth during the first 6 to 8 months after birth   and on tIn addition to the intellectual and attention deficits found among non-FAS alcohol-exposed children, researchers also have documented behavior problems that resemble but are less severe than those found among FAS children. The socialization deficits associated with FAS include poor interpersonal skills and an inability to conform to social conventions . StreissRelatively limited information is available regarding behavioral effects in alcohol-exposed non-FAS children. Using the Achenbach Child Behavior Checklist-Teacher’s Report Form (TRF), average effect may be small, researchers have recently begun to examine whether the effects of moderate drinking are severe enough in certain children to affect their ability to manage on a day-to-day basis at school, home, and with peers.The effects of moderate prenatal alcohol exposure on children’s intellectual performance and behavior have been established. When examining the results of psychological tests, however, children with ARND often appear to have relatively subtle impairments . Although the To evaluate whether a specific deficit is severe enough to impair a child’s day-to-day function, researchers must establish criteria to indicate which test scores are low enough to be functionally significant . For example, an IQ below 70 indicates mental retardation, but little consensus exists regarding the functional importance of a 5- or 10-point decrement when scores fall within the normal range. Moreover, for most psychological tests, such as those focusing on attention, no criteria for functional significance have been established, limiting the ability to evaluate the effect of ARND on children’s everyday function. In the absence of established criteria, To determine whether the ARND children in one study had deficits that could be considered functionally significant, the researchers evaluated the children’s performance at approximately 12 months of age on four measures: (1) the Bayley Mental Index, which assesses simple fine motor and prehensile coordination  and imitation of a model; (2) the Bayley Psychomotor Index, which assesses walking and balance; (3) elicited play, which determines the most complex play with toys a child can imitate ; and (4) cognitive processing speed, a measure of how quickly a child processes information, which is assessed by measuring the average length of the glances the child directs at an object or photograph . ChildreThe researchers then examined the association between the mothers’ alcohol consumption and the rates of functionally significant impairment in the children of both younger and older mothers see . For theThese findings are consistent with data from case studies of multiparous alcohol-abusing mothers of FAS children, which have shown that each successive child is almost always more severely impaired than the previous one. Similarly, animal experiments in which the doses of prenatal alcohol exposure were carefully controlled have documented markedly greater impairment in offspring born to older mothers.Most existing data on the effects seen in non-FAS alcohol-exposed children have been based on each of the mother’s alcohol intake averaged across her pregnancy. However, animal experiments indicate that this average is probably misleading, because ingesting a given dose of alcohol over a short time period  generates a greater peak blood alcohol concentration (BAC) and greater neuronal and behavioral impairment than does ingesting the same dose gradually over several days .The authors reanalyzed the infant data to examine the effects of dose  and frequency  on developmental outcome . ChildreNinety-one percent of the mothers in the study drank infrequently . Among the 11 infants in the sample whose mothers drank frequently  during pregnancy, functional impairment was seen in 4 of the 5 infants whose mothers averaged at least 5 drinks per occasion but in none of the 6 infants whose mothers drank frequently at lower levels (ranging from 1.3 to 4.6 drinks per occasion). The one mother who drank daily was an alcoholic, and her infant was born with FAS. However, the infant of the frequently drinking mother who averaged only 1.3 drinks per occasion showed no evidence of neurodevelopmental impairment. The median drinking pattern of the mothers of the 20 children in Several studies have found that moderate prenatal alcohol exposure has statistically significant effects on children’s cognitive and behavioral development. Using the IOM-proposed terminology, many of these children would be diagnosed as having ARND. ARND differs from FAS, however, in that FAS is characterized by reduced IQ scores and more severe socialization problems. Nevertheless, evaluations of the specific domains in which deficits occur reveal important parallels between FAS and ARND. In the cognitive domain, arithmetic, attention, and working memory are most severely and consistently affected in both disorders. In the behavioral domain, both disorders are marked by increased impulsivity, aggression, and social problems. Researchers are only beginning to address the importance of these deficits for the day-to-day functioning of the ARND child. The aforementioned data suggest that although some non-FAS alcohol-exposed children are only minimally affected by prenatal alcohol exposure, other more susceptible children are impaired to a degree likely to interfere with their ability to function normally. Detailed information about the functional significance of each of the deficits found among ARND children is needed to fully understand the implications of prenatal alcohol exposure for child development.More attention also should be devoted to determining the specific drinking levels and patterns