Erectile dysfunction (ED) is a common complaint in hypertensive men and can represent a systemic vascular disease, an adverse effect of antihypertensive medication or a frequent concern that may impair drug compliance. ED has been considered an early marker of cardiovascular disease. The connection between both conditions seems to be located in the endothelium, which may become unable to generate the necessary dilatation in penile vascular bed in response to sexual excitement, producing persistent impairment in erection. On the other hand, the real influence of antihypertensive drugs in erectile function still deserves discussion. Therefore, regardless of ED mechanism in hypertension, early diagnosis and correct approach of sexual life represent an important step of cardiovascular evaluation which certainly contributes for a better choice of hypertension treatment, preventing some complications and restoring the quality of life. Erectile dysfunction (ED) has been defined according to National Institute of Health from 1993 as the persistent inability to reach or maintain and penile rigidity enough for sexual satisfaction . With thCommon risk factor categories associated with sexual dysfunction exist for men and women including individual general health status, diabetes mellitus, cardiovascular disease, other genitourinary disease, psychiatric/psychological disorders, other chronic disease, and sociodemographic conditions. Actually, vasculogenic ED is considered part of a systemic vasculopathy and has a known relation with cardiovascular risk factors such as hypertension, diabetes, dyslipidemia, and smoking. ED has been considered an early marker of cardiovascular risk that could precede traditional clinical manifestations of atherosclerosis, indicating the presence of vascular disease. In addition, ED could alert clinicians to the presence of unknown risk factors and an increased cardiovascular risk. Thus, ED could offer the opportunity to implement adequate therapeutic efforts to minimize the burden of major cardiovascular disease such as myocardium infarction and stroke .As ED is highly prevalent and deeply impacts overall health of sexually active men, sexual function should be part of anamneses in all hypertensive subjects, especially those over 50 years. Ideally, such investigation could be held before starting therapeutic. To stimulate this attitude, the main objectives of this paper are to review some aspects linking ED and hypertension, including arterial hypertension as a risk factor for ED, ED as a marker of cardiovascular risk, ED and antihypertensive drugs, its possible negative impact in therapeutic adhesion, and lastly, actual therapeutic approach of hypertensive men with ED.ED prevalence was surprisingly high at the end of the classic Massachusetts Male Aging Study (MMAS) reaching almost 40% among men at theirs 6th decade of life. Those numbers were similar in more recent publications over the world  and alsoPrevalence's numbers vary according to characteristics of the population studied and the method used to access erectile function. Some trials have used a single question about sexual satisfaction while others have adopted validated questionnaires like International Index of Erectile Function (IIEF) that could check all five major domains of sexuality: sexual desire, erectile function, orgasmic function, intercourse satisfaction, and overall satisfaction with sexual life .Besides this high prevalence, before oral therapy with phosphodiesterase-5 (PDE5) inhibitors, less than 1/4 of men with ED search for medical help . The mosAfter introduction of sildenafil in therapeutic market in 1998 , their cAs a consequence of PDE5 inhibitors basic development studies, erectile process was better understood and several papers from the last decade stressed the association between ED and vascular disease identified by functional and structural changes related to atherosclerosis process . These eIt seems important to remember the complexity of erection physiopathology as well as of the hypothetical link with cardiovascular disease—endothelial dysfunction—since multiple factors could cause ED and interfere in the delicate balance of mediators released from endothelium . But psyNew therapeutic strategies and molecular targets will help to improve quality of erections and sexual satisfaction. In order to cure ED, if it is really possible, some recent studies propose regular use of drugs with proved endothelial action such as statins or PDE 5 inhibitors, taken daily instead of on demand , in ordeSeveral traditional risk factors were related to ED in medical literature with some evidences coming from well designed epidemiological trials. Age seems to be the clearest risk factor with strong association with the presence and severity of ED . After aErection is a complex psiconeurovascular process and involves several system interactions that converge to an increase in hypogastrian-penian blood flow and subsequent activation of veno-occlusive mechanism of corpus cavernosum . It is wIn addition, penile erectile tissue's integrity depends on oxygen tension fluctuations that occur during physiologic erections. As a consequence of regular erections, several cytokines, vasoactive, and growth factors keep a suitable environment for erectile tissue with a protective effect over stroma and muscular cells of this region . In a paSome trials have shown the presence of vascular disease in men suffering from vasculogenic ED but without traditional risk factors, pointing out ED as a clinical early cardiovascular risk marker . ParticuOn the other hand, as a consequence of its multifactorial aspect, several conditions could promote ED without systemic vascular involvement such as pelvic surgeries, depression, Peyronie's disease, and prostatism. Probably this aspect is one among others to explain the lack of additional contribution of ED over traditional risk factors (Framingham score) during cardiovascular evaluation in some reports . Other pOther risk factors have been related to ED. Sedentarism, obesity, and smoking have been implicated in the etiology of ED, and an approach of these risk factors has been able to reverse ED and restore normal erectile function . MetabolThe Treatment of Mild Hypertension Study) [Hypertension is considered one of the most hazardous cardiovascular risk factors and it is a frequent comorbidity of men with ED . One of n Study)  and its n Study)  probablyOn the other hand, Jensen et al. observed that the main reason for ED among hypertensive individuals were penile circulation disability (found in 89%), probably due to atherosclerosis . BurcharThe association of ED and vascular risk factors including hypertension raises the hypothesis that endothelial dysfunction is the common link between erectile dysfunction and cardiovascular disease. But a possible association between ED and hypertension is much more intricate issue involving other aspects, such as the hemodynamic interferences caused by antihypertensive drugs.There is a complex relationship among arterial hypertension and erectile dysfunction that is explained by the multifactorial pathophysiological process that take place in both conditions . So, conIn this sense, it is an usual popular belief to blame medical therapy for hypertension as the main reason of erectile problems, especially when there is a temporal coincidence of starting symptoms and use of antihypertensive drugs, in particular when including the “old” diuretics and beta-blockers . In almoDevelopment of erectile dysfunction in connection with betablockers might be biased by psychological effects derived from the awareness of being treated with a certain substance. This is an important point since patient concerns about the adverse effects of drugs on erectile function might limit the use of essential medications in cardiovascular high-risk patients .In the same way, data with diuretics and ED are not conclusive. A small number of patients and inadequate evaluation of erectile function limit the trials results. On the other hand, the erectile dysfunction substudy in ONgoing Telmisartan Alone and in combination with Ramipril Global Endpoint Trial/Telmisartan Randomized AssessmeNt Study in ACE iNtolerant subjects with cardiovascular Disease (ONTARGET/TRANSCEND) did not demonstrate a significant association of pretreatment with diuretics with erectile function in high-risk patients .nondipper” pattern on ambulatory blood pressure monitoring among treated hypertensive patients were strongly related to worst erectile function independently of the number or the class of antihypertensive drugs in use [It is important to consider that drugs used for treatment of cardiovascular diseases have often been accused of influencing erectile function, and such belief could influence drug compliance . Some aus in use . On the s in use , angiotes in use , and nebs in use .There is no clinical trial evaluating the effect of calcium channel antagonists on erectile function with an adequate assessment of ED, but they are reported to have no relevant effect on erectile function . For druFavorable effects on nitric oxide synthase and oxidative stress have been shown with nebivolol pointing out a mechanism for improvement of erectile function. Experimental studies have demonstrated an enhancement of endothelial function in aorta and corpus cavernosum with a significant reduction in penile oxidative stress and collagen content , protectAlthough no class of antihypertensive agents presents a clearly superior effect over the others in terms of quality of life, the current impression is that nebivolol, ACE inhibitors, and angiotensin II receptor antagonists may offer some advantage, at least in regard to effects on cognitive function and sexual activity .The presence of comorbidities and concomitant drugs, a common situation in older hypertensive subjects, and lack of diagnostic standardization concerning tools to access erectile function impair a reliable analysis of trials about the relationship between ED and hypertension as well as any robust conclusion about deleterious action of antihypertensive drugs on erectile function . In thisSeveral hypotheses try to explain the pathophysiology of ED in hypertensive individuals. Since the pioneering work of Jeremy et al.  who highOld and more recent experimental studies have pointed out the role of nitric oxide (NO) and other possible mediators in endothelial dysfunction of hypertensive rats . In sponCorroborating this link among hypertension and ED, Vlachopoulos et al. identified that hypertensive men had greater carotid intima-media thickness, lower brachial flow-mediated dilation, and higher levels of serum inflammatory mediators than normotensive individuals with similar cardiovascular risk . They suDuring COBRA  trial, Montorsi et al. evaluated 285 subjects with proved coronary arterial disease and identified that ED symptoms began 2 to 3 years before clinical manifestation of coronary disease . Robust As previously commented, the link between both conditions seems to lie in the endothelium which performs many functions that contribute to homeostasis and prevention of atherosclerosis. Endothelial