Functional magnetic resonance imaging (fMRI) plays a key role in modern psychiatric research. It provides a means to assay differences in brain systems that underlie psychiatric illness, treatment response, and properties of brain structure and function that convey risk factor for mental diseases. Here we review recent advances in fMRI methods in general use and progress made in understanding the neural basis of mental illness. Drawing on concepts and findings from psychiatric fMRI, we propose that mental illness may not be associated with abnormalities in specific local regions but rather corresponds to variation in the overall organization of functional communication throughout the brain network. Future research may need to integrate neuroimaging information drawn from different analysis methods and delineate spatial and temporal patterns of brain responses that are specific to certain types of psychiatric disorders. The human brain is the most mysterious and vital organ. Recent neuroimaging techniques, including functional magnetic resonance imaging (fMRI), electroencephalography (EEG), and magnetoencephalography (MEG), now allow us to probe the brain at unprecedentedly high temporal or spatial resolution without the use of invasive techniques. Since the first fMRI brain scans of the 1980s, scientists have achieved great progress not only in technical procedures employed to acquire brain imaging data but also in data processing methods which subsequently reveal an inspiring understanding of the brain drawn from various data perspectives. fMRI has become the dominant technique in neuroimaging due to its noninvasiveness, lack of radiation exposure, a relatively good spatial and temporal resolution, and relative ease to acquire.In this paper, we will review popular data processing methods used in task-based fMRI and resting-state fMRI  has gained growing popularity in task-related fMRI analysis since its introduction into the neuroimaging community by Friston et al.  in 1994,n matrix . It has β values). Within-group activation  and intergroup differences can be compared by including the BOLD response variations of all subjects in each group in a second-level random effects analysis. However, GLM has also undergone some criticism focusing primarily on the assumptions the model makes [GLM is the dominant method used in task-based fMRI. Studies on psychiatric disorders have used this method to compare brain activities induced by certain experimental manipulations in the patient group and in the control group. For example, Juckel et al.  scanned el makes . GreaterOne important goal of neuroimaging research is to describe the pattern of brain connectivity among different regions. Functional connectivity refers to undirected associations between brain regions while effective connectivity reveals a directed and causal relationship. Psychophysiological interaction, in a clever use of the GLM, measures how functional connectivity is affected by psychological variables without specifying the directions of such influences . It examThe structural equation model, which was developed in the field of econometrics and first applied to neuroimaging data in 1991 , is anotSimilar to SEM, the dynamic causal model is also an approach to estimate effective connectivity and how this connectivity is influenced by experimental variables. However, underlying SEM and DCM are two very distinct generative models , MVPA takes into account multivoxel patterns of brain activity or connectivity. Information contained in these patterns can then be decoded by applying powerful pattern-classification algorithms. This method thus incorporates spatially distributed patterns of activity into the analysis, unlike univariate methods which treat every brain voxel independently. MVPA is often presented in the context of “brain reading” applications reporting that specific mental states or representational content can be decoded from fMRI activity patterns after performing a “training” or “learning phase.”MVPA has been successfully applied to identify functional connectivity difference between males and females , patternBrain regions which are active when our minds wander may hold a key to understanding neurological disorders and even consciousness itself. Resting-state fMRI, which measures spontaneous low-frequency fluctuations (<0.1 Hz) in the BOLD signal, is a relatively new pathway for evaluating regional interactions in the absence of tasks –41. For The seed-based approach extracts BOLD time-series data from a “seed”—a priorly selected voxel or ROI—and assesses the correlation between the average BOLD signal of the seed and the time course of all other brain voxels . Seed-baReHo is another straightforward technique that uses Kendall's coefficient concordance (KCC) to measure the similarity of a given voxel with its nearest neighbors based on the BOLD time-series  Figure . MultiplALFF is an index that reflects the intensity of regional spontaneous brain activity by calculating the voxel-wise magnitude within a defined low-frequency range . In ordePCA is a data-driven method that does not require the input of any prior information about the connectivity pattern. It has been found useful in estimating whether there are functional regions with correlated signal responses in human brain mapping . It was As an extension of PCA, ICA is likewise a data-driven method that has been successfully used in describing fMRI data –96. WithA hot recent method used in resting-state fMRI is graph theory. Graph theory is a mathematical theory and approach to studying graphs made up of nodes and edges and how these nodes connected by edges interact with each other , 104 Fi. The braOver the past decades, the development of fMRI techniques has made great contributions to our understanding of the neural mechanism underlying psychiatric disorder. In the present review, we summarize several major MRI methods widely used in psychiatric neuroimaging. Some methods such as ReHo and VBM focus on regional changes, whereas others take a systematic approach and emphasize the whole brain network. These methods together can reveal the abnormalities in brain structures and functions in psychiatric disorders. However, the functional significance of many measures such as ReHo and ALFF is still not well understood. Psychiatric disorders may be associated with very subtle changes in the brain. One single method may not be enough to fully capture the nature of such alternations. A systematic approach using multimodal neuroimaging and a variety of analysis methods has the potential to identify reliable biomarkers for specific psychiatric disorders. With ongoing progress being made in neuroimaging methods, neuroimaging holds clear promise in helping to diagnose and quantify psychiatric diseases.


There is increasing evidence that fluid overload and acute kidney injury (AKI) are associated but the exact cause-effect relationship remains unclear. Wang and colleagues analysed patients admitted to 30 intensive care units in China and found that fluid accumulation was independently associated with an increased risk of AKI and mortality. This commentary focuses on the close pathophysiological link between AKI and fluid overload and discusses the implications for clinical practice. It outlines some of the challenges, including the difficulty in diagnosing fluid overload reliably with current methods, and stresses the importance of personalised fluid therapy with physiological end-points to avoid the deleterious effects of fluid overload. In a recent article in Critical Care, Wang et al. analysed the data of 2526 patients admitted to 30 intensive care units (ICUs) in China and showed that even relatively small degrees of fluid overload were independently associated with an increased risk of AKI and mortality [Fluids are the second most common intervention in acutely ill patients (after oxygen). Whilst the benefits of early fluid resuscitation in patients with shock and acute kidney injury (AKI) have been accepted, uncertainty remains about the optimal type, volume and timing of fluid delivery. There is growing evidence that fluid administration beyond the correction of hypovolaemia is ortality . Others Although there is now little doubt that fluid overload is associated with AKI, it is difficult to disentangle the cause-effect relationship. Furthermore, translating the results into clinical practice is very challenging, especially since some important issues are still unresolved.First, defining fluid overload is not straightforward. By convention, most studies in the literature, including the study by Wang et al. , definedSecond, whether the consequences of fluid overload depend on the specific type of fluid has not been studied. It is plausible that fluid overload as a result of excessive crystalloid administration has a different impact compared with fluid accumulation following infusion of colloids or massive transfusion of blood products. More data are necessary. The timing of volume administration may also be relevant. A positive fluid balance of 5 litres over an initial 24-hour period followed by no further fluid gain may have a different outcome compared with the same net balance over a period of days.Third, fluid overload may result from overzealous fluid administration or oliguria or a combination of the two. The differentiation is important since fluid overload caused by excessive fluid therapy is potentially avoidable whereas fluid overload as a result of oliguria may reflect AKI and may not be easily modifiable without RRT . Most stFinally, the fact that fluid overload is associated with AKI does not prove causality. One key problem is that the effects of volume overload and the effects of AKI are similar. Both lead to multi-organ dysfunction. They also often have the same denominator, including endothelial dysfunction due to inflammation or ischaemia/reperfusion injury with decay and shedding of the glycocalyx and subsequent capillary leakage . PatientNevertheless, the study by Wang et al. does confirm what other studies have shown previously that a positive fluid balance is associated with worse outcomes. The crucial question for clinical practice is how to ensure appropriate and timely correction of hypovolaemia without causing fluid overload. Unfortunately, the tools to guide fluid management and to inform the clinical team when fluid administration is no longer beneficial are inadequate. Central venous pressure (CVP) or dynamic techniques like fluid responsiveness or echocardiography are often used. However, the CVP does not accurately reflect intravascular volume status and can be misleading . MeasuriIn the absence of any sophisticated tools to confirm euvolaemia or diagnose fluid overload, initial fluid resuscitation should be guided by an assessment of fluid responsiveness followed by fluid therapy titrated against individualized end-points , 14. TheThere is some prospect that novel biomarkers may also have a role to prevent fluid overload. Mekontso Dessap et al. randomly assigned 304 mechanically ventilated patients to a strategy driven by B-type natriuretic peptide (BNP) or usual care . PatientFluid overload, AKI and poor outcomes are clearly associated. However, whether AKI has caused fluid overload or vice versa can be difficult to determine. Better tools are necessary to measure intravascular volume status. Until then, fluid therapy needs to be personalised with physiological end-points, guided by dynamic tests like PLR manoeuvre, and monitored by using cumulative fluid balance coupled, where possible, with daily weights to avoid the adverse outcomes associated with fluid overload.