associated with functionally significant developmental impairment. Research has documented functionally significant deficits in infants whose mothers drank, on average, five or more drinks per occasion once or twice per week. Although considered excessive for a pregnant woman, this level of drinking falls short of the rate usually associated with having a serious drinking problem. Given the marked individual differences in alcohol metabolism and fetal vulnerability, five drinks per occasion may be too high a threshold for many women. Functional deficits may occur in some children who are repeatedly exposed prenatally to only three or four drinks per occasion, especially if the alcohol is consumed on an empty stomach. In evaluating the risk associated with exposure to environmental and food contaminants, a safety margin is usually incorporated to allow for individual differences in sensitivity. Where human data are available, a safety factor of 10 is used for this purpose . Using tThese data demonstrate a statistically significant association between moderate drinking during pregnancy and children’s adverse neurobehavioral outcomes. The data also demonstrate that these effects may be severe enough in some children to affect their day-to-day functioning. Although children exposed to moderate levels of alcohol during pregnancy are not mentally retarded, they show attention deficits and behavioral problems that are similar to, although less severe than, those found in FAS children. These data also demonstrate that as with most neurotoxicants, the human organism is markedly more vulnerable to alcohol exposure during the prenatal period than at any other point in the lifespan. Because of this heightened vulnerability and the apparently long-term, permanent nature of alcohol-related deficits, the best advice continues to be abstinence or, at most, minimal consumption of alcohol during pregnancy.


Urban health indicator (UHI) tools provide evidence about the health impacts of the physical urban environment which can be used in built environment policy and decision-making. Where UHI tools provide data at the neighborhood (and lower) scale they can provide valuable information about health inequalities and environmental deprivation. This review performs a census of UHI tools and explores their nature and characteristics  to increase understanding of their potential use by municipal built environment policy and decision-makers. We searched seven bibliographic databases, four key journals and six practitioner websites and conducted Google searches between January 27, 2016 and February 24, 2016 for UHI tools. We extracted data from primary studies and online indicator systems. We included 198 documents which identified 145 UHI tools comprising 8006 indicators, from which we developed a taxonomy. Our taxonomy classifies the significant diversity of UHI tools with respect to topic, spatial scale, format, scope and purpose. The proportions of UHI tools which measure data at the neighborhood and lower scale, and present data via interactive maps, have both increased over time. This is particularly relevant to built environment policy and decision-makers, reflects growing analytical capability and offers the potential for improved understanding of the complexity of influences on urban health . The relation between urban health indicators and health impacts attributable to modifiable environmental characteristics is often indirect. Furthermore, the use of UHI tools in policy and decision-making appears to be limited, thus raising questions about the continued development of such tools by multiple organisations duplicating scarce resources. Further research is needed to understand the requirements of built environment policy and decision-makers, public health professionals and local communities regarding the form and presentation of indicators which support their varied objectives.The online version of this article (10.1007/s11524-018-0228-8) contains supplementary material, which is available to authorized users. Both the global increase in non-communicable diseases and improved understanding of the social determinants of health have contributed to an increased awareness of the influence of built environment policies on health and wellbeing –3. EstimBenchmark progress at local, regional, national or international levelsSet targets for improvementDemonstrate performance to residentsPrioritise funding allocation/bid for fundingAct as an ‘early warning’ of potential problemsInvolve the public in prioritisation and definition of policy goalsIdentify strengths and weaknesses in a communityUrban health indicator (UHI) tools seek to provide built environment policy and decision-makers with information to develop policies, make decisions and monitor impacts. These metrics can demonstrate the impact of the built environment on health and expose health inequalities within cities. Urban health is a complex system with many interconnected parts –10 whichThe intended use of indicator tools is likely to inform their composition and characteristics, elements which are often represented in a taxonomy . TaxonomFirst, indicator researchers have tended to focus on the development and validation of indicator tools, rather than investigating how such tools are used by policy- and decision-makers . The proSecond, despite the large amount of research on indicator development, there is still a lack of consensus on how to measure the urban environment’s impact on health and related concepts. Standardising the development of urban health indicators is a topic of ongoing debate , 24. DesTo our knowledge, there exist three reviews of relevant indicators. The Prasad et al. systematic review of urban health metrics highlighted