dysfunction is considered one of the earlier steps of atherosclerosis process, preceding angiographic, ultrasonographic, and clinical evidences of vascular disease . These cThe role of NO as a major mediator of erectile pathways such as NO-GPMc and RhoA/Rho-kinase was well documented . Some exClinical evidences seem to confirm this increased vulnerability of cavernosous circulation. Kaiser et al. observed that NO-GMPc pathway was earlier damaged in atherosclerosis process when they followed ED men without cardiovascular disease and compared them with men with normal erectile function. They found that vasodilation of the brachial artery by both mechanisms, endothelium-dependent and independent, was significantly compromised in the group suffering from ED, which illustrates that vascular alterations involved in ED are a generalized process . AccordiAlthough aging and atherosclerosis are recognized risk factors for ED development, according to some experimental studies the mechanism under those situations is distinct . While EDue to the lack of large clinical trials with long followup period designed for ED identification and with cardiovascular morbidity and mortality as endpoints, it is premature to assure that ED identifies an increased cardiovascular risk among hypertensive patients that extends beyond the risk represented by blood pressure elevation . Studies Firstly, it is important to provide an adequate room to talk about sexual dysfunction. It is crucial to listen carefully what patient would like to explain and also to make some questions to check what was understood. The usefulness of standard questionnaires  during dInitial efforts should identify and treat all modifiable risk factors. Next step is a trial with PDE5 inhibitor for those hypertensive subjects without contraindications for sexual activity or for this class of drugs . Thus, kTo assess the individual risk for cardiovascular diseases, repeated measurement of blood pressure and biochemical profile, including fasting serum glucose, creatinine, and lipids, is necessary. Body mass index, according to weight and height, assessment of lifestyle, actual level of physical activity, and potential genetic predisposition should also be obtained. A resting electrocardiogram, should also be documented. If a patient has three or more atherogenic-risk factors, an exercise electrocardiogram should be considered as per the Princeton Consensus Panel. In some high risk patients, a Doppler-sonographic examination of the carotid arteries and lower extremity arteries might also be included into the workup .Cardiovascular disease is not a contraindication to sexual activity in patients who have been properly assessed and treated. In well controlled hypertensive subjects who take one or two antihypertensive drugs and have no other risk factors, indicating low cardiovascular risk, PDE5 inhibitors could be tried. However, among those without treatment or with inadequate blood pressure control or with severe hypertension, it is recommended the cardiologic approach and initial cardiovascular therapy before the prescription of specific drugs to improve erectile function .Patients with diabetes suffering from ED are at special risk for silent cardiovascular disease . TherefoConsidering PDE5 inhibitors mechanism of action, it is reasonable to expect for vascular hemodynamic interferences, effects on blood pressure and coronary circulation alterations, especially among hypertensive patients or those on several antihypertensive drugs. However, blood pressure lowering effects of the current PDE5 inhibitors are low, although oral administration of sildenafil was able to reduce systolic and diastolic blood pressure by 7–10 mmHg in a non-dose-dependent manner . When PDAlthough PDE5 inhibitors have been extensively used for more than one decade, inadequate use is still one major cause of therapeutic failure. Several studies showed that more than a half of ED men leave offices with prescription of PDE5 inhibitor but without correct information about its posology . Most coAfter cardiovascular risk stratification and adequate control of modifiable risk factors, ED patients should be oriented about the right way of sexual medication usage. Although it could sound obvious for doctors, it is prudent to emphasize basic orientation  as improTake PDE5 inhibitor at least one hour before sexual intercourse .Do not take PDE5 inhibitor near (less than two hours) high fat meal or excessive alcohol consumption.Sexual stimulation is essential for PDE5 inhibitors action.Try more than once and ideally in different situations before giving up.Even when patients return claiming nonresponse to PDE5 inhibitor, it is recommended to check all steps where medication were tried and review psychological conditions, partner collaboration, blood pressure control, glucose and cholesterol levels . Low totWhen clinical failure with PDE5 inhibitor is confirmed, urologist should participate and evaluate possible options to allow an active and satisfactory sexual life. Intracavernosous self-injection and penile prostheses constitute the most common path for men refractory to oral therapy . But bes Management of hypertension should take into account, especially in elderly, the possible negative impact of antihypertensive drugs on the patient's quality of life, the deterioration of which may reduce treatment compliance . AlthougKnowledge about sexuality seems important as men and women increased their life expectance and want to keep quality of life during aging. Erectile function preservation is essential for sexually active man while having a lower weight for other who has no intercourses. A sexual evaluation in GP visits could detect ED earlier and also help in risk factors therapy customization, avoiding certain drug classes that could disturb sexuality . BesidesAnother interesting aspect of ED knowledge in hypertensive subjects approach is that some evidences point out to an improvement in blood pressure control maybe as a consequence of better therapeutic compliance after PDE5 inhibitor therapy . LikewisRecent guidelines establish that patients without obvious etiology for ED, such as anatomical disorders, neurological disease, or endocrine causes, should be evaluated for cardiovascular risk factors, arterial hypertension, and arterial disease . The relDespite high prevalence of ED among hypertensive men, it is not often matter of discussion during consultation with general practitioners. An adequate approach to identify sexual health in primary care is needed to identify sexual dysfunctions earlier and also to individualize treatment, to prevent that this situation contributes to a reduction of therapeutic adhesion and even worsens the quality of life among people who already suffer from AH and other traditional risk factors.Sexual function knowledge by general practitioners and cardiologists is relevant and should be ideally accessed before starting therapy for hypertension. In addition to the high prevalence of ED among hypertensive men, its association with the severity of hypertension, and possible interference after the introduction of antihypertensive medications justify the importance of including a brief sexual anamnesis during clinical consultations.After ED diagnosis and for those without contraindication for sexual activity or PDE5 inhibitors usage, doctors must take time to adequately inform their patients about the correct way to take sexual medication. Oral therapy failure could represent a deeper vascular damage and worst cardiovascular risk and generally requires the aid of the urologist in order to restore hypertensive man sexual life.


Obesity in the childbearing population is increasingly common. Obesity is associated with increased risk for a number of maternal and neonatal pregnancy complications. Some of these complications, such as gestational diabetes, are risk factors for long-term disease in both mother and baby. While clinical practice guidelines advocate for healthy weight prior to pregnancy, there is not a clear directive for achieving healthy weight before conception. There are known benefits to even moderate weight loss prior to pregnancy, but there are potential adverse effects of restricted nutrition during the periconceptional period. Epidemiological and animal studies point to differences in offspring conceived during a time of maternal nutritional restriction. These include changes in hypothalamic-pituitary-adrenal axis function, body composition, glucose metabolism, and cardiovascular function. The periconceptional period is therefore believed to play an important role in programming offspring physiological function and is sensitive to nutritional insult. This review summarizes the evidence to date for offspring programming as a result of maternal periconception weight loss. Further research is needed in humans to clearly identify benefits and potential risks of losing weight in the months before conceiving. This may then inform us of clinical practice guidelines for optimal approaches to achieving a healthy weight before pregnancy. Obesity in pregnancy is increasingly common in developed nations –3. This There is well-established animal research which focuses on the topic of periconceptional undernutrition that illuminates these potential risks to offspring. Corroborating this are data from cohorts of people conceived in the Dutch Famine during World War II and in seasonal famines in The Gambia suggestive of long-term health consequences. With the exception of these epidemiological studies from the Dutch Famine, human studies examining offspring outcomes following periconceptional undernutrition are lacking. It is important to determine whether benefits of weight loss during this period are in conflict with potential risks to the long-term health of offspring. Clarifying the critical windows of early development may then lead to the development of evidence-based guidelines for safe weight management in the obese obstetric population, with long-term offspring health in mind.Overweight and obesity are increasingly common in the obstetric population, affecting up to one-third of childbearing women in developed countries , 3, 13. The consequences of obesity for reproductive health range from reduced fertility to increased incidence of gestational disease and adverse neonatal outcomes.Obesity is associated with reduced fertility  and is aSome pregnancy complications associated with obesity are themselves risk factors for subsequent disease in mothers. Women who have had gestational diabetes incur at least a sevenfold increased risk of type 2 diabetes  and preeNeonatal morbidity associated with maternal obesity includes hypoglycemia, respiratory distress, macrosomia, birth defects, and greater rate of admission to neonatal intensive care , 5, 49. Several of the complications associated with maternal obesity are not only a risk to health at the time of birth but are also risk factors for adult disease in the offspring . MacrosoMechanisms for how obesity predisposes to adverse outcomes are not yet clearly understood. Developmental pathways likely to be involved include central mechanisms