It is believed the immune system can contribute to oncogenic transformation especially in settings of chronic inflammation, be activated during immunosurveillance to destroy early neoplastic cells before they undergo malignant outgrowth, and finally, can assist growth of established tumors by preventing clearance, remodeling surrounding tissue, and promoting metastatic events. These seemingly opposing roles of the immune system at the different stages of cancer development must all be mediated by innate signaling mechanisms that regulate the overall state of immune activation. Recently, the cytosolic nod-like receptor (NLR) pathway of innate immunity has gained a lot of attention in the tumor immunology field due to its known involvement in promoting inflammation and immunity, and conversely, in regulating tissue repair processes. In this review, we present all the current evidence for NLR involvement in the different stages of neoplasia to understand how a single molecular pathway can contribute to conflicting immunological interactions with cancer. Anthropology of Immunology, Martin eloquently describes “the body as nation state at war over its borders, containing internal surveillance systems (encompassed in the immune system) to monitor foreign intruders” , and caspase-1  to inhibit anti-tumor immunosurveillance. Related to the gut studies in the first section, IL-1β over-expression in the stomach was shown to induce inflammation and cancer . This wamor site . Howevermor site . The resThis evidence clearly implicates the NLRs and inflammasome pathway in tumor-promotion and defense. They directly facilitate tumor cell growth and metastasis, and help prevent any anti-tumor immune responses. It is curious to speculate how accurate the analogy of tumor to “unhealing wounds” is with regards to NLR involvement. Are NLRs engaged in the same way by malignant disease as they are by damaged tissues prior to malignant transformation, in both cases inducing repair and protective properties? Fitting with the tumor editing hypothesis, any pro-inflammatory DAMPs or other signals resulting from initial transformation that would trigger tumor clearance have in theory been selected away, leaving only those characteristic of damaged self in need of repair. Inflammasome involvement in such diverse functions as tissue repair, immune suppression, and inflammation warrants a search for more inflammasome-activated targets besides IL-1β and IL-18 that could fine-tune downstream effector mechanisms. Are these two cytokines alone able to control such diverse effects, or are they working in collaboration with many other pathways, the overall milieu defining the result? Concerted efforts to consolidate information across tumor models and treatments, being mindful of cell-type specificity, will help clarify these points.We have now seen how NLRs switch roles in every stage of cancer progression Figure . In eachThe 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.


Despite high levels of naturally-acquired immunity (NAI) within local communities in malaria high transmission settings in Africa, such people often experience clinical disease during peak transmission months due to high parasite challenge. Major recruiters of unskilled labour in high-transmission malaria settings in Africa generally withhold chemoprophylactic medication from this large component of their labour force, which if administered during peak “malaria season” could reduce incidence of clinical malaria without unduly affecting NAI.Naturally acquired immunity confers protection against severe clinical disease and death, but does not prevent mild clinical disease and, therefore, still results in worker absence and worker debilitation. Evidence exists that NAI persists despite periodic parasite clearance and therefore provides opportunity for drug prophylaxis during peak transmission months, which contributes to broader malaria elimination objectives, community well-being, and reduced absence from work. Such chemoprophylaxis could be by way of standard daily or weekly supervised administration of prophylactics during peak transmission months, or occasional intermittent preventive treatment (IPT), all aimed at reducing parasite burden and clinical disease. However, challenges exist regarding compliance with drug regimens over extended periods and high parasite resistance to recommended IPT drugs over much of Africa. Despite withholding chemoprophylactics, most large companies nevertheless pursue social responsibility programmes for malaria reduction by way of vigorous indoor residual spraying and bed net provision.The lack of clear understanding regarding functioning of NAI and its role in malaria elimination campaigns, concerns about drug resistance and appropriate drug choice, lack of studies in the use of IPT in people other than pregnant women and small children, plus lack of guidance regarding drug options for IPT in the face of widespread resistance to sulfadoxine–pyrimethamine, means that large contractors in malaria endemic settings will likely continue to withhold malaria prophylactic drugs from locally-recruited workers, with adverse consequences on workforce well-being. Nevertheless, if the point of chemoprophylaxis is to reduce clinical malaria by way of reducing parasite challenge without significantly impacting NAI, then a comparable result can be achieved by implementation of effective vector reduction programmes which minimize parasite transmission but maintain NAI. Plasmodium falciparum; doubts about compliance with drug-taking regimens among workers is also a major factor in withholding such malaria prophylactic drugs. NAI does not prevent clinical malaria, but reduces the frequency and severity of such events [Mining and other large, labour-intensive companies operating in remote areas of Africa often employ hundreds of local rural residents for unskilled labour. Medical staff at such facilities routinely make available or recommend malarial prophylactics to expatriate staff, but withhold it from local employees, which form the bulk of their establishment. This is not done for cost-saving reasons, but more usually as a precautionary measure out of concern for disrupting naturally-acquired immunity (NAI) among local people, particularly against the lethal and most common parasite h events . HoweverPlasmodium parasites in the blood, but rather a state of sub-clinical asymptomatic disease with low persistence of parasites in the blood, in effect producing a constant immunological challenge and thereby maintaining NAI.Naturally acquired immunity is a complex phenomenon which is still not fully understood –3. Neversustaining malaria, despite the development of some transmission-blocking immunity reducing the efficiency of sexual stage gametocyte transmission to mosquitoes [Telephonic interviews were conducted with medical representatives of six large international mining and construction operations in high transmission malaria settings in eight African countries Table . Withoutsquitoes .Table 1GThe concerns of mining and similar major operations in malarious areas of Africa regarding the use of malaria prophylactics, and the unintended encouragement of community parasitaemia as a consequence, does not occur in a vacuum. Broader national and international interests are aimed at eradicating malaria and much success has been achieved in reducing malaria case numbers over the preceding decade . The thrThe contradictions and complexities now become apparent. If people spending their lives in areas of high malaria transmission have NAI which provides them safety from severe clinical malaria and death, should the world then stop what might be misguided humanitarian efforts in eliminating malaria? Of course not. The most obvious reason is the very high fatality rate among children below 5 years of age as a direct consequence of malaria—despite the ameliorating effects of NAI—and also the debilitating impact of even mild malaria in subsequent years on child schooling plus the disproportionate impact on women by way of negatively affecting capacity for family care, income-generating activities and subsistence food production.The apparent contradiction of countries engaging in malaria control and thereby potentially reducing NAI is probably not as counterproductive as may seem. Low levels of circulating parasites appear to be sufficient to maintain NAI and even periodic short-term clearance of infections is not necessarily bad , 8; suchFurther complicating the matter is the issue of ethics. However well-intentioned, a unilateral decision by any large international company to selectively prescribe healthcare within its workforce—especially when the decision is largely based on imperfect evidence and leads to one segment of workers being consciously allowed to contract disease—can expose that company to accusations of unfair discrimination. Clearly the issue is complex, but society demands resolution of even complicated situations with solutions that are equitable and not open to hints of benevolent patronage. That is the purpose of this paper, to explore ways in which such companies can fulfil their social responsibility in a fair and even-handed way.One avenue for defensible compromise that addresses company concerns yet addresses societal expectations is to offer informed choice. Locally-employed workers could be given appropriate presentations by trusted local health workers to fully inform them of the options and then offered a choice. Those that opt for prophylaxis during peak transmission months could then be provided on-site supervised consumption of malaria prophylactic drugs to improve compliance. However, this means a daily or weekly regimen over a period of two or 3 months and again raises the same concerns within medical practitioners about the practicality of adequate compliance among workers who work shifts, take vacation or have days off, and sometimes even hide medication to hand to family members; poor compliance is a serious challenge that fosters development of drug resistance, a major global concern in the face of limited anti-malarial drug choices , 11.If the primary objective of malaria chemoprophylaxis in local workers is to reduce the incidence of clinical episodes by reducing parasite challenge in peak transmission season and yet not unduly affecting NAI, then a more practical option is to implement IPT. This means administering a full therapeutic parasite clearance dose once or twice during the high transmission season to fully-informed, signed-consent local workers. The practice of malarial IPT is well-established and widely-applied among pregnant women and children –18 withoHowever, implementation of either of the two prophylactic options presented above—daily or weekly anti-malarials during peak transmission months or occasional IPT—will present the challenge “So which drug should we use?”. The historical preferred choice of chloroquine and pyrimethamine is no longer available due to near universal parasite resistance to these drugs, and even the current choice of sulfadoxine–pyrimethamine for IPT is rapidly approaching the end of its practical usefulness because of widespread resistance . One posParasite reduction and lowered incidence of clinical malaria in local inhabitants of malaria high-transmission areas can nevertheless be achieved in other ways, without necessarily adversely impacting on NAI Table . Many coTaking all the above factors into consideration, it appears that medical practitioners at mining and similar companies in high-transmission African countries have considerable justification for not advocating malaria prophylactic drugs to locally-recruited staff as a general rule, although exceptions will exist such as immune-compromised persons with HIV/AIDS. Some proof that these mining and other large companies are not withholding malaria prophylactic medication as a mere cost-saving exercise is the near-universal practice of such companies to engage in other extensive and costly malaria control practices, in particular provision of insecticide-treated bednets and indoor spraying of residential quarters with mosquito-combatting insecticides. Such vector control programmes that result in reduced parasite transmission fundamentally achieve the same desired objective for locally recruited workers, that being to reduce parasite challenge during peak transmission season which in turn leads to reduced incidence of clinical malaria, but allows persistence of NAI. Nevertheless, the use of IPT as a more directly targeted method for periodic controlled parasite reduction in local communities deserves serious consideration for improved community well-being, provided appropriate anti-malarial drugs are available.