the lack of available data for metrics in low and middle income countries and questioned the translation of evidence gained through using such metrics into policy and decision-making . RothenbThis systematic review examines a specific type of indicator compilation which could inform municipal built environment policy and decision-makers about the social determinants of health, defined as ‘urban health indicator tools’. The review has two distinct parts, as outlined in a previously published protocol , 29. ParSystematic Reviews including a completed PRISMA-P checklist ndicators are used to simplify information about complex phenomena, such as sustainable development or, in this case, QOL, in order to make communication easier and quantification possible’ . This waThree UHI tools referred to the complex process of policy and decision-making, sometimes in recognition that indicators may not inform policy due to this complexity. For example, Hunt and Lewin commented that ‘policy action may not easily follow the identification of environmental health problems [through indicators], which is due both to the large numbers of other factors that also affect health and to the complexity of the policy process’ .UHI tools rarely explained strategies used to help account for complexity. Feneri et al. used Multi-criteria Decision Analysis to ‘conceptualize the complex issue of evaluating quality of life’ . They spintend to inform policy and decision-making, yet it is unclear whether a significant number achieve this aim. Third, we found that the majority of UHI tools are evidence-based and therefore provide a potential route from research through to policy. Fourth, we have explored the nature of how UHI tool methodologies address complexity, identifying specific strategies. Finally, we have shown that there is a degree of similarity in the domains measured across UHI tool topics.This review identified great diversity in the purpose and characteristics of urban health indicator tools making it difficult to draw simple conclusions. However, the review generated novel findings about UHI tools as they relate to the needs of built environment policy and decision-makers. Our taxonomy demonstrates the importance of considering users’ needs when developing indicator tools to ensure they can be used to support built environment practitioners. Our main findings are summarised here with implications discussed below. First, we found that the proportion of tools with data aggregation/measurement at the neighborhood and lower scale and presentation of data via digital interactive maps have both increased over time. Second, we highlighted that the majority of UHI tools In comparison to existing reviews of indicators which measure the urban environment’s impact on health, this review casts a wider net by including measures of health, QOL, liveability, wellbeing, and walkability/physical activity. This has enabled a detailed analysis of a large number of indicator tools and their respective characteristics, including 8006 individual indicators. The review was limited to English language publications, potentially excluding many UHI tools from non-English language countries. The method used to classify whether a tool had been used beyond research was simplistic and may have underestimated those tools which were indeed used beyond research.The increasing number of UHI tools with data aggregation at neighborhood or lower scale is of significance for built environment policy and decision-makers. In 2002, Talen questioned the usefulness of indicators to inform urban planning because the majority were comparing cities (inter-city) rather than neighborhoods (intra-city) . NeighboA number of tools  did not explain the evidence used for indicator selection, creating questions over the suitability of their use in policy and decision-making. Although the validity of individual indicators (association between exposure and outcome) was not assessed by this review, the range of methods for selecting indicators demonstrated that this process was not always informed by evidence about environmental exposures and health effects. Badland et al. called for further research about the validity of indicators within UHI tools  . HoweverThe distinction of whether UHI tools are used beyond research is of interest when considering transfer of research knowledge to practitioners. We were unable to confirm whether 45/120 tools (37.5%) which intended to inform policy/decision-making achieved this aim. There could be a delay between research and use or this may also point to other knowledge translation issues. UHI tool producers should consider the needs of their audience and may benefit from wider strategies to increase research use by policy and decision-makers (see ). The apStandardisation of UHI tools may be aided by our finding that there is significant overlap across domains measuring health-related topics such as QOL, liveability and wellbeing. Rothenberg et al. also found similarities in indicator domains across urban health indicator compilations . GuidancA standardised set of global indicators would mean that rather than developing new UHI tools, researchers and practitioners could choose from an internationally published set of evidence-based indicators. Local selection of indicators would likely be based on data availability, health priorities and community opinion. The WHO’s Urban Health Index provides methods for local public health data analysts to produce local indices (including instructions for mapping the results) , 53. SucWhilst some indicator producers recognised that indicators could help explain complex phenomena, other authors noted that they may not be effective at influencing a complex policy and decision-making process. This topic will be explored further in a subsequent paper related to this systematic review which will synthesise qualitative data from studies exploring the use of UHI tools in the built environment policy and decision-making process.Observing the similarity across indicator measures, there is a question about whether some data are included simply because they are easy to measure (or commonly measured as a part of routine statistics), whilst other more difficult topics are excluded. For example, although noise is known to impact multiple health outcomes , it is lESM 1(DOCX 122 kb)