regulating appetite and peripheral mechanisms involving insulin sensitivity and cardiovascular regulation . On the Given the evidence for poorer pregnancy outcomes associated with obesity, current National Institute for Health and Care Excellence (NICE), American Congress of Obstetricians and Gynecologists (ACOG), and the Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG) guidelines advocate for a BMI within the normal range prior to falling pregnant –15. The Weight loss as a means for improving health in overweight and obesity has been widely validated, but there are currently no randomized controlled trials evaluating pregnancy outcomes after weight loss prior to pregnancy. A population-based study showed that a moderate reduction (4.5 kg) in prepregnancy weight is associated with lower risk of gestational diabetes . Other sThe period of maximal weight loss following Roux-en-Y procedures is typically 12–18 months, after which weight plateaus. Retrospective studies have found no difference in rates of obstetrical complications between those who conceive during this period of weight loss and those who conceive after this period , 62. LonIn order to confidently advocate for weight loss prior to pregnancy, the health profession must consider the potential risks associated with this behaviour change. Up to half of pregnancies are unplanned  and many“Periconception” refers to “developmental stages which include some or all of the following early events: oocyte maturation, follicular development, conception, and embryo/blastocyst growth up until implantation” . The per IGF gene compared with their unexposed siblings of the same sex [Epidemiological data from the Dutch Famine provide insight into potentially life-long consequences of undernutrition in the periconceptional and early gestational periods . This 5-same sex .Interesting patterns relating to maternal undernutrition and gestational length have also been reported from The Gambia , 83 Tab. PregnanResearch on animals, primarily ewes, provides support for many of the findings in epidemiological studies and highlights additional consequences of maternal undernutrition . Some ofPericonceptional undernutrition is associated with changes in offspring HPA axis function from early gestation to adulthood. The programming and subsequent function of the HPA axis has broad implications for health, as disturbed HPA axis function is associated with obesity and cardiovascular-related mortality , 87.Activation of the HPA axis in sheep is essential for initiating parturition . MaternaAbnormal exposure to maternal glucocorticoids potentially explains altered fetal HPA axis function. In the same study that reported earlier activation of fetal HPA axis following periconceptional undernutrition, maternal ACTH and cortisol levels were suppressed during undernutrition and for several weeks following refeeding . PlacentFunctional differences in the HPA axis persist postnatally following exposure to undernutrition around conception and early gestation, despite adequate nutrition throughout the remainder of gestation. There are variable findings reflecting different methods of undernutrition, although there consistently appears to be a difference in HPA axis function from controls. In offspring undernourished from preconception into the first 30 days of gestation, cortisol response is suppressed following arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) stimulation . Convers IGF2/H19 differentially methylated region (DMR) [ IGF2 as adults compared to their unexposed sex-matched siblings [Alterations to the growth and function of the HPA axis that persist postnatally following periconceptional undernutrition are potentially mediated by epigenetic modifications to molecules implicated in adrenal growth and steroidogenesis. Specifically, insulin-like growth factor 2 (IGF-2) and its receptor IGF-2R are parentally imprinted genes involved in these processes . Zhang eon (DMR) . Interessiblings .Thus, the periconceptional period is an important stage in which nutrition status may influence programming of the HPA axis.Regulation of fetal growth and postnatal body composition appear to be at least partially programmed by the maternal reproductive environment in very early development.Periconceptional undernutrition alters the normal relationships of early gestational weight gain with placental and fetal growth . FollowiIn male offspring only, periconceptional undernutrition increases percent fat mass, restricts linear growth, and leads to smaller relative organ weights in adult sheep . This isAlterations to the HPA axis following periconceptional undernutrition are not limited to adrenal gland structure and function. The hypothalamus is an important locus of control in energy homeostasis, particularly via feeding and activity drives. Persistent epigenetic modification of the hypothalamic proopiomelanocortin (POMC) and glucocorticoid receptor (GR) genes in late gestation offspring exposed to periconceptional undernutrition is likely to cause abnormal feeding regulation, as well as energy metabolism and expenditure in later life , 96. OffA potential pathway of altered fetal growth following periconceptional undernutrition is glucose metabolism. While there is evidence for the influence of late gestational nutritional status on later-life glucose metabolism , the perInsulin resistance is a normal adaptation to pregnancy, allowing for greater availability of glucose to the fetus . PreconcAltered insulin response to glucose is evident in late gestation , and at In adult offspring, periconceptional undernutrition in normal weight ewes results in impaired glucose tolerance . This isEpigenetic modification of hepatic insulin-signalling molecules may explain these fetal and postnatal differences in glucose metabolism following periconceptional undernutrition. Nicholas et al.  found a In addition to maladaptive metabolism, adult disease is largely influenced by impaired cardiovascular function. Hypertension is an important component of the metabolic syndrome and is strongly related to cardiovascular disease .Cardiovascular adaptations to periconceptional undernutrition are apparent in late gestation, demonstrated by increased fetal blood pressure . In twinTorrens and colleagues  found thWhile there are well-established links between overtly elevated basal cortisol and hypertension , there iPericonceptional undernutrition is associated with a number of differences in fetal and postnatal measures, many of which appear to be related to the HPA axis. Some of these modifications may act as compensation in the immediate period to protect and prepare the offspring for an adverse environment. Further development in an environment with abundant energy availability may then predispose the offspring to metabolic and cardiovascular disease.Further clarification is required as to which periods of development are most important and therefore most sensitive to undernutrition. With respect to human pregnancy, which is rarely diagnosed until several weeks gestation, it is likely that undernutrition at any point in periconception will have consequences.The mechanisms by which the nutrition insult results in long-term changes in the offspring are still unclear. Ongoing research into the role of epigenetics will probably contribute to further understanding of programming response to adversity.Finally, human studies are lacking in the field of weight loss prior to pregnancy. Given the animal data, both the benefits and risks of losing weight prior to conception need to be identified in clinical populations. This is necessary to design evidence-based practice guidelines for managing obesity in the obstetric population.


Rafael Dal-Ré and colleagues discuss how incidental findings are likely to be viewed as potential benefits of research participation in genomics trials, and investigators should implement mechanisms to ensure provision of timely and appropriate care. Ensuring provision of such interventions in countries lacking a universal public health care system may prove challenging.Please see later in the article for the Editors' Summary Genome/exome data are likely to play an increasing role in clinical trials, and incidental findings are likely to be viewed as potential benefits for individuals of research participation.Participants in clinical trials across differing trial sites utilizing genome/exome sequencing information should be afforded the same standard of care, including return of incidental genomic findings.Participants may opt in to receiving incidental genomic findings, and clinical trial investigators should implement mechanisms to ensure provision of timely and appropriate care to prevent or ameliorate conditions associated with incidental findings.Ensuring the provision of such interventions in countries lacking a universal public health-care system may prove more challenging than in countries with public health-care support.Three basic principles are key in assessing the ethics of any research conducted on humans: respect for persons (or autonomy), beneficence, and justice Increasingly, clinical trials to develop new drugs and biologics involve whole genome or exome sequencing (WGS/WES), including for biomarker characterization, for identification of genomic risk factors, and for population-based research In response to growing recognition that actionable incidental genomic findings could be of value for patient care, the American College of Medical Genetics and Genomics (ACMG) published recommendations for management of incidental genomic findings obtained in clinical practice Example 1 Genes/diseases:BRCA1, BRCA2/breast and ovarian cancersAge of onset: Breast cancer, ≥25 years old; ovarian cancer, ≥40 years oldPrevention interventions for women:Breast cancer: <25 years old: annual clinical breast exam; >25 years old: surveillance ; clinical breast exam (every six months) or prophylactic bilateral mastectomy; chemoprevention: not provenOvarian cancer: 30–35 years old: periodic screening ; >35 years old: prophylactic bilateral salpingo-oophorectomyExample 2 Genes/diseases:PKP2, DSP, DSC2, TMEM43, DSG2/arrhythmogenic right ventricular cardiomyopathy/dysplasiaAge of onset: Usually from adolescence onwards (4–64 years old)Prevention interventions:Prevention of sudden cardiac death by dramatically reducing exercise and discontinuing competitive athleticsPrevention or delay of disease progression: beta-blockers and angiotensin-converting enzyme inhibitorsElectrocardiogram monitoring Example 3 Genes/diseases:LDLR, APOB, PCSK9/familial hypercholesterolemiaAge of onset: homozygous: adolescence; heterozygous: men—heart attacks in ≥40 s (85% had one by age 60); women—heart attacks in ≥50 sPrevention interventions:Children and adolescents: dietChildren and adults: lifestyle interventions Drugs to be considered : statins, bile and sequestrant resins, niacin, ezetemibe, gemfibrozil, fenofibrate, and othersEmploying WGS/WES in clinical trials could advance the development of new treatments and diagnostic tools for many disorders and conditions, for instance, in oncology In recent years, the idea that investigators have “ancillary