Sociodemographic inequalities in the stage of diagnosis and cancer survival may be partly due to differences in the appraisal interval . A number of symptom appraisal models have been developed describing the psychological factors that underlie how people make sense of symptoms, although none explicitly focus on sociodemographic characteristics.We therefore conducted a conceptual review synthesising all symptom appraisal models, and focus on potential links with sociodemographics that could be the focus of future research.Common psychological elements across nine symptom appraisal models included knowledge, attention, expectation and identity, all of which could be sensitive to sociodemographic factors. For example, lower socioeconomic status (SES), male sex and older age are associated with lower health literacy generally and lower cancer symptom knowledge. Limited attentional resources, lower expectations about health and lack of social support also hamper symptom interpretation, and would be likely to be more prevalent in those from lower SES backgrounds. Symptom heuristics (‘rules of thumb') may lead to symptoms being normalised because they are common within the social network, potentially disadvantaging older populations.A better understanding of the processes through which people interpret their symptoms, and the way these processes differ by sociodemographic factors, could help guide the development of interventions with the aim of reducing inequalities in cancer outcomes. In England there are notable socioeconomic inequalities in cancer survival . Lower sThe majority of cancers are diagnosed following symptomatic presentation in primary care . The ModThere is also evidence demonstrating that lower SES, male sex and older age are associated with delayed presentation for cancer symptoms  are considered ‘an outcome of a perceptual process' , and intWe used a keyword search of electronic databases  to identify relevant models using the terms symptom, somatic, illness, perception, appraisal, interpret and model. Additional models were found from reference lists. Inclusion criteria for models were broad and included the following: (1) the model has been published in English journals or books (no date limits were set); (2) the model started from before the presence of noticeable symptoms; and (3) the model was predominantly about how people notice and interpret symptoms. We did not include the Model of Pathway to Treatment because it is not a unique model of symptom appraisal in its own right but rather a framework in which existing psychological theories of appraisal and help-seeking are integrated. We do, however, include those symptom appraisal models that The aim was to provide a conceptual framework to help understand sociodemographic differences in symptom appraisal. We focussed principally on SES because lower SES is a risk factor for advanced stage at diagnosis across several cancer types . If the interpretation process has resulted in a benign hypothesis , this will prolong the appraisal interval for persistent symptoms , or findAll of the symptom appraisal models identify factors that broadly resemble ‘knowledge', ‘attention', ‘expectation' and ‘identity' as determinants of progress along the detection–interpretation–response pathway see . ExampleOverview. Across models the concept of ‘knowledge' is described as ‘exemplars', 'schemes', ‘schemas', ‘mental representations', ‘prototypes' and ‘awareness', but in all cases is conceived of a cognitive factor that guides the interpretation process. When bodily changes are detected, people use their existing knowledge to aid interpretation.Sociodemographics differences. Population-based surveys in the United Kingdom using the cancer awareness measure  (ressors) .In line with the Cue Competition Theory, the theme of ‘competing demands' has been associated with lower SES, because demanding social environments often characterise lower SES households . CompetiWomen are shown to report more symptoms and visit the doctor more often than men . PennebaNegative effect may also contribute to SES differences in attention. According to the reserve capacity model, lower SES individuals experience more depression, anxiety and hostility compared with high SES individuals . NegativTargeting of attentional components may require a multi-faceted approach. For example, people low in consideration of future consequences could benefit from emphasis on the short-term benefits of presenting early with symptoms  alongside the potential long-term gains (earlier diagnosis). If evidence supports the notion that there are individual differences in body vigilance, campaigns could be targeted at groups who are less likely to notice changes in their body, offering practical action plans to encourage them to make time to attend to their body.Overview. Expectation-based processes feature in a number of symptom perception models and refer to the contribution of beliefs and emotions in guiding the perception and interpretation of bodily changes  is influenced by characteristics, including their sociodemographic profile. Self-identity is constructed within a social and cultural reality  may also influence interpretation  , which mOne possibility is that negative expectations of future health (based on self-identity) may contribute to inaction when people are faced with potential cancer symptoms. Modifying identity, particularly based on a sociodemographic group, is likely to be difficult. However, one possibility would be to increase the public's confidence in cancer as a curable and survivable disease, resulting in higher future expectations. Another possibility would be to harness the positive influence of social support in aiding symptom interpretation. Both of these suggestions may be well served at the community level, with involvement of potential users of the intervention from the start.A synthesis of nine models of symptom appraisal identified common processes, which included knowledge, attention, expectation and identity. In some cases there is evidence of sociodemographic variation in these elements, which could explain the sociodemographic differences in responses to possible cancer symptoms. Lower symptom awareness and health literacy in men, older people and people from lower SES backgrounds may contribute to misinterpretation of possible cancer symptoms. Attentional aspects may have an impact because higher external demands and negative effects, particularly in those from lower SES backgrounds, make interpreting bodily changes challenging. Expectation-based biases resulting from co-morbidity, stereotypes and the age–illness rule influence symptom interpretation, and sociodemographic differences in identity and life experience may hamper the detection or appropriate interpretation of symptoms. Conversely, social sanctioning may facilitate recognition of symptom seriousness and encourage help-seeking in higher SES groups.The next step is to design studies within specific cancer types to test the mediation of sociodemographic differences by symptom appraisal processes. This cancer-specific approach will also allow for differential effects of sociodemographics to be observed according to cancer type. The resulting evidence base can be used to inform the development of targeted interventions, designed, for example, to make it more likely that people appraise their symptoms accurately (knowledge), are more vigilant for bodily changes (attention), are less pessimistic (expectation) and have a better understanding of their personal risk (identity), with the goal of reducing time to help-seeking.This review provides a synthesis of symptom appraisal models to draw out common elements that may help understand inequalities in potential cancer symptoms. The primary aim was to highlight links between the psychological constructs from the models and demographic characteristics that could be the focus of future research. As the focus was on stimulating a research agenda, this review was not amenable to other types of review such as meta-analysis or meta-synthesis. In addition, because the literature review was not exhaustive, we may have omitted studies that would have provided a valuable contribution. The conceptual framework was developed with cancer in mind, but, as with models of help-seeking behaviour, may also be applicable to other illnesses , This conceptual review has synthesised models of symptom appraisal and highlighted elements that may help explain inequalities in the appraisal interval. It provides a framework to design studies that will provide a better understanding of the mechanisms through which people interpret their symptoms, and the relationship of these mechanisms to sociodemographic factors. This understanding should help guide the development of future intervention strategies to reduce inequalities in symptomatic presentation.


This paper is a country case study for the Universal Health Coverage Collection, organized by WHO. Abebe Alebachew and colleagues illustrate progress towards UHC and its monitoring and evaluation in Ethiopia.Please see later in the article for the Editors' Summary This paper is part of the PLOS Universal Health Coverage Collection. This is the summary of the Ethiopia country case study. The full paper is available as Supporting Information file This country case study is part of the Universal Health Coverage Collection. The World Health Organization defines universal health coverage (UHC) as a situation in which all people who need health services receive them, without incurring financial hardship The objectives of this paper were to document the availability of globally proposed UHC indicators Ethiopia has not yet promulgated an official definition of UHC, although numerous strategies, policies, and guidelines are being implemented to achieve universal access to primary health care and reduce impoverishment due to health spending Among the 61 proposed indicators Ethiopia measures most of the major service coverage indicators related to reproductive, maternal, and child health and key infectious disease services using routine information systems and population-based surveys. However, indicators of chronic, non-communicable disease service coverage are generally not available. Three indirect measures of financial coverage are available from National Health Accounts estimations, but direct measures of catastrophic spending and impoverishment due to health care spending have not been calculated.Ethiopia has shown significant progress in reducing under-five, infant, and neonatal mortality rates over the last decade. These rates have declined by 47%, 39%, and 25%, respectively  [16]–[18Immunization rates and the delivery of other child health services have improved substantially since 2000 The total number of health care facilities, particularly primary care centers, has increased nearly 10-fold since 2005 and distribution on a per capita basis is largely equitable; on the other hand, secondary and tertiary level service capacity has not improved significantly Ethiopia is moving to expand financial protection through various financing initiatives. A social health insurance law was recently passed and a national health insurance agency established. Pilot community-based health insurance schemes have also been initiated. Nonetheless, out-of-pocket spending remains a major source of health financing for Ethiopia's population and is a significant barrier for accessing and utilizing health services Some of the WHO's proposed UHC measurement indicators may not yet be applicable or feasible in a low-income context like Ethiopia, particularly those requiring frequent, large population-based household surveys as well as those related to chronic conditions. Local stakeholders expressed a preference for indicators that are more programmatically relevant to their context and less resource-intensive to collect. If UHC is included in a post-MDG agenda, involving country representatives in selecting these indicators would harness political commitment towards UHC implementation.Countries like Ethiopia should be assisted in defining and developing UHC strategies; technical support should also be given to build their capacity to collect, analyze, and use routine and survey-based information. Such capacity in Ethiopia is growing but still limited, and this has negatively impacted the quality and availability of data.Text S1The full country case study for Ethiopia.(DOCX)Click here for additional data file.