Streptococcus pneumoniae. In this mSphere of Influence article, she reflects on how the paper entitled “Role of the manganese efflux system mntE for signalling and pathogenesis in Streptococcus pneumoniae”  homeostasis in  Streptococcus pneumoniae. In this mSphere of Influence article, she reflects on how the paper entitled “Role of the manganese efflux system mntE for signalling and pathogenesis in Streptococcus pneumoniae”  has impacted her thinking and research direction toward investigating the molecular underpinnings of why and how bacteria maintain optimal intracellular Mn levels.Julia E. Martin works in the field of metals in biology, with a focus on manganese (Mn) homeostasis in  Bacteria must acquire these essential metal ions exogenously in sufficient quantity to fulfill their cellular demand. This metal essentiality is exploited by the mammalian host immune system through nutritional immunity, in which transition metal ions are sequestered by the host in an effort to limit growth of invading bacteria. Historically, many studies have focused on metal uptake and other mechanisms of bacterial adaptation to host-imposed metal starvation. This is particularly true of Mn, an important micronutrient that is largely recognized for its role in oxidative stress resistance and in enhancing virulence of bacterial pathogens. Although elusive, it was revealed almost half a century ago that excess Mn can impair bacterial growth . Today,S. pneumoniae 1552 , encoding a cation diffusion transporter. Mn became the prime suspected substrate after metal cation sensitivity profiles and intracellular metal composition measurements obtained by inductively coupled plasma-mass spectrometry of null-mntE mutants showed reduced growth and elevated Mn concentrations, respectively, compared to unstressed wild-type cells. Mn isotopic uptake assays further demonstrated that the mntE mutant deficient in metal export accumulates significantly higher levels of intracellular Mn, consistent with the hypothesis that MntE functions in Mn efflux. A role for Mn efflux in host pathogenesis was observed by assessing the virulence of the mntE mutant in the intranasal murine mouse model of infection. Given that the mntE mutant was attenuated, Rosch et al. analyzed the global transcriptome of pneumococcus grown under various conditions of Mn stress. The transcript profiles obtained were used as a mechanism to elicit which metabolic pathways might be important for pneumococcus in establishing disease. Overall, these data highlight that perturbations in Mn homeostasis may alter multiple cellular processes, including iron (Fe) uptake. Intracellular cross talk between Mn and Fe homeostasis is not unprecedented. These two metal ions share many similar properties, and it was previously proposed that the Mn/Fe ratio may serve as an effective reporter for microbial resistance to host defenses during pathogenesis. The discoveries presented in the article by Rosch et al. support further research pursuits in the development of alternate metal-based antimicrobial therapeutics as a means to strategically treat infectious diseases.Briefly, on the basis of sequence homology, the authors of that article identified a conserved gene sequence, The article by Rosch et al. was published during my Ph.D. research and has since impacted my thinking and research direction toward investigating the molecular underpinnings of why and how bacteria maintain optimal intracellular Mn levels. Similarly to other transition metals, intracellular Mn in bacteria is buffered by the absolute binding affinities of the metallobiomolecules that regulate the expression of the downstream genes which often encode metal efflux transporters like MntE. These regulatory metallobiomolecules sense bioavailable “free” Mn, which is a weakly competitive metal ion compared to other transition metals. Although collaborative efforts between microbiologists, structural biologists, and biophysical chemists have provided insight into the fundamental parameters that influence intermetal competition and mismetallation of these metallobiomolecules, relatively little information is available regarding the molecular mechanisms of how intracellular Mn is trafficked to target metalloenzymes in cells and why cells must avoid cellular Mn toxicity. Transport mutants incapable of Mn efflux permit researchers like myself to perturb Mn homeostasis and examine its cellular effects. Although my research is focused on the single metal ion Mn, I am mindful that Mn is part of a bigger metal milieu. It is apparent that intermetal competition or cross talk occurs between Mn and several other metal homeostasis pathways, including Fe, Zn, and Cu. This competition becomes substantially more complex at the host-pathogen interface, in which bacteria respond differently to distinct host responses. Continued collaborative efforts and interdisciplinary approaches are necessary to advance our understanding of metal cross talk within and among organisms. I foresee that transcriptomic profiles, such as those presented in the article by Rosch et al., and evolving proteomic approaches will play a big role in this area in the future.