care” obligations to their study participants has emerged Although the potential benefit associated with identification of an incidental genomic finding resides largely in its informational value, for individuals to receive the full benefit of learning of an incidental genomic finding, subsequent preventive interventions would need to be undertaken to prevent or ameliorate the manifestation of the associated condition. Ensuring that this benefit is distributed fairly to all participants in a clinical trial involving WGS/WES presents particular challenges.Because justice demands that similar people be treated similarly and that irrelevant differences between them should not result in differential benefit If the standard of care for clinical WGS/WES evolves to include actively seeking a list of genetic variants associated with actionable medical conditions, then participants in clinical trials involving WGS/WES may justifiably expect to receive sequencing information to promote their health Varying types of health-care systems at trial sites could present a significant challenge with regards to the health-care delivery response to incidental genomic findings. If an incidental genomic finding is to be reported to a trial participant, the investigator would be responsible for provision of comprehensive counseling including discussion of available intervention choices. While, ideally, all treatment and care choices resulting from participation would be provided free of charge to a trial participant, in reality, access to interventions will vary based on the clinical trial site. If the country where the trial is conducted has a universal public health-care system, it would seem reasonable that the public system would provide treatment in advance of disease manifestation. This is reasonable since if the trial participant had not undergone the WGS/WES, and hence had not received any intervention to prevent the development of the disease, the public health care system eventually would have had to address treatment of the disease when it manifested.However, in regions lacking a universal public health-care system, a clinical trial sponsor could justifiably be held responsible for implementing mechanisms to ensure the provision of the interventions needed by all participants facing medically actionable incidental genomic findings. Institutional Review Boards/Research Ethics Committees (IRB/RECs) would be expected to approve trial protocols with WGS/WES only if all potential participants are assured in the informed consent process that any incidental genomic findings will be properly managed, as per the standard of care. Although important in any trial, this is especially relevant in international clinical trials, where the sponsor has an obligation to fulfill the requirements of the principle of justice across all countries and settings. Inadequate intervention in response to incidental genomic findings resulting from WGS/WES should not be a means of discrimination among trial participants within the same or across different countries. Relevant IRB/RECs should review the mechanisms of providing the appropriate standard of care and the nature, duration, and limits of interventions to be provided.Because most conditions associated with actionable genetic variants (such as those listed by the ACMG) will require long-term management, sponsors could be tempted to conduct trials involving constitutional WGS/WES only in countries with universal public health-care systems. This would have an unethical discriminatory effect, both depriving members of some countries or communities of the potential benefits of clinical trial participation and failing to demonstrate the relevance of trial outcomes for members of those populations. To avoid this potential injustice, sponsors should consider securing a health-care insurance agreement for trial participants to assure access to the interventions needed to manage any reported incidental genomic findings.All participants in clinical trials, including international trials, have the right to be treated with the standard of care. In countries—including low- or middle-income countries—where the standard of care may be very different from that of a wealthy country (or may not even exist), it has been suggested in other circumstances Finally, some commentators The number of trial participants likely to be affected is relevant for estimating the likely cost of managing incidental genomic findings as an obligation of ancillary care. One (or more) mutations—if the ACMG list would be applied—is presumed to be present in 1% of all trial participants The ACMG recommendation that incidental genomic findings be reported regardless of a patient's preference regarding whether to receive this information Emerging data show that some individuals want to receive all types of WGS/WES information—including those genomic results that cannot be interpreted We feel that in a research context, participants' autonomous decision not to know genomic information should be respected. To respect the autonomy of clinical trial participants, explanation of the possibility that WGS/WES will result in incidental genomic findings should be disclosed as part of the consent process. Participants should receive comprehensive pre-sequencing counseling and be informed of the options to learn or not learn of any resulting incidental genomic finding. They should also have the option of changing their minds with regard to learning of incidental genomic findings at any point during the trial In anticipation of genome sequencing becoming commonplace in clinical trials, from the beginning, the scientific community should be respectful of the rights of all participants, regardless of where they are recruited. Among these rights is the right to receive the standard of care regarding reporting of incidental genomic findings and provision of interventions necessary to prevent or ameliorate medical conditions associated with those findings. The challenges of developing a common approach for clinical trials involving WGS/WES warrant an open debate among stakeholders  in the clinical research enterprise.Alternative Language Summary Points S1Spanish translation of the Summary Points by R D-R.(DOCX)Click here for additional data file.


Inflammatory bowel diseases (IBD)—and in particular Crohn's disease—are immune-mediated processes that result in denuded intestinal mucosa and can produce decreased appetite, weight loss, and systemic inflammation. Current treatments include anti-inflammatory medications, immunomodulators, and feeding interventions. Ghrelin is an endogenous orexigenic hormone that directly stimulates growth hormone release, increases gut motility, and has cardiovascular and anti-inflammatory properties. Although ghrelin levels are elevated in active IBD, administration of ghrelin in most  animal models of colitis has produced improvements in disease activity and systemic inflammation. The mechanism for these effects is not known but may relate to decreased inflammation, increased motility, increased appetite, and increased colonic blood flow. Human trials have not been performed, however, and more research is clearly needed. Inflammatory bowel diseases (IBD)—including Crohn's disease and ulcerative colitis—are immune-mediated processes that result in denuding of intestinal mucosa . TreatmeOne intriguing agent that has been shown to ameliorate IBD in animal models is the orexigenic hormone ghrelin –6. FolloThis paper will focus on data regarding the physiology of ghrelin in the setting of IBD, its effects in the setting of animal models of IBD treatment, and potential mechanisms whereby ghrelin may act to improve pathophysiology of IBD. As we will see, though the exact mechanism of ghrelin's action in IBD is uncertain, its efficacy in reducing local inflammation in animal models warrants consideration as a possible future treatment of IBD in humans.While small amounts of ghrelin are expressed in the hypothalamus, small intestine, and other tissues, ghrelin is produced primarily by gastric endocrine cells. Ghrelin is traditionally thought of as a meal-initiating hormone, given that levels increase just prior to meals and fall after meals , though Ghrelin is the only O-octanoylated peptide in the body, with a preference for C8 fatty acids added to a serine moiety by the enzyme ghrelin O-acyl transferase (GOAT) , 21. TheGrowth Hormone Stimulation. Ghrelin acts on receptors in the pituitary to directly stimulate growth hormone release ..69].Ghrelin has been shown to decrease levels of inflammatory cytokines, when administered to isolated leukocytes and when given in models of disease. Systemic administration of anti-inflammatory agents such as infliximab in IBD has clearly decreased local disease activity as well as systemic activity. The decrease in systemic inflammation seen following ghrelin administration in these models of colitis could be a primary effect or due to a decrease in local disease activity mediated by another effect of ghrelin.None of the reports of ghrelin administration in colitis have included levels of growth hormone or IGF-1, though studies of GHS-1a agonist treatment in cachexia have reported increased levels of IGF-1 . Use of Ghrelin's increase in intestinal motility and decrease in transit time could theoretically decrease exposure of intestinal mucosa to agents responsible for local inflammatory reaction. These agents could include products of commensal bacterial or—in the setting of animal models of colitis—treatment agents such as TNBS and DSS.As mentioned previously, enteral feeding continues to be used in some settings of human IBD. While increased food intake in the setting of ghrelin treatment is a plausible mechanism by which ghrelin may improve inflammatory bowel disease, none of the current reports on ghrelin administration have reported increases in food intake, even in cases of increased body weight.Konturek et al. noted an increase in colonic blood flow among colitic rats treated with ghrelin . While gClearly many unanswered questions remain regarding the potential role of ghrelin as a treatment for IBD. Chief among these is whether ghrelin would have any benefit among humans—and if so, at what dose and route. Preliminary studies of ghrelin in humans with cachexia have been promising regarding the lack of significant side effects and the emergence of small molecule GHSR-1a agonists that have a longer half-life than ghrelin itself . A key dIn conclusion, ghrelin is an endogenous hormone that is involved in appetite stimulation, growth hormone release, and gut motility and which appears to have anti-inflammatory properties. Though ghrelin levels appear to be elevated in Crohn's disease, treatment with exogenous ghrelin in animal models has been shown in some—but not all—studies to improve disease course. While this efficacy is promising, it remains to be seen whether ghrelin treatment in humans with IBD could overcome the adverse processes leading to appetite suppression, inflammation, and gut motility. Also uncertain are the mechanisms by which ghrelin may produce these effects. Clearly, more investigation is necessary.