Cardiovascular and autoimmune diseases are among major health concerns in developed countries, and both represent a significant source of morbidity, mortality and economic costs. Despite they are thought to affect subjects at different ages, most of the deaths of patients affected by autoimmune diseases are represented by cardiovascular deaths. Several manifestations of cardiovascular diseases can be observed in patients with autoimmune diseases, such as endothelial dysfunction, accelerated atherosclerosis and an increase in the rate of acute coronary syndromes. Thus, people with autoimmune diseases have an increased cardiovascular risk and a worse outcome in the case of cardiovascular events. In this review, we will describe the correlations between the two spectra of diseases. Cardiovascular diseases are the main cause of morbidity and mortality and autoimmune diseases are among the leading causes of death among young and middle-aged women in developed countries . DespiteInflammatory and autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematous (SLE), suffer from increased cardiovascular morbidity and mortality owing to accelerated atherosclerosis and premature coronary artery disease , 3, and Systemic sclerosis (SSc), an autoimmune disease with a less prominent inflammatory component but significant vascular abnormalities , has alsMany observational studies in cohorts of RA patients with different characteristics reported a wide range of incidence rates for cardiovascular events and mortality. Some studies show a more than doubled risk compared to general population, which is comparable to the cardiovascular risk in diabetes mellitus , 14, whiAnother factor that may link atherosclerosis to RA is a common genetic background. An increasing number of studies report gene polymorphisms that are associated with a higher risk of cardiovascular diseases in RA . Human lHowever cardiovascular risk is increased before the clinical onset of RA. In fact several studies showed that the first signs of atherosclerosis are already present in patients with recent-onset RA , with siOver the past few decades, the survival rate for patients with lupus has improved from 50% to between 80% and 90% at 10 years. However, the mortality rate for patients with SLE is three times that in the general population . Most ofA review by Karrar et al found that the prevalence of coronary artery disease among women aged 35 - 44 years with SLE is 50 times higher than among age- and sex-matched controls .Multiple mechanisms account for the increased atherosclerotic burden in this spectrum of diseases. In previous studies, an abnormal lipid profile was reported in 46-56% of SLE patients . Renal iCardiovascular diseases are chronic diseases associated with an inflammatory component and inflammation is considered a novel risk factor for coronary heart disease with growing importance. Despite still debated, this inflammatory component is currently seen as not directly induced by an autoimmune process. A non-autoimmune primary pathological process such as apolipoprotein B-containing lipoproteins deposition may result in a vicious cycle of inflammation promotion and perpetuation. Subendothelial retention of apolipoprotein B-containing lipoproteins triggers a maladaptive, non-resolving inflammatory response that drives atherogenesis . A systeIn addition, chronic steroid therapy (a mainstay of treatment for SLE) worsens hyperglycemia and hyperlipidemia, contributing to atherosclerosis. The role of corticosteroids in the atherosclerosis of SLE has not been fully defined. It has been argued that steroids may be a double-edged sword, such that at low dosages, they may exert beneficial anti-inflammatory effects, while at high dosages, steroids may cause harm by exacerbating metabolic cardiovascular risk factors , 31.Two previous studies of subclinical atherosclerosis in SLE showed that long-term treatment with corticosteroids was not associated with a significantly increased risk , 33. IndBesides glucocorticoids, other immunosuppressive agents have been implicated in the pathophysiology of atherosclerosis . AzathioCyclosporin A can increase low-density lipoprotein (LDL) cholesterol levels and induce glucose intolerance, both of which can cause atherosclerosis. In addition, cyclosporin A has been shown to be associated with endotheliitis , increasIt is worth noting the possible positive and adverse effects of anti-inflammatory drugs in atherosclerosis. In the case of acute myocardial infarction (MI), the rate of recurrence is higher in the first few months. This heightened risk is at least in part driven by a post-MI monocytosis. The inhibition of this monocytosis might reduce the risk of recurrent events  but it aIn addition, patients with autoimmune diseases often have antiphospholipid syndrome, predisposing them to thrombosis and MIs. The literature also reports patients treated for spontaneous coronary artery dissections secondary to SLE . Other pRecent studies demonstrate that, in addiction to epicardial vessel plaques, also CMD can be detected in patients affected by chronic inflammation disorders . The cli2-) combines almost instantaneously with NO to form peroxynitrite that has a dual effect of decreasing NO bioactivity while promoting protein and lipid oxidation [2- and peroxynitrite have been shown to be increased in SLE [Previous studies demonstrated diffuse microvascular dysfunction in SLE. This is caused by increased sympathetic outflow  and incrxidation  and bothd in SLE .Cardiac disease is a common finding also in SSc even if it is often clinically occult. In fact, myocardial disease is evident in 20-25% of patients with SSc, while at post mortem pathologic studies report a myocardial involvement in up to 80% of patients , 51. TheCoronary flow reserve (CFR) has been considered a useful diagnostic index for the functional and physiologic assessment of coronary circulation . The CFRCFR has been demonstrated to be impaired in most patients with SSc, even in the absence of clinical signs of cardiac disease . CFR impCMD can be also detected in RA. There is accumulating evidence that cardiovascular risk is increased before the clinical onset of RA. Several studies showed that endothelial dysfunction can be detected in patients with recent-onset RA . GonzaleThere is an additional concern related to CMD. The most impressive expression on CMD is the occurrence of no reflow (NR). NR is responsible for the inability of a previously ischemic region in the territory of a recanalized infarct-related artery to be reperfused. NR is caused by functional and structural damage  of coronDifferent pathogenetic mechanism predispose to NR. First there could be an irreversible damage of cellular components of microvascular circulation in the ischemic area when flow is restored. In fact prior to cell death there is a period during which the ischemic myocyte is viable, but vulnerable to further injury if blood flow is restored. Reperfusion causes the reintroduction of oxygen and energy into an abnormal cellular environment and triggers additional events that produce further myocyte damage. Either there could be a functional impairment of the microvascular coronary circulation with myocyte hypercontracture , plateleThe presence of a pre-existing CMD poses patients affected by autoimmune diseases to an increased risk of NR occurrence. It is worth noting that NR occurrence is strongly related to a worse prognosis .Despite the association between autoimmune disease and NR has not been addressed in the literature, in our experience these patients often show NR after both primary and non-primary percutaneous coronary revascularization, with even increased rate after stent deployment.Studies from the general population demonstrate association between markers of inflammation and increased levels of matrix metalloproteinases, markers of oxidant stress, and tissue factor, all of which are purported to have a role in plaque destabilization and thrombosis . PatientA previous study showed a 50 times greater of MI in women affected by SLE aged 35 - 44 compared to women of similar age from a population-based sample . In contUrowitz et al suggested for the first time that SLE mortality followed a bimodal pattern. In fact, whereas deaths early in the course of the disease are most often due to active lupus, later deaths are frequently secondary to atherosclerotic coronary heart disease and acute MI .Although Ward et al found similar in-hospital outcomes for MI between SLE and non-SLE patients, the only systematic study examining coronary artery bypass grafting (CABG) in patients with connective tissue diseases found lower survival and a higher frequency of reintervention (mean follow-up: 35 months) when compared to the general population , 70.A recent study on SSc patients showed that they had a significantly increased risk of MI and stroke after a mean follow-up of 5.5 years, with a hazard ratio of 1.97 for MI and 2.56 for stroke . In thisA population-based cohort study of RA patients reported an increased risk of coronary heart disease and MI 2 years prior to RA diagnosis using the American College of Rheumathology 1987 criteria . Almost In addition antithrombotic therapies are less likely to be prescribed at discharge in patients with autoimmune diseases as reported by Maksimowicz-McKinnon et al . Lower aAutoimmune diseases predispose to accelerated atherosclerosis and various manifestations of cardiovascular diseases. It is therefore of main importance in these patients to have cardiovascular risk factor assessment and to manage optimally known risk factors. Moreover screening for cardiovascular diseases should be performed in advance in patients affected from autoimmune diseases, since cardiovascular deaths are among the main causes of mortality in these patients.