Wolbachia are alpha-proteobacteria known to infect arthropods, which are of interest for disease control since they have been associated with improved resistance to viral infection. Although several genomes for different strains have been sequenced, there is little knowledge regarding the relationship between this bacterium and their hosts, particularly on their dependency for survival. Motivated by the potential applications on disease control, we developed genome-scale models of four Wolbachia strains known to infect arthropods: wAlbB , wVitA (Nasonia vitripennis), wMel and wMelPop (Drosophila melanogaster). The obtained metabolic reconstructions exhibit a metabolism relying mainly on amino acids for energy production and biomass synthesis. A gap analysis was performed to detect metabolic candidates which could explain the endosymbiotic nature of this bacterium, finding that amino acids, requirements for ubiquinone precursors and provisioning of metabolites such as riboflavin could play a crucial role in this relationship. This work provides a systems biology perspective for studying the relationship of Wolbachia with its host and the development of new approaches for control of the spread of arboviral diseases. This approach, where metabolic gaps are key objects of study instead of just additions to complete a model, could be applied to other endosymbiotic bacteria of interest. Wolbachia pipientis, an obligate intracellular bacteria known to provide pathogen-blocking capabilities to its host, has been used to this end. Wolbachia-infected mosquitoes have been released into the environment as a strategy for controlling the mosquito population and hence the spread of arboviral-derived diseases. However, there is little knowledge regarding the specific interactions that occur between Wolbachia and its host, particularly those associated with its obligate intracellular nature. In this work we studied Wolbachia endosymbiosis from a systems biology perspective. By analyzing the gaps in the metabolic networks of four Wolbachia strains we were able to identify key interaction points between this intracellular bacteria and its host, which play a crucial role in their metabolic relationship. This approach focuses on what is missing to allow unveiling new information regarding the interplay between interacting metabolic networks, and it is especially useful in the case of intracellular bacteria where experimental information is scarce. This additional insight on the metabolic interactions between host and parasite hold great potential for the development of arboviral disease control strategies.The expansion of the geographic distribution of arthropods has led to an increase in the number of infections of Zika and Dengue. This motivates the search for new alternative approaches for disease control.  Wolbachia are obligate intracellular alpha-proteobacteria, member of the Rickettsiales order known to infect nematodes and arthropods by developing diverse complex interactions with their hosts, such as supplementation with vitamins, cytoplasmic incompatibility and parthenogenesis [ogenesis –4.Wolbachia has shown to impart a fitness advantage to arthropod host such as better reproductive traits or improved resistance to virus infection [wMelPop [The nature of these interactions is influenced by the strain and organism involved and they have been reviewed extensively . In somenfection . Particunfection , riboflanfection , to path[wMelPop .Wolbachia interactions with their hosts driven by potential applications in development of novel control strategies for the spread of arbovirus-derived diseases such as Yellow fever, Zika, Chikungunya and Dengue [Wolbachia strains have been sequenced [Several studies have been developed towards the understanding of d Dengue . Genomesequenced ,10–12 anequenced  in orderWolbachia are known to have a reduced genome size as a result of their adaptation to depend on other organism to their survival. Thus, it is expected that they exhibit a small and rather incomplete metabolic network, as it has been previously observed for other endosymbiotic bacteria [Wolbachia metabolism could be achieved by analyzing a representation of their wide metabolic capabilities given by genome-scale models.bacteria ,14. We hGenome-scale models (GEMs) have emerged as a powerful tool for studying cellular metabolism since they provide a global representation of all biochemical transformations that could be carried out by a specific organism based on their genome annotation .Wolbachia strains found in insect arthropods, wAlbB  [wVitA (from Nasonia vitripennis) [wMel [wMelPop [Drosophila melanogaster). We hypothesized that the analysis of the metabolic gaps in the curation stage could reveal potential candidates to explain the endosymbiotic nature of Wolbachia as shared metabolites between both species.In this work, we developed genome-scale models for four opictus) , wVitA (ipennis) , wMel [1s) [wMel  and wMel[wMelPop  , wVitA (Nasonia vitripennis) and wMel and wMelPop (Drosophila melanogaster) genomes were used to generate draft genome-scale models using modelSEED [Wolbachia pipientis' symbiotic relationship with its hosts.Four strains of odelSEED . Gap filWolbachia based on phylogenetic information and predicted pathways in the annotation process [Wolbachia, Rickettsia and Escherichia coli [We proposed a draft biomass composition for  process . The obthia coli –19. Comphia coli .Wolbachia metabolic reconstructions was achieved using COBRApy [Integration of the four obtained  COBRApy . Gene pr COBRApy .Wolbachia strains were selected based on the following characteristics: wVitA due to its reported ability to cause cytoplasmic