In recent years, polypharmacology is becoming an increasingly important aspect in drug design. For example, pharmaceutical companies are discovering more and more cases in which multiple drugs bind to a given target (promiscuous targets) and in which a given drug binds to more than one target (promiscuous ligands). Both of these phenomena are clearly of great importance when considering drug side-effects. Given that screening drugs against all the proteins expressed by the human genome is infeasible, several computational techniques for predicting the pharmacological profiles of drugs have been developed, ranging from statistical analyses of chemical fingerprints and biological activities  to 3D doHere we present a novel shape-based approach which uses spherical harmonic (SH) representations ,3 to comet al.[In this contribution, we present details of our approach applied to a subset of the MDL Drug Data Report (MDDR) database containing 65367 compounds distributed over 249 diverse pharmacological targets for which experimental binding information is known. The similarity of each ligand to each target’s ligand set is quantified and used to predict promiscuity. To our knowledge, this is the largest all-against-all polypharmacological study to have been carried out using shape-based techniques. We compare our promiscuity predictions with computational and experimental results obtained by Keiser


There is currently no cure for muscular dystrophies, although several promising strategies are in basic and clinical research. One such strategy is cell transplantation with satellite cells (or their myoblast progeny) to repair damaged muscle and provide dystrophin protein with the aim of preventing subsequent myofibre degeneration and repopulating the stem cell niche for future use. The present review aims to cover recent advances in satellite cell/myoblast therapy and to discuss the challenges that remain for it to become a realistic therapy. Muscular dystrophies comprise a large group of heterogeneous genetic disorders characterized by progressive muscle weakness and degeneration, which vary with respect to severity, the muscle groups affected and the involvement of the heart Satellite cells are the principal skeletal muscle stem cell. They reside between the sarcolemma and basal lamina of muscle fibres and are mitotically quiescent until required for growth or repair. Upon receiving activation signals, they rapidly proliferate to produce a pool of myoblasts that fuse with each other to form nascent myofibres and/or with damaged fibres to repair them. A small minority do not differentiate but, instead, re-enter quiescence to maintain the stem cell pool There is a body of experimental evidence to support the hypothesis that not all satellite cells are functionally equivalent. Only a minority of satellite cells contributes to muscle regeneration et al. The findings that stem cells other than satellite cells  could also contribute to muscle regeneration led to studies moving away from using satellite cells/myoblasts, towards atypical stem cells et al. Cell therapy (i.e. the delivery of myogenic cells to enact muscle repair) has been considered as a potential therapy for DMD for many years, ever since Partridge Strategies to overcome some of these problems include improved immunosuppression, the injection of more cells but in smaller volumes to prevent ischaemic necrosis, and high-density injection protocols to aid migration Clinical trials, using autologous myoblasts, for muscular dystrophies that affect only subsets of muscles, namely oculopharnygeal muscular dystrophy  and facioscapulohumeral muscular dystrophy  are currently underway (initiated in 2005). These have the benefit of not requiring immunosuppression. Oculopharnygeal muscular dystrophy is characterized by late onset eyelid drooping (ptosis) and dysphagia  as a result of dystrophy of the pharyngeal muscle. Myoblasts from unaffected limb muscles were grafted into the pharyngeal muscle of patients, following on from promising preclinical trials conducted in the beagle dog A major disadvantage of lentiviruses is that they can carry only a relatively small DNA insert of up to 10 kb in vivoWhy is it that myoblasts do not perform as well as satellite cells? When placed in tissue culture, the majority of satellite cells proliferate rapidly, although a minority divide slowly and it is the latter that contribute more extensively to muscle regeneration The ability to guide the behaviour and fate of stem cells in culture is hindered by a limited understanding of the niche composition and the regulation that it imposes on satellite cell fate. The niche comprises both biochemical  and biophysical  factors that direct stem cell fate It is now well recognized that oxygen tension is an important component of stem cell niches. Most tissue culture is performed using atmospheric levels of oxygen (20%) when, in reality, tissue levels are much lower, usually 2–9% 14.4–64.8 mmHg) depending on the tissue; even within a tissue, there is considerable variability depending on the proximity of cells to blood vessels .8 mmHg det al. et al. in vivo in cardiotoxin injured mdx mouse muscles Two recent studies highlight the benefits of using more physiological levels of oxygen in the cultivation of myoblasts. Duguez in vitro, has largely been ignored. Using a bioengineering approach, Gilbert et al. in vitro and, more importantly, result in extensive muscle regeneration in vivo compared to traditional culture on plastic The importance of physiological tissue rigidity has long been suspected but, as a result of the complexity of reflecting this et al. Most satellite cell research is carried out using mouse cells because only very low numbers of human satellite cells can be obtained by muscle biopsy, which are then cultured to increase the cell number and thus become myoblasts. Recently, Latil Satellite cells are absolutely necessary for muscle regeneration −/−γC−/− immunodeficient mice improves donor-derived muscle regeneration by extending the window of proliferation, increasing migration and delaying differentiation in vivo to stimulate differentiation of the donor myoblasts in vivo evidence for pro-inflammatory macrophages having a supportive role in the regulation of myoblast behaviour after engraftment into pre-injured muscle Coinjection of pro-inflammatory (but not anti-inflammatory) macrophages, along with human myoblasts, into regenerating muscle (injured by cryodamage) of Rag2et al. Another vital component of the regenerating niche is muscle connective tissue (MCT) cells , including fibroblasts and dual potential fibro/adipoprogenitors (FAPs) The effect of ageing on satellite cell function is a matter of much debate because the loss of skeletal muscle mass and function with increasing age (sarcopenia) is of great importance in ageing western populations. However, despite evidence that the satellite cell niche deteriorates with age et al. http://www.acceleronpharma.com/products/ace-031/; ClinicalTrials.gov Identifier: NCT01099761).There is a plethora of studies in mice examining ways of augmenting the regenerative potential of myoblasts. Preventing cell death, increasing proliferation and/or migration, and decreasing early differentiation have all been shown to have a positive impact on mouse and human myoblast transplantations in immunodeficient mice. For example, upregulating the heatshock response (Hsp70 protein) improves both mouse and human myoblast survival, leading to increased engraftment in vivo, several studies have linked this with the limited proliferation of the injected myoblasts. When human myoblasts are injected into the cryodamaged muscles of Rag2−/−γC−/− immunodeficient mice, in medium containing serum, which is rich in growth factors, rather than NaCl/Pi, the window of proliferation is extended from 3 to 5 days. This increases migration, leading to enhanced regeneration as a direct result of slower myoblast differentiation in vivoConcerning the limited migratory capacity of human myoblasts A major problem with the intramuscular injection of myoblasts in the human clinical trials was that regeneration (dystrophin positive fibres) appeared to be limited to damaged muscle along the injection trajectory. This was also seen in primate experiments et al. et al. et al. et al. et al. in vitro differentiation, and notably generating a large number of muscle fibres upon intramuscular transplantation into immunodeficient dystrophic mice et al. et al. Sgca, encoding α-sarcoglycan) to correct the genetic defect and with MyoD to induce myogenic differentiation. Importantly, donor cell engraftment into Sgca-null immunodeficent mice, was obtainable using both intramuscular and inter-arterial injections, as indicated by α-sarcoglycan expression iPSCs in vivo. In particular, the effect of factors such as microRNAs, growth factors and extracellular matrix components produced by host cells, including myofibres, blood vessel-associated, stromal and inflammatory cells, and the effect of the host satellite cell niche on donor satellite cell engraftment are particularly relevant to improving donor cell engraftment. We envisage that a combination of tissue culture conditions to promote or retain the ‘stem-like’ myoblasts, with modification of the host muscle environment to enhance donor satellite cell migration, proliferation and self-renewal, will be the way forward.In recent years, there has been both an improved understanding of the biology of satellite cells themselves, together with increasing knowledge on the effect of the host skeletal muscle environment on their function Because satellite cells and their progeny myoblasts Even in the era of molecular therapies, myoblast or other stem cell therapies are still highly relevant. Although potential treatments for DMD such as exon skipping are promising, exon skipping is neither applicable to all DMD patients, nor will it restore lost muscle fibres. An effective stem cell-based treatment will therefore be a powerful alternative, or adjunct, to other therapies.