This paper focuses on the recent advances on radiolysis-assisted shape-controlled synthesis of noble metal nanostructures. The techniques and protocols for producing desirable shapes of noble metal nanoparticles are discussed through introducing the critical parameters which can influence the nucleation and growth mechanisms. Nucleation rate plays a vital role on the crystallinity of seeds while growth rate of different seeds’ facets determines the final shape of resultant nanoparticles. Nucleation and growth rate both can be altered with factors such as absorbed dose, capping agents, and experimental environment condition to control the final shape. Remarkable physical and chemical properties of synthesized noble metal nanoparticles by controlled morphology have been systematically evaluated to fully explore their applications. Recently, interest in metallic nanoparticles has been growing steadily due to their unique and fascinating properties and potential applications compared to their bulk counterparts , 2. It iMany unusual properties that have been observed by shape variation arise as a result of the spatial confinement of electrons and phonons, their large surface-to-volume ratios, and electric fields around the nanoparticles –10. ThesAs a result of strong plasmonic properties, noble metal nanoparticles have attracted great interest for many applications , 18. In This work is focused on the recent advances in the radiolytic synthesis of shape-controlled noble metal nanoparticles. In addition, several strategies on controlling the nucleation and growth process to obtain the shape-controlled metal nanoparticles and their remarkable properties will be reviewed.The ideal techniques for preparation of metallic nanoparticles should be reproducible and possess ability to control the shape of the particles with monodispersity yields. Moreover, avoiding use of toxic precursors, using of environmentally friendly solvents, keeping the reaction temperature close to room temperature, and also minimizing the quantities of generated by-products are great advantages that make this method outstanding.In bottom-up routs, most of the preparation techniques are based on the liquid phase category with the potential to synthesize large quantity of nanoparticles with good control of size, shape, morphology, crystallinity, composition, and surface chemistry at a reasonable low production cost . Chemica−15 s), physico-chemical stage (~10−15–10−12 s), and non-homogeneous chemical stage (~10−12–10−6 s) .Fig.Fig55].FIt is clearly observed from Fig. Okamoto et al. showed the shape dependency of UV-visible absorption of Au nanoparticles synthesized by gamma-ray irradiation with various dose rates Fig.  57]. As. As57]. Since physical and chemical properties of nanomaterials strongly depend on their size and morphology, therefore, shape-controlled nanoparticles can introduce ideal candidates for devising new applications. For example, different metal nanostructures including nanospheres, nanotriangles, nanorods, and nanowires have been examined as SERS substrates to improve the sensitivity of conventional sensors. Yang et al. have studied on dependency of Ag nanoparticles’ shapes on SERS enhancement . They haAnother optical property that can be affected by shape transition of metal nanoparticles is photoemission spectrum. It has been reported that triangular colloidal silver nanoparticles are highly photoluminescent compare to their spherical shape . Figure As it is clearly observed from Fig. We have introduced a number of useful strategies that can be tuned to control the radiolytic synthesis of noble metal nanoparticles with a specific shape in a solution phase. The nucleation process of seed and growth rate of different facets presents important role in determining the final shape of nanoparticles. Both nucleation and growth processes directly depend on coalescence and reduction rate which can be controlled by experimental parameters such as absorbed dose, dose rate, capping agents, and type of metal precursors which presented with several examples of recent researches. It is important to consider that the manipulation of the growth processes in the most attainable strategies is built for shape-controlled synthesis rather than the nucleation process. It is also illustrated how controlling the final shape can tune the optical properties of metal nanoparticles.


Targeted therapy of cancer is often associated with clinically significant diarrhoea; however, the mechanisms underpinning this adverse effect are currently unknown. Diarrhoea following treatment with tyrosine kinase inhibitors (TKIs) of EGFR is particularly troublesome. Until recently, understanding of EGFR TKI-induced diarrhoea has been limited to clinical observation. However, our group has recently developed the first rat model of EGFR TKI-induced diarrhoea. This paper reviews the published and unpublished findings. Molecularly targeted cancer agents are emerging as an effective approach to the treatment of a range of different tumours. They have ushered in a new age of personalised medicine in cancer therapy and have been made possible due to significant advances in genetics and molecular biology in the last two decades. However, despite improved specificity for cancerous cells compared to traditional cytotoxic chemotherapy, these agents are still associated with a number of important clinical adverse effects, particularly rash and diarrhoea. Importantly, the mechanisms underlying these toxicities and the significance to patient outcomes are poorly understood and have received little attention to date. In response to this gap in knowledge, our research group has developed a clinically relevant rat model of diarrhoea in response to treatment with the small molecule tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER2), lapatinib. This review will reflect on the rationale for embarking on the project, the series of experiments that established the model, and the challenges encountered along the way.Diarrhoea is a troublesome side effect of almost all systemic cancer treatments and has been researched intensely –3. WhilsDiarrhoea is one of the most common adverse events recorded following treatment with all TKIs and is a dose limiting toxicity for TKIs that block EGFR signalling . DiarrhoA summary of diarrhoea associated with TKIs for EGFR is shown in Despite diarrhoea being extremely common and in some cases dose-limiting, there has been almost no clinical research investigating the underlying causes of this side effect. Possible reasons may include the misconception that diarrhoea is an unimportant toxicity combined with the difficulties in directly assessing the gastrointestinal tract. Although there is yet to be any defined pathogenesis, some authors cautiously speculate that TKI-induced diarrhoea is a specific toxicity separate from chemotherapy-induced mucosal injury , which aEGFR is expressed by cells of epithelial origin, including the skin and gastrointestinal tract, so it is not surprising that toxicities affect these systems. The prevailing hypothesis is that inhibition of EGFR signalling will lead to reduced growth and healing of the intestinal epithelium, leading to mucosal atrophy, due to the stimulatory effect of the EGFR pathway on enterocyte proliferation . An alteDiarrhoea is often worsened during TKI and chemotherapy combination treatment and may be due to shared metabolic and drug efflux pathways between drugs. For example, coadministration of lapatinib and paclitaxel results in 20% increased exposure to both agents, suggested to be through downregulation of CYP3A4 . PazopanRecognising and understanding this major lack of research, we decided to establish a platform to investigate mechanisms and test interventions for EGFR TKI-induced diarrhoea which led to the development the first rat model of targeted therapy-induced diarrhoea.The use of animal models to investigate treatment-related toxicity has provided a wealth of knowledge in regard to injury pathogenesis and has enabled relatively high throughput testing of interventions matched to pathobiology, which has been particularly evident in the mucositis field –27.min similar to clinical studies. Conventionally housed male albino Wistar rats received an oral gavage of study drug (TKI) or vehicle (0.5% methylcellulose/0.1% Tween 80) every 24 h for 28 days. Daily measurements of weight loss, diarrhoea, and distress were recorded. Groups of rats were sacrificed each week to assess circulating lapatinib levels and gastrointestinal tissue changes at the morphological and molecular level. Full experimental details can be found in Bowen et al. [In contrast, there is a paucity of models available to specifically investigate mechanisms of TKI-induced diarrhoea. As such, our group has developed a rat model of targeted therapy-induced diarrhoea that uses a four-week schedule of daily oral lapatinib treatment to induce mild to moderate diarrhoea, whilst achieving a CLapatinib (GW572016/Tykerb GlaxoSmithKline) was chosen as the initial TKI for testing in the model since it is a highly promising drug in clinical trials for the treatment of multiple solid tumours , but optIn our rat model, we found that lapatinib in a dose range of 100 to 500 mg/kg induced diarrhoea in a majority of rats, without being excessively toxic over the month of treatment. At 240 mg/kg lapatinib, roughly 70% of rats experienced diarrhoea during the treatment period, with diarrhoea episodes characterised by intermittent and repeated presentation . This reAlthough our model created symptomology similar to the clinical situation, somewhat surprisingly there was no macroscopic or microscopic tissue injury seen within the jejunum or colon. As such, lapatinib does not cause epithelial atrophy at clinically relevant doses in the rat, and the mechanism of diarrhoea does not rely on this change occurring. In contrast to these findings, studies in mice examining various EGFR TKIs have shown gastrointestinal damage with significant epithelial atrophy. Mice administered gefitinib twice daily for ten days had prominent changes in the small intestine . The autOur rat model of TKI-induced diarrhoea also assessed the effect of combination therapy with paclitaxel due to clinically important diarrhoea seen in patients treated with both lapatinib and chemotherapy. Paclitaxel was chosen as the chemotherapy agent to investigate in our model since it is the current drug of choice for neoadjuvant breast cancer therapy . PaclitaGiven that we found no measurable intestinal pathology following lapatinib treatment alone, our study did not support the theory of TKI-induced direct mucosal damage leading to diarrhoea . It is wIn contrast, combined lapatinib and paclitaxel treatment did cause gut tissue alterations, including epithelial apoptosis, crypt hyperplasia, and inflammatory infiltrate to the lamina propria. Previous research by our group using conventional chemotherapy drugs including methotrexate, 5-fluorouracil, and irinotecan has shown that apoptosis is a key early marker of chemotherapy-induced gastrointestinal injury, and crypt hyperplasia occurs during the regenerative phase following severe injury , 43–46. Development of this model is the first step in creation of a clinically relevant platform to examine mechanisms and test treatments for EGFR TKI-induced diarrhoea. We have built upon our group's collection of established chemotherapy-induced models of diarrhoea and believe this is a significant addition to the field. However, our preliminary findings have identified a number of aspects that need further clarification to improve our understanding and management of lapatinib-induced diarrhoea. Firstly, given that lapatinib bioavailability is relatively low and highly variable between patients , a closeThe outlook for the clinical use of TKIs is to increasingly include them in first line treatment of curable disease. Therefore, it is extremely important to have a thorough understanding of the adverse effects associated with their use and the best approaches to minimise toxicity. Animal models such as the one described herein are an invaluable source of knowledge and will provide the platform to conduct studies that improve our knowledge of the mechanisms underlying diarrhoea as well as to uncover the best intervention targets.