incompatibility, wAlbB was known to infect the mosquito Aedes albopictus, wMel due to its reported effects on pathogen virus blocking and wMelPop because of its pathogenicity to their host. Based on their genome annotation, four genome-scale models were obtained , a component known to be part of the protein fraction of the insect cuticule and wMelPop includes 2 unique reactions associated with tetrahydrofolate metabolism, which are not associated with its pathogenic nature and present gaps of its metabolic nature. We then propose that wMelPop detrimental effect on its host lies in regulatory processes that determine cell density inside its host rather than in specific metabolic reactions that could be associated with pathogenicity.On the other hand, wMel strain, which has been described to rely mainly on amino acids to support their energetic requirements, having limited carbohydrate synthesis capacity and being unable to transport ATP directly from its host and to synthesize Lipid A [Wolbachia metabolic reconstruction exhibiting a complete glycolysis pathway starting from fructose 6 phosphate towards phosphoenolpyruvate and a complete TCA (tricarboxylic acid) cycle.We analyzed previously reported metabolic features for the  Lipid A ,22. Our Wolbachia presents a highly conserved pentose phosphate pathway (PPP) for sugar nucleotide synthesis, contrary to what has been reported for close organisms such as Rickettsia [Additionally, ckettsia  and an aribD reported to be found in wMel, wRi, wHa, and wAu [The riboflavin synthesis pathway, another putative symbiosis-determining pathway, is highly conserved with one missing step that is common to all studied strains, FMN hydrolase/5-amino-6-(5-phospho-D-ribitylamino) uracil phosphatase. However, this reaction has been found to be associated with the gene  and wAu . TherefoWe also analyzed the biomass reaction given by modelSEED which is generated from phylogenetic information and the predicted pathways based on their genome annotation . ParticuWolbachia is closer to the Rickettsia genus rather than Escherichia coli, the exemplary Gram-negative species. Subsequently, the biomass composition was modified to include specific phospholipid and fatty acid composition derived from studies made in Escherichia coli and particularly in Rickettsia prowazekii [However, owazekii –19, in oWolbachia and its hosts . The obtained metabolic reconstruction is able to synthesize S-adenosyl methionine (SAM) from methionine but includes gaps associated with SAM metabolization for heme synthesis, which was initially reported to also be missing in the nematode infecting strain wOo [Wolbachia [ pathway , contrarrain wOo  but thenolbachia .Wolbachia and Rickettsia have also been identified. The obtained Wolbachia model has reactions without associated genes involved in the synthesis of isopentenyl phosphate (IPP) and 4-hydroxybenzoate from lanosterol  whilst Wolbachia conserves metabolic pathways for metabolic provisioning to its hosts.Alternative strategies to study metabolism of endosymbionts have been published recently. Driscoll et al., (2017)  reconstrWolbachia on its host for their survival, from annotation gaps originated from experimental errors in the process of sequencing and annotation of the DNA of the analyzed strains. Particularly, of the 1,174 genes found originally in the annotation process for wMel [Wolbachia gene annotation, there is a considerable number of genes associated with regulatory processes that are not represented by this approach. Widely studied Wolbachia genes as the cytoplasmic incompatibility genes  [There is no available automatic method to distinguish between true annotation gaps, which have biological meaning and correspond to gene losses that explain the dependency of for wMel , only 31A, cifB)  and ankyA, cifB)  are not We propose that the inclusion of information of multiple strains could help minimize this problem and provide the scientific community with a robust tool for metabolic studies of this intracellular bacteria. Additionally, this approach, where metabolic gaps are key objects of study instead of just additions to complete a model, could be expanded for other endosymbiotic bacteria of interest.Wolbachia genome-scale model for four strains known to infect arthropods. We propose that the metabolic gaps present in this model are key to study metabolic interactions with their hosts and could potentially influence processes that lead to cytoplasmic incompatibility and pathogen blocking.In this work we present Wolbachia metabolism is characterized by the import of amino acids for energy and growth, and also particularly the import of isopentenyl phosphate and 4-hydroxybenzoate for ubiquinone 8 biosynthesis. Although, Wolbachia share several characteristics with Rickettsia, the metabolism of the analyzed Wolbachia strains presents highly conserved pathways for nucleotide synthesis and riboflavin, which can be ascribed to their mutualistic rather than parasitic behavior.Wolbachia metabolism to improve the understanding on the relationship with its host and the development of new approaches towards the control of spread of arboviral diseases.We have provided a systems biology analysis framework to study S1 FileWolbachia genome-scale models in SBML for the four analyzed strains wMel, wMelPop, wAlbB, and wVitA.(ZIP)Click here for additional data file.S2 FileWolbachia genome-scale models.Spreadsheet (xlsx format) listing all the included reactions and gene associations for the four (XLSX)Click here for additional data file.S3 FileSpreadsheet (xlsx format) listing new annotated genes and filled metabolic gaps derived from this work.(XLSX)Click here for additional data file.