Despite considerable efforts to tackle childhood obesity, it is recognized as one of the biggest health problems globally. Childhood obesity is a leading cause of many comorbid conditions such as metabolic syndrome and insulin resistance as well as type 2 diabetes. A strong body of evidence suggests that regular exercise without calorie restriction or weight loss is associated with reduced insulin resistance as well as improved insulin sensitivity in overweight and obese adults. However, despite the well-known benefits associated with regular exercise alone, the independent role of exercise training without calorie restriction on insulin resistance is still uncertain in youth. Some studies observed that both the aerobic and resistance type of exercise training without calorie restriction resulted in meaningful changes in insulin sensitivity, suggesting that exercise alone is an effective therapeutic strategy for reducing insulin resistance in overweight and obese youth. However, only few studies are available on the optimal dose of exercise training without calorie restriction or preferred exercise modality for reducing insulin resistance, which warrants further investigations in the pediatric population. The prevalence of childhood obesity has been increasing throughout the world during the past few decades . Accordiβ-cell function, thereby developing type 2 diabetes in youth [Independent of ethnicity, age, and sex, obesity represents the most important risk factor for insulin resistance as well as increased circulating levels of insulin, leading to decreased insulin sensitivity and impaired in youth . In addiin youth . TherefoIn adults, a number of well-controlled studies have reported that insulin resistance is improved with regular exercise training even without calorie restriction or weight loss –16. AlthThus, in this review, we present the available evidence regarding the role of exercise training without calorie restriction for reducing insulin resistance in children and adolescents. In addition, we distinguish the exercise modality for enhancing insulin sensitivity, which should be recommended for the treatment of insulin resistance in this age group.in vivo insulin sensitivity, in nondiabetic children aged 10–16 years (n = 357). They observed significant relationships between physical activity and lower fasting insulin (r = −0.12) and insulin sensitivity (r = 0.18) even after accounting for BMI, percent body fat, and/or waist circumference. In a recent report by Thomas et al. [In adults, it is well established that physical activity and cardiorespiratory fitness are significantly associated with the constellation of diabetes-associated risk factors  as well s et al. , accelerWith regard to the relationship between cardiorespiratory fitness and insulin sensitivity, previous cross-sectional studies in youth have reported conflicting results. Some studies –32 have It is well known that a single bout of aerobic exercise with sufficient duration and intensity can increase insulin-stimulated glucose uptake in contracting skeletal muscles and insulin sensitivity via increased translocation of glucose transporter  to the cell surface , 18. HowA single bout of resistance exercise is also known to enhance insulin sensitivity at least 24 hours after the last exercise session in healthy individuals . HoweverCombined together, the above observations clearly suggest that people should add regular exercise in their daily routine to increase or at least to maintain insulin sensitivity, which may further reduce the increased incidence of insulin resistance or associated risk factors in children and adolescents.r = −0.48) in this study [Although many interventional studies have focused on the beneficial impact of exercise and calorie restriction-induced weight loss on insulin resistance and body composition, growing evidence also suggests that exercise training without substantial weight loss can induce meaningful improvements in insulin resistance in adults –16. Dunc2 peak) resulted in decreased insulin resistance in postpubertal obese adolescents. Indeed, the decreased fasting insulin was significantly correlated (r2 = 0.40) with the decreased visceral adiposity in this study.Despite extensive observations and experiments in adults demonstrating the beneficial effects of aerobic exercise alone on insulin resistance, only limited evidence is available on this issue in children and adolescents . Nassis To the best of our knowledge, there are 6 randomized controlled trials wherein the effects of aerobic exercise on insulin sensitivity were examined in children and adolescent . Meyer eSavoye et al.  examinedTaken together, these observations provide some evidence that engaging in regular aerobic types of exercise in the absence of weight loss or calorie restriction is beneficial to improve insulin resistance in overweight and obese youth.Although majority of studies have implemented aerobic exercise training as an efficient exercise modality for reducing insulin resistance, considerable evidence also suggests a beneficial effect of carefully supervised resistance (or combined) exercise training without calorie restriction on insulin resistance in adults –45. DaviPreviously, Treuth et al.  have demn = 22). The subjects were randomly assigned to either resistance training group  or control group. After the intervention, insulin sensitivity, determined by FSIVGTT, significantly increased (45.1%) in the exercise group, as compared to the controls. In this study, the increased insulin sensitivity was induced independent of cardiorespiratory fitness or body composition changes. Recently, Lee et al. [r = −0.43) and visceral adipose tissue (r = −0.42), suggesting that improvements in body composition may play an important role to enhance insulin sensitivity in obese adolescents. In addition, the authors also noted that as compared with aerobic exercise, resistance exercise may be a better exercise modality to enhance body composition and insulin sensitivity particularly in obese male adolescents.To the best of our knowledge, there are 6 randomized controlled studies wherein the effects of resistance (or combined) exercise training without calorie restriction on insulin sensitivity were examined in children and adolescent . Shaibi e et al.  conductee et al. . FurtherTaken together, despite the lack of conclusive evidence in the pediatrics, the observations demonstrating that resistance exercise training is associated with improvement in insulin resistance are clearly encouraging children and adolescents to participate in resistance type of exercise on daily basis. However, future studies are also required to explore the long-term effects of resistance exercise training on insulin resistance as well as determining the optimal exercise modality, as the effective therapeutic strategy to reduce insulin resistance in this population.The dramatic increase in childhood obesity in recent years is paralleled by decreased physical activity as well as cardiorespiratory fitness levels, which is also attributed to increase insulin resistance and diabetes-associated risk factors in children and adolescents. As observed in adults, an increasing body of evidence suggests that acute physical activity and chronic exercise have a beneficial effect on reductions in insulin resistance through multiple adaptations such as improved insulin sensitivity and glucose uptake of skeletal muscles and body composition changes in overweight children and adolescents. Studies also demonstrated that both aerobic and resistance type of exercise alone  resulted in significant improvement in insulin sensitivity, suggesting that exercise alone is an effective therapeutic strategy for reducing insulin resistance in overweight and obese children and adolescents. However, future studies are required to explore the optimal dose of exercise and efficacious modality to elicit meaningful reductions in insulin resistance in youth.


Rhinopithecus and Nasalis) has likely evolved as single-male harem systems coalesced, whereas the multilevel system of papionins  and hominins most likely arose as multimale–multifemale groups split into smaller units. We hypothesize that, although ecological conditions acted as preconditions for the origin of multilevel systems in all three clades, a potentially important catalyst was intraspecific social threat, predominantly bachelor threat in colobines and female coercion/infanticide in papionins and humans. We emphasize that female transfers within bands or genetic relationships among leader males help to maintain modular societies by facilitating interunit tolerance. We still lack a good or even basic understanding of many facets of multilevel sociality. Key remaining questions are how the genetic structure of a multilevel society matches the observed social effort of its members, to what degree cooperation of males of different units is manifest and contributes to band cohesion, and how group coordination, communication, and decision making are achieved. Affiliative and cooperative interunit relations are a hallmark of human societies, and studying the precursors of intergroup pacification in other multilevel primates may provide insights into the evolution of human uniqueness.Multilevel (or modular) societies are a distinct type of primate social system whose key features are single-male–multifemale, core units nested within larger social bands. They are not equivalent to fission–fusion societies, with the latter referring to routine variability in associations, either on an individual or subunit level. The purpose of this review is to characterize and operationalize multilevel societies and to outline their putative evolutionary origins. Multilevel societies are prevalent in three primate clades: papionins, Asian colobines, and hominins. For each clade, we portray the most parsimonious phylogenetic pathway leading to a modular system and then review and discuss likely socioecological conditions promoting the establishment and maintenance of these societies. The multilevel system in colobines (most notably  Among primates, multilevel social systems comprise several hierarchical tiers that are perhaps better referred to as modular or nested systems. The basal unit is typically a spatiotemporally cohesive one-male unit (OMU) with one adult male and one to several females. These core units aggregate at varying temporal scales and in that way form at least one second (the band) or even higher grouping level (the troop or herd) (Grueter and Zinner Rhinopithecus spp.), proboscis monkeys , and the papionin species hamadryas baboons (Papio hamadryas) and geladas (Theropithecus gelada) as well as humans. Circumstantial evidence indicates that multilevel systems may occur in a few more taxa such as mandrills (Mandrillus sphinx) and uakaris (Cacajao spp.)  Table . HoweverPan troglodytes). However, we would argue that multilevel systems and fission–fusion are different phenomena. In multilevel societies , subunits have stable membership over longer periods and bands are also quite stable in composition. In mm–mf societies with fission–fusion, however, only the higher social grouping level is stable whereas subunits are flexible and unpredictable in terms of size and composition  have also been included in the category of species with fission–fusion  societies with fission–fusion tendencies, as seen in chimpanzees  and the larger social group (band), e.g., Papio hamadryas. On the other hand, when OMUs congregate on an irregular basis and bands are more fluid and not as consistently assembled or behaviorally integrated as in strict modular systems, this would constitute a flexible system, e.g., Theropithecus gelada  normally live in large and relatively cohesive mm–mf groups that are female bonded: males usually disperse out of their natal group while females remain philopatric. Females are arranged into stable linear dominance hierarchies  intermediate systems between autonomous OMUs and modular societies, in which OMUs have various relations with other OMUs, i.e., friendly relations with some OMUs and antagonistic relations with others), and show a tendency toward spatial amalgamation, i.e., home range boundaries become blurred as units share the same space and under some circumstances or in some species organize daily activities in a cohesive manner. Such intermediate modularity has been documented in Trachypithecus pileatus (Stanford T. geei (Mukherjee and Saha Semnopithecus spp.) form large coherent mm–mf/mixed-sex groups (Borries Asian colobines (Presbytini) exhibit several distinct types of social organization  and their social relationships, and hence on the social system of a particular taxon . It could be argued that ecological conditions are not merely permissive, but actually drive the nested nature of the multilevel social system of colobines . First, resources must permit the formation of very large groups, i.e., an abundant and uniformly distributed resource base is required for the formation of bands  that form AMUs of substantial size and are frequently