Specifically, the accession number PRJNA238968 is obsolete, and the accession number PRJEB1698 should be used to access the sequencing data deposited in the EBI-ENA database (


Gene duplication is a recurring phenomenon in genome evolution and a major driving force in the gain of biological functions. Here, we examine the role of gene duplication in the origin and maintenance of moonlighting proteins, with special focus on functional redundancy and innovation, molecular tradeoffs, and genetic robustness. An overview of specific examples-mainly from yeast-suggests a widespread conservation of moonlighting behavior in duplicate genes after long evolutionary times. Dosage amplification and incomplete subfunctionalization appear to be prevalent in the maintenance of multifunctionality. We discuss the role of gene-expression divergence and paralog responsiveness in moonlighting proteins with overlapping biochemical properties. Future studies analyzing multifunctional genes in a more systematic and comprehensive manner will not only enable a better understanding of how this emerging class of protein behavior originates and is maintained, but also provide new insights on the mechanisms of evolution by gene duplication. Moonlighting proteins are thought to originate by evolutionary “tinkering” or “co-option” whereby functional novelties arise neutrally in pre-existent scaffolds and eventually result in biochemical advantages . Howevercis or in trans may also enhance a new function without an associated evolutionary tradeoff. In other words, mutations leading to novel functions may occur not only within the open reading frame of a gene, but also in its promoter region or in an interacting gene . Therefore, it is tempting to speculate that duplication dynamics have played a role in the origins and maintenance of gene multifunctionality. In what follows, we present different scenarios of evolution of moonlighting proteins by gene duplication, starting from ancestral monofunctional or multifunctional states and driven by neutral or adaptive evolution .With the evolutionary models of gene duplication in mind, we examined available databases of moonlighting proteins described to date . Many ofgene-dosage amplification . For instance, gene-dosage amplification selection and stoichiometric balance seem to influence the maintenance of moonlighting activities in duplicate. At least three yeast cytosolic ribosomal proteins show additional roles related to the regulation of their own RNA stability and expression (The scenario in which both paralogs moonlight after gene duplication seems to be quite common in yeast (pression . Strong ENO2 is an essential gene and is expressed in the presence of glucose at a 20-fold higher level than its paralog. Eno1 and Eno2 have specialized in their catalytic properties and expression patterns, while their moonlighting behavior has been maintained (In addition to gene-dosage amplification, subfunctionalization seems prevalent in the conservation of moonlighting behaviors after gene duplication. For example, Sis2 and Vhs3 participate in the same coenzyme-A synthesis protein complex and are negative regulators of a serine/threonine phosphatase . These pintained .HXK2 is expressed in growth under glucose, whereas HXK1 is expressed under different carbon sources (hxk2 gene-regulation phenotype (LYS21 suppresses the histone-acetyltransferase mutant phenotype, but to a lesser degree than LYS20 (Even for duplicate pairs in which only one copy has been defined moonlighting, it is common to find evidence of some degree of multifunctional behavior in its paralog. The yeast hexokinases Hxk1 and Hxk2 provide such an example. Both duplicates catalyze the phosphorylation of hexoses under different biological conditions:  sources . Besideshenotype . Likewishenotype . Althoughenotype . Moreovean LYS20 . Other mGAL1/GAL3 pair is perhaps the only example presented in Table 1 with conclusive evidence that one paralog has lost the multifunctional character of the ancestral moonlighting protein (GAL3 and during diauxic shift (In the examples provided above, we assume that the paralogs originated from a moonlighting ancestor. The  protein . These g protein . It is iic shift . TherefoIn conclusion, many duplicates seem to preserve partially overlapping ancestral and moonlighting functions despite long evolutionary time. Dosage selection and incomplete subfunctionalization seem to be key mechanisms of functional preservation after gene duplication. Since subfunctionalization relies on the partition of ancestral properties, this mechanism is expected to play a role in the evolution of complex genes-that is, pleiotropic genes or genes with different expression patterns-or multifunctional genes .Molecular functions of moonlighting paralogs may be conserved by selection of gene-dosage amplification. Although genes under such pressure evolve slowly , functiocis-regulatory regions, resulting in paralogs with the same biochemical properties but different expression patterns (Incomplete subfunctionalization also leads to functional conservation of moonlighting paralogs. Rapid gene divergence is considered to occur typically in patterns . Regulatpatterns . An initThe “problem” of genetic redundancy is the apparent paradox that seemingly dispensable gene copies are retained at the long term . Proteinparalog responsiveness is exposed under particular genetic or environmental contexts. Interestingly, Hxk1 and Lys21 respond to the absence of their paralogs even under conditions in which the catalytic activities are not needed (Several duplicate moonlighting genes from yeast show mechanisms of buffering that can provide genetic robustness. For instance, Eno2, Hxk1, and Lys20/21 are up-regulated in response to deletion of their paralogs . Such pat needed , suggestEscherichia coli (Other sources of redundancy and genetic robustness are also likely to play a role in the evolution of protein moonlighting. For instance, protein multifunctionality may originate even if mutations favoring the emergence of a new activity have a strong underlying tradeoff with the pre-existing function, but are tolerated at the organismic fitness level due to compensatory mechanisms . While ghia coli  suggest cis or in trans. In conclusion, an interplay between the strength of the molecular tradeoffs between functions, the selection for their conservation mediated by dosage selection or rapid changes in gene expression, and genetic robustness shape the evolution of moonlighting duplicate genes for long evolutionary times.Different scenarios of evolution of moonlighting proteins by duplication and amplification are not mutually exclusive and may coexist with previously proposed mechanisms, such as repurposing or tinkering. The outcomes are likely influenced by the structural properties of proteins and of the mutations that occur in The evolutionary pathways leading to the generation, retention, and loss of moonlighting proteins remain largely unknown. The actual extent to which gene duplication has contributed to their evolution may be elucidated as we gain insights into additional examples in yeast and in other organisms. Genetic complementation with the paralogous sequences of known moonlighting proteins could shed light into the conservation and volatility of multifunctional behaviors. Given that molecular roles are usually exposed under specific cellular contexts, challenging organisms systematically with different environmental and genetic perturbations will help to identify novel examples of moonlighting proteins. In addition, analyses of sequenced genomes could provide a means to predict and annotate this emerging type of functional behavior, especially in non-conventional species that are closely related to model organisms. For instance, lineage-specific duplicated genes with particular functional profiles may be good starting point to look out for proteins with extra biological roles.As with most studies of genes and gene functions, the focus on moonlighting proteins and their evolution necessarily touches on the more fundamental question of how we define and detect biological “function.” Approaching the problem in more comprehensive and systematic ways will ultimately impact our understanding of the evolution of both duplicate genes and moonlighting proteins.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.