Hormones of the hypothalamic-pituitary-gonadal (HPG) axis that regulate reproductive function have multiple effects on the development, maintenance and function of the brain. Sex differences in cognitive functioning have been reported in both health and disease, which may be partly attributed to sex hormones. The aim of the current paper was to provide a theoretical review of how sex hormones influence cognitive functioning across the lifespan as well as provide an overview of the literature on sex differences and the role of sex hormones in cognitive decline, specifically in relation to Alzheimer’s disease (AD). A summary of current hormone and sex-based interventions for enhancing cognitive functioning and/or reducing the risk of Alzheimer’s disease is also provided. Hormones of the hypothalamic-pituitary-gonadal (HPG) axis that regulate reproductive function are also potent neurosteroids and have multiple effects on the development, maintenance and function of the brain . Sex difSex steroid hormones, including estrogens, progesterone and androgens are regulated by the HPG axis. While the neuroendocrine control mechanisms are complex, a simplified account of the HPG axis suggests that the hypothalamus releases gonadotropin-releasing hormone (GnRH) which stimulates the anterior pituitary to produce and secrete gonadotropins, including luteinizing hormone (LH) and follicle-stimulating hormone (FSH), into the bloodstream. LH travels through the blood stream to stimulate the release of the sex steroids, androgens and estrogens, from the gonads. These sex steroids complete the endocrine feedback loop by inhibiting the release of GnRH. It is well known that the roles of gonadal hormones extend beyond the regulation and development of reproductive functions. Evidence accumulated over the last four decades, from animal studies, cell culture studies and studies in humans support the influence of sex hormones on brain processes involved in cognitive functioning.There is a relatively large knowledge base, primarily derived from rodent studies, demonstrating that estrogens can facilitate higher order cognitive functioning via a range of likely interrelated functions that involve multiple signalling pathways . EstrogeLike estradiol, progesterone exerts trophic effects on brain development throughout adolescence and adulthood . EstrogeGonadotropin levels, including LH and FSH have also been connected with cognitive functioning and implicated in cognitive decline ,21. EndoSex differences in cognitive functioning have been widely reported across a number of species, including healthy adults. On average, males outperform females on spatial abilities and females outperform males in verbal abilities. It is important to point out that these sex differences are based on averages and do not apply to all individuals. In addition to these differences there are many cognitive domains without sex differences (for a review on ‘gender similarities and differences’ see ). SocietThe effect of sex hormones on cognition is thought to begin in utero when brain development diverges in males and females in response to androgen production. Human males have a surge in fetal testosterone between weeks 8 and 24 of gestation  . During Prenatal androgens are hypothesised to influence lateralisation of brain and behaviour, with males developing a right hemisphere dominance that may aid performance on spatial tasks . SeveraFollowing the influence of prenatal sex hormones on behavioural development, the second wave of sex hormone-dependent neural organisation is during adolescence . FollowiClear and consistent evidence linking pubertal hormones or pubertal timing to cognitive functioning is lacking (as reviewed by Berenbaum and Beltz ). Beltz For reproductive-age females, menstrual cycle studies and contraceptive pill studies provide an opportunity to further study how endogenous and exogenous levels of sex hormones may effect cognition. For naturally cycling women, the average menstrual cycle length is somewhere between 25 and 31 days . TypicalIn their comprehensive review of the topic, Sundstrom Poromaa  identifiContraceptive pill studies provide an opportunity to explore the influence of exogenous hormones on cognitive functioning in reproductive-age females. Oral contraceptives contain synthetic analogues of estrogen and progesterone. The most commonly used estrogen analogue is ethinylestradiol. The progestin component in oral contraceptives is more diverse, with varying degrees of androgen receptor activity. In our systematic review of 22 studies assessing the contraceptive pill and cognition , it was Collectively, prenatal and puberty-related increases in sex hormone levels appear to contribute to sex differences in cognitive functioning, with the most convincing evidence linking androgens to mental rotation abilities. Menstrual cycle study results do not provide convincing evidence to support significant cognitive changes across menstrual cycle phase. More research is needed