on the outskirts of the group in most multilevel settings  AMUs of substantial size are more or less permanently on the outskirts of the reproductive group in most multilevel settings . We outline two scenarios, the ecological and the social model, of how large groups might have become substructured. The ecological model assumes that splitting was caused by ecology/environment and social factors came subsequently into play. This model has been generally applied to hamadryas baboons. The alternative, the social model, which we favor here, posits that it was mainly social factors that triggered substructuring, but that ecology was a necessary precondition. An alternative to the ecological model is the time constraint model, and an alternative to the social model is the social brain model, both of which provide other possible explanations for substructuring in papionins.sensu Anderson sensu Smuts It is generally thought that a combination of ecological and social factors promoted the emergence of modularity in papionins Bergman . After het al. et al. A similar social feature to the male–female bonds in hamadryas also exists in savanna baboons, i.e., long-term special relationships (“friendships”) between males and females in which the female benefits from male protection  and investment in her infant, while the male may benefit from increased mating opportunities with the female, but the evidence for the latter is inconclusive  are larger than groups of mm–mf baboons .The following hypothetical scenario can be envisaged for hamadryas baboons , which are determined by various ecological needs, such as predation avoidance or optimal habitat use and foraging , may result in an insecure social environment. It is then conceivable that large groups split into OMUs because females started to form clusters with closely related females within a larger group, and later a male attached himself to them. Dunbar argues that increased aggression and reduced reproductive output associated with growing group size resulted in females tending to bond together into small matrilineal groups for mutual protection  to buffer themselves against the stresses and harassment imposed on them by living in large groups. The alternative, that higher levels of sociality in geladas evolved via a merger of OMUs cannot be discounted, but does not seem to fit well with the phylogenetic relationships. Ohsawa  because it determines the ability to process complex information about social relationships , with many societies exhibiting more than one pattern simultaneously. Thus although dispersal is often sex-biased, it may be bisexual, an uncommon feature in other primates Chapais . Patriloet al. In contrast with the situation in hamadryas baboons and geladas, bonds between multifamily groups in humans are strong, as are alliances between groups. Basically, this is because individuals maintain bonds with their natal kin even after joining another group to breed contrary to the situation in other primates (Rodseth Pan paniscus), and gorillas (Gorilla spp.). We briefly review those social systems to prepare the ground for the phylogenetic reconstruction of human modular societies. Chimpanzees form dynamic mm–mf societies, with communities comprising up to 150 members. Members move around in continually changing parties within the community range  that early hominins  were anatomically more similar to chimpanzees than to gorillas in terms of absolute body size and relative brain size supports the ancestral male kin group hypothesis Chapais . The simet al. et al. Further evidence for the ancestral male kin group hypothesis comes from strontium isotope analysis of tooth enamel in hominins. The strontium contained in plants is incorporated into the teeth of mammals ingesting them during the period of enamel mineralization so that the strontium composition of teeth in mature individuals is a likely marker of the geographical location where those individuals grew up  and Paranthropus (3.0–2.5 Ma) probably lived in rather open mosaic habitats, with open woodlands, bushlands, riverine forests, and seasonal floodplains  where ancestral mm–mf troops are hypothesized to have split into smaller foraging units in response to ecological conditions. The smallest units of the foraging parties may have been were lone individuals (as is sometimes the case among human foragers), labile parties or stable subunits, as in hamadryas. It is unlikely that foraging parties were made up of lone individuals because of the considerable predation threat , and made it possible for females to retain a protector male (bodyguard hypothesis). Moreover, male monopolization of isolated units was also facilitated in a habitat of dispersed resources . LCA parties/subunits were prone to coalesce around localized drinking/feeding areas and scarce refugia in an open habitat , factors that promoted band formation. Polygynous subunits subsequently evolved into mostly monogamous units owing to the evolution of constraints on polygyny. Pair-bonding might have later operated as a preadaptation for the evolution of paternal care and the sexual division of labor in response to the evolution of protracted juvenile dependency and high costs of maternity Chapais .In this article, we have attempted to typify modular societies as a distinct form of primate sociality. We have also emphasized that fission–fusion and modularity should be treated as distinct phenomena that can be coupled, but do not have to be. We have developed a theoretical framework based on socioecological theory to picture parsimonious scenarios for the evolution and maintenance of these most complex social systems in a variety of primate lineages. One main point is that the precursors to modularity seem to have been large mixed-sex groups in hominins and baboons that subsequently underwent internal fission, but autonomous OMUs that coalesced into bands in colobines (summarized in Fig. Two unifying elements favoring the maintenance of modular societies are, depending on the species, male cooperation and female dispersal. That males cooperate to compete against other males is widely accepted as a universal human attribute Brown , and it et al. et al. et al. et al. The other unifying element is female dispersal. In colobines, female dispersal within bands likely contributes to interunit tolerance and sporadic affiliation among females and thereby fortifies the maintenance of a modular society. In hamadryas, female dispersal, mainly within band and forcefully executed by males (Swedell


Plasmodium infection do not acquire complete protection against the liver stage of this parasite despite prolonged and frequent exposure. However, sterile immunity against Plasmodium liver stage can be achieved after repeated exposure to radiation attenuated sporozoites. The reasons for this different response remain largely unknown, but a suppressive effect of blood stage Plasmodium infection has been proposed as a cause for the lack of liver stage protection.Human populations that are naturally subjected to Plasmodium yoelii 17XNL, the response generated in mice subjected to daily infective bites from normal or irradiated mosquitoes was compared. The effect of daily-infected mosquito bites on mice that were previously immunized against P. yoelii liver stage was also studied.Using Plasmodium infection. Exposure to daily infected mosquito bites did not eliminate the protection acquired previously with a experimental liver stage vaccine.It was observed that while the bites of normal infected mosquitoes do not generate strong antibody responses and protection, the bites of irradiated mosquitoes result in high levels of anti-sporozoite antibodies and protection against liver stage P. yoelii infected mosquitoes is essentially different, probably because of the blood stage infection that follows normal mosquito bites, but not irradiated. While infective mosquito bites do not induce a protective liver stage response, they also do not interfere with previously acquired liver stage protective responses, even if they induce a complete blood stage infection. Considering that the recently generated anti-malaria vaccines induce only partial protection against infection, it is encouraging that, at least in mouse models, immunity is not negatively affected by subsequent exposure and infection with the parasite.Liver stage immunity generated by irradiated versus normal  Plasmodium sporozoites grants protection against inoculation with non-attenuated sporozoites in mice models and humans [Plasmodium liver stage infections even if exposed to more than two infective mosquito bites daily [Malaria is a widespread mosquito-borne infectious disease that kills almost one million persons per year, most of which are children. Growing scientific knowledge and the recent malaria research trend provide unparalleled opportunities to develop a malaria vaccine. Although the subunit vaccines are the most advanced in research pipelines and field clinical trials , an effed humans ,6. Howeves daily ,8, as ises daily . Using aes daily . In thises daily .In this work, it was observed that daily bites of non-irradiated infected mosquitoes do not induce liver stage protection, while the daily bites of irradiated mosquitoes do. This result again confirms the concordance of observations in mice and in humans regarding liver stage immunity and focuses the questions about generation of liver stage protection on the differences between infection generated by irradiated versus normal sporozoites.Since the use of partially protective malaria liver stage vaccines in the field may soon be a reality , how wouAnopheles stephensii mosquitoes were maintained as described [P. yoelii 17XNL. This parasite line has been grown for over ten years without cloning and is the same used in previous work [P. yoelii 17XNL irradiated (12 krad) sporozoites dissected from the salivary glands of infected mosquitoes, followed two weeks later with a dose of 50,000. For daily mosquito bites, each mouse had two mosquitoes feed on the tail for 3 min. This was repeated daily for 6 weeks. Parasitaemia was followed regularly using thin blood smears from a drop of tail blood.escribed  and infeous work . IrradiaP. yoelii-specific antibody levels, 104 salivary-gland sporozoites in each well were air-dried on glass multiwell IFA slides. Mouse serum was titrated and primary antibody bound to sporozoites was detected using FITC-labelled antimouse IgG (Sigma). A monoclonal P. yoelii CS-specific antibody (2F6) was used as a positive control .For titration of 4 (non-irradiated) P. yoelii 17XNL GFP sporozoites [Challenge of mice was performed in groups of five mice by i.v. injection of 10rozoites , freshlyP < 0.05.Data were analyzed using Prism . ANOVA test was performed as mentioned. Statistics were considered significant if P. yoelii 17XNL are irradiated was followed [To compare the responses of mice to daily normal or irradiated mosquito bites, a protocol previously shown to induce protection in mice when mosquitoes infected with the non-lethal parasite followed . The firfollowed ,15. The To study whether the immune response induced by a vaccine can be affected by subsequent bites of infected mosquitoes, we immunized mice with two doses of radiation-attenuated sporozoites. This immunization protocol that induces only partial liver stage protection was purposely chosen to reflect the situation with current human liver stage vaccine prototypes . After iP. yoelii sporozoites to determine liver stage protection.One week after the last mosquito bite a small volume of serum was obtained from each mouse to determine anti-sporozoite antibody levels and seven weeks later mice were challenged with non-irradiated Parasitaemia was measured in each mouse weekly, confirming that irradiation of sporozoites and mosquitoes was effective; no blood stage infection was found in the group subjected to the bites of irradiated mosquitoes or the group immunized with irradiated sporozoites that did not receive mosquito bites. The data also show that mosquito bites induce a delayed development and lower levels of parasitaemia in mice that were previously immunized with irradiated sporozoites compared to non-immunized mice, probably reflecting the lower liver stage infection levels in the partially immunized mice Figure .When the levels of anti-sporozoite antibodies were determined, we observed that mice subjected to the bites of irradiated, infected mosquitoes develop significantly higher levels of antibodies compared to mice subjected to the bites of normal infected mosquitoes. It is important to note that both groups of mice had received equal numbers of mosquito bites and it is, therefore, expected that each group had received equal numbers of sporozoites.Antibody levels in the groups of mice that were immunized with two doses of the irradiated sporozoite vaccine followed or not by the bites of normal infected mosquitoes showed no significant differences in their anti-sporozoite antibody levels, suggesting that after immunization, the antibody levels are not affected by the bites of