Whereas previous research has indicated that sleep problems tend to co-occur with increased mental health issues in university students, relatively little is known about relations between sleep quality and mental health in university students with generally healthy sleep habits. Understanding relations between sleep and mental health in individuals with generally healthy sleep habits is important because (a) student sleep habits tend to worsen over time and (b) even time-limited experience of sleep problems may have significant implications for the onset of mental health problems. In the present research, 69 university students with generally healthy sleep habits completed questionnaires about sleep quality and mental health. Although participants did not report clinically concerning mental health issues as a group, global sleep quality was associated with mental health. Regression analyses revealed that nighttime sleep duration and the frequency of nighttime sleep disruptions were differentially related to total problems and clinically-relevant symptoms of psychological distress. These results indicate that understanding relations between sleep and mental health in university students with generally healthy sleep habits is important not only due to the large number of undergraduates who experience sleep problems and mental health issues over time but also due to the potential to intervene and improve mental health outcomes before they become clinically concerning. The undergraduate years are a period of vulnerability when considering sleep problems and mental health. Sleep problems tend to worsen over time in undergraduate students , a findiPrevious research has indicated that sleep problems are associated with poorer mental health outcomes from childhood to adulthood . ExiPrevious research has established that sleep problems are associated with impaired mental health in childhood and beyond. For example, 2- to 3-year-old children with parent-reported poorer sleep behaviors were more likely to experience increased externalizing  and internalizing  problems relative to those with better nighttime sleep habits . Other rRelations between sleep and mental health have been confirmed and extended in adolescents and young adults. For example, findings from one study indicate that approximately 67% of adolescents with multiple sleep problems  were classified as having a DSM-relevant anxiety or depressive disorder  to examine whether self-reported sleep quality and mental health were associated in university students with generally healthy sleep habits. Students were identified as having generally healthy sleep habits if they went to bed before 2:00am at least four nights per week, obtained six or more hours of sleep per night, and scored 10 or less at enrollment on a sleepiness questionnaire. In addition, students with generally healthy sleep habits did not have a history of diagnosed sleep disorders, they did not have abnormal sleep habits, and they did not take nighttime sleep aids more than three nights per week. Because previous research has focused on the differential contributions of nighttime sleep duration and nighttime sleep disruptions to mental health outcomes , 25–28, As reported previously , the comSeventy-one students at a large public university in the southwest were selected to participate after having been recruited through the undergraduate participant pool or through advertisements placed on campus. The data from one graduate student were excluded from analysis as well as the data from one undergraduate student who failed to comply with study protocol. The data from 69 undergraduate students  are presented . Thirty-four of the participants were of Asian descent, 26 were of Caucasian descent, five were of mixed race, and one was of African American descent; three participants did not report race information. Eleven of the participants identified as Hispanic; one participant did not report ethnicity information. Participants elected to receive either $25 in cash or three points of extra course credit in appreciation for their participation.Participants were selected based on their responses to an online screening questionnaire that was developed for this study based on previous research . The surGiven our interest in examining sleep-mental health associations in university students with generally healthy sleep habits, at least relative to university students recruited more broadly, eligible students went to bed before 2:00am at least four nights per week, obtained six or more hours of sleep per night, and scored 10 or less on the ESS . They alEligible students were contacted by phone or email about continuing their participation in the study. Students who agreed to participate were randomly assigned to arrive at the laboratory for their first session at either 9:00am or 9:00pm, a manipulation that was imposed due to the larger goals of the study from which the presented data were drawn.After informed consent was obtained, each participant completed the questionnaire battery described below. Participants were also provided with contact information for the university counseling center due to the sensitive nature of some items on the questionnaires; participants were instructed to contact them with any mental health issues or concerns.Eligible participants completed a battery of questionnaires that included the ESS , the PitThe ESS  is an eiThe PSQI  assessesResponses to the 19 primary questions on the PSQI  were redWe also examined two additional variables from the PSQI in relation to participant mental health. Information about nighttime sleep duration was obtained from one question that inquired about the amount of sleep obtained at night on average over the past month. Information about the frequency of nighttime sleep disruptions was computed from eight questions inquiring as to how often participants experienced trouble sleeping for various reasons. Participant responses to each question were coded from zero (if participants were not affected by the problem) to three (if participants experienced the problem three or more times per week). The codes on these items were then summed to yield a continuous sleep disruption score.Mental health was assessed using the ASR . The ASRThe data from the ASR  were redz-scored before analysis as recommended in the ASR data reduction manual [The total scores obtained from the ASR were inspected for outliers, identified as values that were three or more standard deviations above or below the mean. Outlying values were truncated to the highest acceptable value in the dataset plus one, as recommended in reference . The totn manual .p ≤ .05 and (b) were correlated with either nighttime sleep duration or the frequency of nighttime sleep disruptions at p ≤ .10.The data were analyzed using SPSS Version 20. The general analytic approach parallels that used to examine associations between participant sleep quality and its components in relation to temperament in this sample . PrelimiDescriptive statistics for the main study variables, including the percent of participants in the clinical range on each ASR variable, are shown in p ≤ .05.Between-subjects analyses of variance (ANOVAs) were conducted to determine whether group assignment (testing at 9:00am or 9:00pm), race , and ethnicity (Hispanic relative to non-Hispanic) were associated with participant sleepiness, global sleep quality, and mental health. Participant sex and age were not considered as covariates, as the ASR output is adjusted for these variables. The analyses revealed no differences by group assignment, whereas group differences were found by race and ethnicity on mental health outcomes but not on participant sleepiness or global sleep quality . Given these significant differences, we included participant race  and ethnicity (0 = non-Hispanic or 1 = Hispanic) as categorical covariates in all subsequent analyses. As a result, the data from three participants without race values and one participant without an ethnicity value were excluded from the following analyses. Significant results are presented when Partial correlations among continuous measures of sleep quality and self-reported mental health are shown in We conducted one regression examining the relation between nighttime sleep duration and the frequency of nighttime sleep disruptions on the syndrome scales pertaining to total problems . We thenNighttime sleep duration was negatively associated with total problems and the frequency of nighttime sleep disruptions was positively associated with total problems. Race and ethnicity were unassociated with total problems.The frequency of nighttime sleep disruptions was positively associated with increased report of anxiety and somatic complaints but not depressive problems; nighttime sleep duration was not associated with reports of anxiety, depressive problems, or somatic complaints. Race and ethnicity were also unassociated with each of these mental health outcomes.Understanding relations between sleep and mental health in university students with generally healthy sleep habits is important because student sleep habits tend to worsen over time  and evenThe conducted analyses indicated that reduced nighttime sleep quality was associated with various aspects of undergraduate mental health. Specifically, poorer global sleep quality was related to increased externalizing problems, particularly when considering scales associated with aggressive and rule-breaking problems (see reference ). PoorerRegression analyses revealed that nighttime sleep duration and the frequency of nighttime sleep disruptions were differentially associated with total problems and certain DSM-oriented scales. In particular, the analyses showed that reduced nighttime sleep duration and increased frequency of nighttime sleep disruptions were associated with self-reported total problems. Examination of clinically-relevant symptoms of psychological distress on the DSM-oriented scales indicated that nighttime sleep disruptions were associated with anxiety problems and somatic complaints but not with depressive problems; nighttime sleep duration was unassociated with mental health on each of the analyzed DSM-relevant scales. Taken together, these findings suggest that although nighttime sleep duration and the frequency of nighttime sleep disruptions may be associated with particular aspects of mental health, other specific sleep-related behaviors not analyzed in this report may be influential as well. As evidence of this, we found significant associations between global sleep quality and DSM-relevant depressive problems, but neither nighttime sleep duration nor the frequency of nighttime sleep disruptions were significant predictors in the conducted regression analysis. Future work is needed to determine which particular aspects of poor sleep quality are associated with reduced functioning in these areas.One of the primary strengths of this research was in our inclusion of the ASR as a measure of participant mental health. This self-report instrument is a clinically-validated measure of mental health that allows for the analysis of both externalizing and internalizing problems as well as more specific behavioral issues, including those directly related to DSM disorders. As mentioned previously, the ASR has sound psychometric properties and has been used extensively in clinical settings . AlthougIn light of the demonstrated associations between poor sleep and mental health problems in this and other research, future studies should be conducted to examine how existing sleep interventions impact mental health, both in students with sleep problems and in those with generally healthy sleep habits. There is a growing body of literature examining the efficacy of sleep interventions in college populations, with most interventions focused on psychoeducational sleep programs, mindfulness or relaxation training, and cognitive behavioral therapy –44. CognGiven the potential implications of better understanding relations among sleep and mental health outcomes in university students, future research should address some of the limitations of this research and the larger literature more generally. One of the main limitations of this work is the relatively small sample size that resulted from the imposed exclusion criteria. As mentioned, participants routinely went to bed before 2:00am, they commonly slept for at least 6 hours per night, they did not routinely take sleep aids, and they agreed to abstain from caffeine, alcohol, tobacco, and illegal substances during their participation in the study. As such, our sample may not be representative of the general population of university students, who likely experience more severe sleep problems. As evidence of this, only 11% of university students in previous research  receivedAlthough future work is needed, the conducted research indicates that poorer sleep quality is associated with reduced mental health in university students with generally healthy sleep habits; regression analyses further revealed the manner in which nighttime sleep duration and the frequency of nighttime sleep disruptions predicts mental health outcomes when controlling for participant race and ethnicity. Although only a minority of the tested students experienced clinically concerning mental health issues, understanding relations between sleep and mental health in university students with generally healthy sleep habits is important because (a) undergraduate students may experience worsening sleep over time  in assocS1 Dataset(SAV)Click here for additional data file.