to understand how different contraceptive pills influence cognitive functioning; however, existing evidence suggests a potential enhancing effect of the contraceptive pill on verbal abilities  as well as an effect on mental rotation that is dependent on the androgenicity of the progestin component.The natural transition to menopause is associated with fluctuating and eventually decreasing levels of ovarian estrogens and progesterone as well as an increase in serum FSH. Menopause can also be induced surgically in cases of medical necessity. The impact of both menopause  and aging on cognition is complex, with substantial interindividual variability . In addiNatural menopause is often associated with subjective cognitive complaints, particularly in the area of memory ,56. WhilSurgical menopause that includes the removal of ovaries, oophorectomy, has been associated with an increased risk of cognitive impairment and dementia later in life . The horAge-related declines in cognition are reported across most cognitive domains . Sex difEndocrine-related changes that accompany aging may also contribute to age-related declines in cognitive functioning, particularly the observed sex differences in brain aging and neurodegenerative processes . In femaFor both males and females, an age-related loss of sex steroid hormones has been associated with an increased risk of cognitive decline . Around More recently, gonadotropin levels, particularly LH, have been implicated in playing a role in cognitive decline ,21,78. FWomen are disproportionately affected by AD—they are more likely to develop AD, have a faster rate of cognitive and functional decline following a diagnosis of AD  and appeAPOE gene risk allele (ε4), the strongest known genetic risk factor for late-onset  AD, may confer differentially higher risk for AD conversion in females as compared to males [APOE-ε4 and CSF tau levels in females compared with males, particularly among amyloid positive females [Met66 allele of the Brain Derived Neurotrophic Factor (BDNF) gene, which has been associated with an increased risk of AD in females as compared to males [Sex also appears to modulate the impact of genetic risk factors in the aetiology of AD. For example, the to males ,91, partto males . A recento males .While the evidence supporting a link between estrogen depletion and risk for AD appears relatively consistent , the infIn the last decade, several observational and randomised controlled trials have provided some evidence to suggest that HT may reduce the risk of AD and that early use is protective and later use is either not protective or detrimental . HoweverIn relation to hormone types, continuous, combined CEE/synthetic medroxyprogesterone acetate (MPA) has not been associated with cognitive benefits (regardless of timing), whereas estrogen alone , as well as estradiol valerate combined with diongest or norehindrone may result in cognitive benefits (as reviewed by ). The tyThe development of selective estrogen receptor modulators (SERMs), such as raloxifene, provide an estrogen therapy with mixed agonist/antagonist properties, thus avoiding some of the adverse risks that have been associated with estradiol therapy . RaloxiIn addition to the biological explanations for the observed sex differences in cognitive decline and AD risk is a range of related health and lifestyle factors, which can be associated with gender differences due to sociological factors as well as sex differences. An increased cognitive reserve/resilience, that is, a higher level of education/occupation and greater engagement in cognitive activities, is considered a protective factor for AD. Education, a key mediator of cognitive reserve, has historically been associated with gender differences and the educational attainment for the older population of today may explain some gender differences between the level of cognitive reserve ,109. StrAPOE and Clusterin) were assessed for sex-specific resilience factors [A longitudinal study of non-demented older adults who were identified as carrying two AD risk genes  to continue to investigate how sex and sex hormones contribute to cognitive health and cognitive decline. Sex hormones  are influential in maintaining neuronal health and promoting neuronal cascades that underpin cognitive processes. As the potential for therapeutic modulation of the endocrine system advances, it is essential we understand how sex hormones influence cognitive functioning in AD. Much can be gained from advancing research in this field. Specifically, determining which hormonal pathways are most relevant to cognitive decline in AD. There is also the capacity for sex-related targeting of modifiable risk factors. A sex-specific focus of cognitive decline and AD is still not mainstream. Given the growing evidence supporting a role for sex hormones in cognitive functioning, combined with the many documented sex differences associated with AD, it appears that much can be gained from advancing research in this field.