infected mosquitoes Figure , even ifP. yoelii-GFP sporozoites. It is important to use a P. yoelii strain that allows us to differentiate the challenge parasites from parasites (in liver or blood stage forms) that were inoculated before by either the immunization protocols or the mosquito bites. This is possible analysing the livers of challenged mice using real-time PCR to amplify the GFP sequence present only the challenge sporozoites. This technique is not as sensitive as the conventional RT-PCR used for detection of liver stage infection, which amplifies sequences within the 18S rRNA that are very abundant in the parasite [To study the level of immunization acquired by the different groups of mice, each mouse was challenged with non-irradiated parasite .P. yoelii-infected mosquito bites per day, which is considered a high transmission rate and, therefore, it is not possible to extrapolate the results to lower frequencies of transmission, to a situation with concurrent bites of infected and uninfected mosquitoes or to infection with different strains of Plasmodium, which would reflect more accurately the conditions in malaria-endemic areas. In particular, the degree of variability of Plasmodium strains that is found in the field may interfere with the protection induced by irradiated infected mosquitoes.Mice were challenged 14 weeks after being subjected to the first infected mosquito bite, when blood stage parasitaemia was undetectable. It was found that, while the liver stages of the GFP challenge sporozoites were not detectable in mice subjected to irradiated infected mosquito bites, mice that had been bitten by normal infected mosquitoes developed a detectable liver stage infection (Table In groups of mice that were immunized with irradiated sporozoites, subsequent infected mosquito bites, which caused a patent blood stage infection Figure , did notPlasmodium liver stage protection [Taken together, these data indicate that the generation of liver stage immunity against malaria is greatly dependent on whether the mosquitoes that transmit infection are irradiated or not. For both mice and humans, it has been extensively demonstrated that irradiated sporozoites induce a protective immune response ,6, howevotection  could beThe analysis of mice that were first partially immunized with irradiated sporozoites and then subjected or not to infected mosquito bites indicates that liver stage protection was not lost after infective mosquito bites that result in blood stage infection. Since the mice were only partially protected, a full blood stage infection took place after mice were bitten by infective mosquitoes. This allowed us to analyze the effect of blood stage infection on the maintenance of protection induced by the vaccination. Contrarily to the immune suppression described when blood stage infection takes place right after the liver stage , the proP. yoelii infected mosquitoes is essentially different: while irradiated mosquitoes induce a protective immune response with high levels of anti-sporozoite antibodies, normal infected mosquitoes do not generate protection and only low levels of specific antibodies. This situation reflects the response of humans observed both in vaccination trials, where irradiated mosquito bites induce protection, and in the field, where years of constant re-infections do not result in sterile protection against the liver stage. The direct comparison of the human immune response to normal versus irradiated mosquito bites is not possible because the conditions of exposure to infective mosquito bites in the field are very different from experimental exposure to irradiated mosquito bites. In directly comparing the response generated by irradiated or non-irradiated infected mosquito bites, this work indicates that the mosquito irradiation is the only essential variable needed to generate protective liver stage response.The results presented show that the liver stage immunity generated in response to irradiated versus normal While infective mosquito bites do not induce a protective liver stage response, they also do not interfere with previously acquired liver stage protective responses, even if they induce a complete blood stage infection. Considering that the recently generated anti-malaria vaccines induce only partial protection against infection, it is encouraging that, at least in mouse models, immunity is not negatively affected by subsequent exposure and infection with the parasite.The authors declare that they have no competing interests.TP performed mice mosquito daily bite infections and measured parasitaemias. RL carried out mice immunizations, real-time PCR experiments and contributed to the writing of the manuscript. CGR carried out antibody quantifications. KAW conceived the study, participated in its design and trained TP. AR conceived the study, participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.


Female signals of fertility have evolved in diverse taxa. Among the most interesting study systems are those of multimale multifemale group-living primates, where females signal fertility to males through multiple signals, and in which there is substantial inter-specific variation in the composition and reliability of such signals. Among the macaques, some species display reliable behavioural and/or anogenital signals while others do not. One cause of this variation may be differences in male competitive regimes: some species show marked sexual dimorphism and reproductive skew, with males fighting for dominance, while others show low dimorphism and skew, with males queuing for dominance. As such, there is variation in the extent to which rank is a reliable proxy for male competitiveness, which may affect the extent to which it is in females’ interest to signal ovulation reliably. However, data on ovulatory signals are absent from species at one end of the macaque continuum, where selection has led to high sexual dimorphism and male reproductive skew. Here we present data from 31 cycles of 19 wild female crested macaques, a highly sexually dimorphic species with strong mating skew. We collected measures of ovarian hormone data from faeces, sexual swelling size from digital images, and male and female behaviour.We show that both sexual swelling size and female proceptivity are graded-signals, but relatively reliable indicators of ovulation, with swelling size largest and female proceptive behaviours most frequent around ovulation. Sexual swelling size was also larger in conceptive cycles. Male mating behaviour was well timed to female ovulation, suggesting that males had accurate information about this.Though probabilistic, crested macaque ovulatory signals are relatively reliable. We argue that in species where males fight over dominance, male dominance rank is surrogate for competitiveness. Under these circumstances it is in the interest of females to increase paternity concentration and assurance in dominants beyond levels seen in species where such competition is less marked. As such, we suggest that it may in part be variation in male competitive regimes that leads to the evolution of fertility signalling systems of different reliability. The study of inter-sexual communication is central to sexual selection theory . One of Over the past decade, several studies of wild and free-ranging primates have tested this evolutionary model, and collectively it has found broad support . TheDuring each cycle from each female we took an average 24 (range 5–63) swelling images. If the swelling was filmed with the video camera, sequences of the swelling were transferred to a Laptop with Video Capture 6.0 and Scenalyzer 3.52. One picture was captured daily from the video sequences of each observed female with ACDSee 5.0.1. If images were taken using the camera, the highest quality image (if multiple images were available) was chosen and images were transferred to Adobe Photoshop CS2. To correct for differences in camera distance and zoom between images, we adjusted each swelling size measurement for the known size of the ischial callosities (see above). Wherever possible, this was done using the left ischial callosity; if the left ischial callosity was not clearly observable for any reason, the right one was used instead. We measured the ischial callosity height in each image using the Adobe Photoshop measurement tool, and expressed this as a fraction of the known ischial callosity height. We then measured the swelling height, and multiplied this by the ischial collosity fraction to get the true swelling size measurement. We undertook two different measurements of swelling size in this way . Data were collected using instantaneous focal animal sampling . Focal fAs female hormone levels may be related to female behaviour and as, independently of this, female behaviour may indicate the timing of ovulation specifically, we assessed relationships between sexual swelling size and behaviour and both hormone levels, and estimates of the timing of ovulation based on progestogen levels, separately. To do this, we first took the progestogen (P) and estrogen (E) levels measured from our faecal samples and tested whether female sexual behaviour was related to the E:P ratio [e.g. ]. When aM. nigra does not exhibit post-conception swellings, see [58]; Engelhardt et al. unpublished manuscript], or if miscarriage was subsequently observed . Cycles were classified as non-conceptive (N = 15) if they were immediately followed by another cycle .We tested relationships between the timing of ovulation and swelling size and behaviour using the 14 day period around ovulation only. We created a scale (Day Relative to Ovulation = DayRO) whereby the two days of the ovulation window were both considered Day 0, and each day before  and after this  were designated accordingly. We did this and analysed data for a period representing the total fertile phase of the cycle , or Our analyses of variation in inter-cycle fertility revealed significant differences between conceptive and non-conceptive cycles. Females expressed significantly larger sexual swellings in conceptive cycles than in non-conceptive cycles, and also exhibited some behaviours less often in conception cycles . Males showed some behavioural differences between cycle types, but consistent with results in some other species [e.g. chimpanzees ], males The apparent relative reliability of female ovulatory signalling is consistent with the low female oestrus synchrony, high mating skew  and high sexual dimorphism  found inOur analysis of relationships between male behaviour and sexual swelling size indicate that male behaviour was well timed to this reliable signal of the timing of ovulation. Interestingly male behaviours such as inspection were not related to the timing of ovulation . The prOur data provide new insights into the evolution of female fertility signals in a taxa in which there is a high degree of variation in the reliability of such signals. Unlike in many multimale multifemale group-living primate species where signals of ovulation appear relatively unreliable, females in this very sexually dimorphic macaque species signal differences in intra- and inter-cycle conceptive probabilities using different types of signal, in this case sexual swellings and female behaviour. Male crested macaques appear to have information about the timing of female ovulation, and time their mating effort appropriately. Together with data on mating skew and the monopolisation of females by dominant males , the present data help build a picture of a highly interesting species in which dominant males fight for rank, monopolize mating and reproduction, and in which females signal fertility to males in a probabilistic but relatively (compared to related species) reliable fashion. Collectively, our data are important for understanding the evolution of female fertility signals because they represent an extreme end of the variation seen across the macaques, some of which do not possess clear signals of ovulation such as sexual swellings at all. Our suggestion that comparative variation in male competitive regimes may be linked to the evolution of fertility signalling systems of different reliability provides testable predictions for other species. Further understanding the selective pressures that have given rise to such comparative variation will provide a wealth of insight into the veracity of current socio-ecological models, as well as our understanding of signal evolution.The authors declare that they have no competing interests.JH processed data, undertook analyses, drew the figures and wrote the paper; MH provided reagents, oversaw the hormonal analyses and helped draft the manuscript; CS processed data and undertook analyses; MA co-conceived the project, oversaw processing of hormone samples and provided logistical and institutional support in Indonesia; DP-F co-conceived the project and provided logistical and institutional support in Indonesia; AE conceived the project, collected and processed data and co-wrote the paper. All authors read and approved the final manuscript.