This paper summarizes pathological changes that affect microglial cells in the human brain during aging and in aging-related neurodegenerative diseases, primarily Alzheimer’s disease (AD). It also provides examples of microglial changes that have been observed in laboratory animals during aging and in some experimentally induced lesions and disease models. Dissimilarities and similarities between humans and rodents are discussed in an attempt to generate a current understanding of microglial pathology and its significance during aging and in the pathogenesis of Alzheimer dementia (AD). The identification of dystrophic (senescent) microglia has created an ostensible conflict with prior work claiming a role for activated microglia and neuroinflammation during normal aging and in AD, and this has raised a basic question: does the brain’s immune system become hyperactive (inflamed) or does it become weakened (senescent) in elderly and demented people, and what is the impact on neuronal function and cognition? Here we strive to reconcile these seemingly contradictory notions by arguing that both low-grade neuroinflammation and microglial senescence are the result of aging-associated free radical injury. Both processes are damaging for microglia as they synergistically exhaust this essential cell population to the point where the brain’s immune system is effete and unable to support neuronal function. Diseased microglia are different from activated microglia, in that they are incapacitated cells whereas activated microglia are cells capably responding to injury. Microglial disability and dysfunction have now captured the imagination of neuroscientists made apparent by a number of excellent recent reviews with different perspectives on the subject [The idea of “diseased microglia” represents a recent conceptual development in neuropathology that was first raised in the context of Creutzfeldt-Jakob disease . Disease subject –6. Follo subject , additio subject , termed  subject . Microgl subject –12. As tin situ assessment of microglial phenotypes which in conjunction with morphological changes will provide information about cell function and dysfunction in a variety of disease states. In situ description and evaluation of microglia in human brain in terms of their morphology and phenotype is essential for elucidating the role these cells play in neurodegenerative disease pathogenesis. Human histopathology is also important for establishing relevance and represents the first line of investigation for neurodegenerative conditions. Together with human genetic studies, as well as experimental transgenic or other animal or cell culture models of disease addressing molecular mechanisms, true insights into disease pathogenesis can be gained.In this paper, we focus on microglial cell morphology and phenotype as seen by immunohistochemical staining of brain sections. Morphological plasticity is a characteristic feature of microglia that is evident even in the static images of traditional microscopy, as shown here. Modern microscopy methods allowing live cell imaging have confirmed what has long been suspected from cell culture studies , namely in vitro under artificial conditions. Neither the time scales nor the microenvironment created in vitro are representative of human brain [in vitro systems may show what microglia are theoretically capable of doing when stimulated to the maximal extent possible, these results are not validated by human neuropathology and they cannot address the complexities of neurodegenerative disease pathogenesis with direct relevance. A major discrepancy between microglial activation in vitro and in vivo is that in vitro experiments use the proverbial sledgehammer method (LPS or similar) to achieve maximal microglial stimulation in vitro, producing maximal and readily measurable effects, while at the same time it is recognized that the intensity of microglial activation in the normally aged and AD brain is consistent with only minimal, low-level neuroinflammation [in vitro constitutes a flaw in scientific reasoning explained possibly by immature judgment or premature ambition.The currently widespread opinion that activated microglia can be both beneficial and detrimental leaves open the possibility that microglial activation is harmful under certain conditions, and destructive microglial neurotoxicity as a possible factor in neurodegenerative disease has been discussed on numerous prior occasions . an brain , and whiammation –32. To eIt is currently thought that chronic, low-level inflammation is associated with major degenerative diseases of aging including those affecting the CNS ,33. HistIt has been hypothesized that free radical damage in the aging brain causes inflammation  but freeRegarding the microglial changes that occur during aging, these can be seen as being consistent with both mild activation and with senescence. While a rapidly changing immunophenotype and dramatic morphological transformations are part of high-intensity microglial activation after acute CNS lesions, such changes occur only gradually during aging reflecting slowly progressive evolution of a senescent morphology and phenotype, which is similar in its manifestation to low-grade activation described in models of mild CNS injury –54. MicrMicroglial mitosis, which is part of microglial activation in acute injury situations when there is a sudden demand for more cells to restore homeostasis, is absent or minimal during normal aging when such impromptu demands do not occur. However, when acute CNS injuries happen in aged animals the ability of microglia to divide is unimpaired and even greater than in young animals  showing Histopathological studies of microglia can be a valuable diagnostic aid to neuropathologists, a fact that was already recognized many decades ago by del Rio-Hortega, who wrote: “It is sufficient to find the microglial alterations to recognize the areas more deeply affected by the disease” . HistoloTherefore, this most current notion of microglial stability combined with the longevity of humans, complex human genetics, and the many influences created by diverse human lifestyles, diets, habits of drug consumption , latent infections, injuries, and systemic diseases, as well as subtle brain pathologies all of which may influence microglial behavior creates a tremendously heterogeneous picture that is reflected in the diverse microglial phenotypes encountered. This morphological heterogeneity of the microglial population can make it difficult to differentiate between non-activated, activated, and senescent (diseased) microglia since some features may not be unequivocal, and thus morphological assessments are best combined with immunophenotypic ones employing several microglial markers to characterize cells.A key issue is the reliable identification of and distinction between activated and diseased microglia, because this ultimately provides clues about the cells’ (dys) functional activities in any given region of interest. Since there are no single histochemical markers to facilitate simple differentiation multiple markers together with morphological assessments comprise a conclusive evaluation. Both microglial activation and microglial dystrophy are defined originally by characteristic morphological appearances ,72. WithNeuroimmunological differences between rodent and human microglia were discussed recently , and we inflammation is inherently tied to injury and pathology, and it would be a stretch (in our minds) to call normal aging a disease. Perhaps more compelling is to point out that these purely aging-related alterations in rodent microglia are not associated with neuronal pathology, unlike in humans where microglial dystrophy and neurofibrillary degeneration appear to be linked.In the same vein, the term neuroinflammation when applied to rodents in the context of normal aging is inappropriate as in vitro and in vivo studies, the difficulty of reliable identification of activated microglia in human brain, and the fact that neuroinflammation in AD is quite mild and therefore unlikely to be destructive [Neurofibrillary degeneration (NFD), or tau pathology, and synapse loss offer the best neuropathological correlates of cognitive impairment and dementia in AD –85, and tructive . Additiotructive –91; b) atructive –94; c) ttructive ; d) tau tructive –98. For tructive . The lintructive –102.Damaged neurons undergoing NFD are not “obvious stimuli for inflammation” as previously thought  since this clear is the ability of microglia to phagocytize neuronal debris [The idea that detrimental microglial activation also contributes to the degeneration of neurons in the substantia nigra in Parkinson’s disease (PD) is firmly embedded in the literature yet much of it is speculative and extrapolated from animal and cell culture models –107. In l debris ,111,112,l debris , and cerl debris –119. Witl debris ,121, a fl debris ,123. It in vitro [Current understanding of the relationship between microglia and amyloid-beta protein (Aβ), as well as the many questions that have arisen from it, have been discussed in detail recently , and in in vitro ,131. We in vitro –135, areOther than plain cellular senescence, our current understanding of mechanisms that cause or aggravate microglial dystrophy and degeneration is in its infancy. As shown in Figure G93A transgenic rat, an animal model of motor neuron disease [G93A rat where they are evident as cellular aggregations and fusions. Most conspicuous among the latter is the formation of Langhans-type multinucleated giant cells (MNGCs), which is a highly unusual phenomenon to observe in rat brain. In humans, MNGCs are almost always found in disease states associated with immune suppression, notably HIV encephalitis but also lymphomas and tuberculous meningitis, and are thought to occur via fusion of virus-infected microglia [G93A transgenic rats MNGCs were coincident with another bizarre event in rat brain, namely the presence of bacillus bacteria  has also been described in another experimental model of severe neuropathology induced in rats through administration of the G-series nerve agent, soman . In thisOverall, it appears then that microglial dystrophy can be induced experimentally in laboratory rodents under certain extreme conditions, such as genetically induced neurodegenerative disease or acute intoxication. Ultrastructural studies showing severe mitochondrial abnormalities  support The study of diseased glial cells and their potential importance for neurodegenerative disease pathogenesis is in its infancy . Specifia priori reason for such impairment. Moreover, in human brain there is no evidence showing that microglia are attempting to remove soluble, non-fibrillized Aβ accumulating in diffuse plaques. The lack of amyloid removal may therefore not be due to impaired phagocytic ability but instead be caused by toughness and indigestibility and of the amyloid fibrils resulting in the aforementioned “frustration” which wears down the cells and contributes to their exhaustion together with other factors including naturally occurring senescence. Thus amyloid accumulation, unlike NFD development, may not be a consequence of microglial dysfunction.Multiple lines of evidence have suggested that amyloid plaques accumulate and persist because dysfunctional microglia are unable to remove them ,161–163.Metaphorically speaking microglia are hybrids between neuroprotective glia and immunocompetent cells and their functional deterioration plays out in diminished neuroprotection as well as in decreased immunological capabilities. The latter is strikingly illustrated in Figure in vitro observations, and this has not been helpful for advancing understanding of AD pathogenesis. Microglial cells are not aggressors; they are victims of free radical damage like all cells. To help curb the ongoing dementia epidemic a major scientific challenge for the future will be to find ways of slowing or minimizing microglial senescent degeneration.There are aging-related morphological and phenotypic changes in rodent and human microglia that are consistent with both cell senescence and low-grade activation. However, it is all but impossible to distinguish between low-grade neuroinflammatory and senescent changes and these seemingly contradictory phenomena are in fact one and the same. Since the focus is on aging when it comes to AD, it would be wise to stop using the term neuroinflammation as this is suggestive of pathology more severe than what really exists, and therefore potentially misleading. In the context of aging it makes sense to speak simply of microglial senescence, which in humans progresses to an advanced, pathological level, called dystrophy, that can be directly associated with neurofibrillary degeneration. Making this purely semantic change from inflammation to senescence would help eliminate the rather widespread notion that microglia are aggressive immune effector cells, which is the unfortunate result of prematurely overinterpreting and extrapolating