USA alone) and it is associated with considerable morbidity. The development of endometriosis is inextricably linked to the process of menstruation and thus the models that best recapitulate the human disease are in menstruating non‐human primates. However, the use of these animals is ethically challenging and very expensive. A variety of models in laboratory animals have been developed and the most recent are based on generating menstrual‐like endometrial tissue that can be transferred to a recipient animal. These models are genetically manipulable and facilitate precise mechanistic studies. In addition, these models can be used to study malignant transformation in epithelial ovarian carcinoma. Epidemiological and molecular evidence indicates that endometriosis is the most plausible precursor of both clear cell and endometrioid ovarian cancer . While this progression is rare, understanding the underlying mechanisms of transformation may offer new strategies for prevention and therapy. Our ability to pursue this is highly dependent on improved animal models but the current transgenic models, which genetically modify the ovarian surface epithelium and oviduct, are poor models of ectopic endometrial tissue. In this review we describe the various models of endometriosis and discuss how they may be applicable to developing our mechanistic understanding of OCCA and OEA. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Endometriosis is a common benign gynaecological condition affecting at least 10% of women of childbearing age and is characterized by pain – frequently debilitating. Although the exact prevalence is unknown, the economic burden is substantial (∼$50 billion a year in the  Endometriosis is defined as the presence of endometrial tissue (glands and stroma) outside the uterine cavity; it is found primarily in the pelvis and can involve all organs but particularly the ovaries and pelvic peritoneum. It is a common condition affecting an unknown number of women of reproductive age. Typical symptoms include pain and subfertility, and the complexity and severity of the pain can present major management challenges Definitive diagnosis requires laparoscopy or laparotomy and histological confirmation, but there is often a protracted time to diagnosis  The concentration of a range of inflammatory mediators – cytokines, growth factors, eicosanoids, and angiogenic factors – is altered in the peritoneal fluid of women with endometriosis At present, there is no definitive answer for the origin of endometriosis, but the most widely accepted theory is that it arises from endometrium that has disseminated to ectopic sites, most commonly by retrograde menstruation Endometriosis has high prevalence, significant morbidity, and considerable economic impact. The wide range of treatments used in clinical practice underscores our limited understanding of the pathogenesis of endometriosis and approaches that exploit new models and analytical techniques could have high utility. Furthermore, although endometriosis itself is a benign disease, several epidemiological studies have identified it as a risk factor for some types of malignancies, most notably ovarian cancer, and particularly ovarian clear cell adenocarcinoma (OCCA) or ovarian endometrioid adenocarcinoma (OEA). Models which allow the underlying mechanisms to be elucidated offer the prospect of patient stratification and over the longer term, new therapies.Epithelial ovarian cancers (EOCs) are not a single disease but rather a group of diseases with distinct morphological and molecular features, all of which may affect the ovary. The principal subtypes of EOCs are high‐grade serous carcinoma, low‐grade serous carcinoma, mucinous carcinoma, clear cell adenocarcinoma (OCCA), and endometrioid adenocarcinoma (OEA). These tumours likely have different cells of origin, distinct histopathological features, and exhibit different molecular profiles. However, all subtypes of EOCs are treated by surgical debulking and platinum‐based chemotherapy. OCCA and OEA have high rates of primary resistance to platinum‐based chemotherapy which contributes to their poor prognosis when presenting with late stage or reoccurrence.Endometriosis is now recognized as a likely precursor of some subtypes of epithelial ovarian cancer. As early as 1925, Sampson made the observation that cancer developed in ectopic endometrial tissue p < 0 · 0001; OEA odds ratio 2.04, 95% CI 1.67–2.48, p < 0.0001; and low‐grade serous odds ratio 2.11, 95% CI 1.39–3.20, p < 0.0001). However, the history of endometriosis in these case–control studies was self‐reported and this may predispose to recall bias. While it is presumed that cancer cases may be more likely to report a history of endometriosis than controls, it is likely that this bias is random and may not account for the difference in association between the different subtypes of ovarian cancer.A pooled analysis of 13 case–control studies (23 144 women) was used to examine the consistency and degree of association between endometriosis and the different subtypes of ovarian cancer PTEN) occurred in 42.1% (8 of 19) OEAs, 27.3% (6 of 22) OCCAs, and 56.5% (13 of 23) solitary endometrial cysts. Importantly, in five OEAs and seven OCCAs with synchronous endometriosis, three cases in each group showed identical LOH events in both tumour and endometriosis; one case in each group had LOH in the tumour only. The remaining endometriosis cases (one OEA and three OCCAs) did not show LOH events. None of the endometriotic lesions showed LOH events that were not also present in the co‐existent tumours PTEN function by LOH or mutation is an early event in the development of endometriosis‐related cancers of the ovary.The consistent finding of increased risk in the above epidemiological studies supports a two‐ to three‐fold increase in the risk of OCCA and/or OEA in patients with endometriosis. However, it is plausible that a common characteristic, such dysregulation of cytokine production or steroid metabolism within the peritoneal environment, predisposes to both conditions. However, there are several lines of molecular evidence that suggest that OCCA and OEA arise directly from endometriotic lesions. The first published studies used information from loss of heterozygosity (LOH) surveys in OCCA and OEA cases to test whether identical changes were also present in adjacent endometriosis. In a study of 19 OEAs, 22 OCCAs, and 23 solitary endometrial cysts, LOH at 10q23.3  specimens. Two of the patients who had OCCA samples with ARID1A mutations also had contiguous atypical endometriosis . One of these patients had a truncating mutation and the other had two somatic mutations predicting loss of BAF250a protein (encoded by ARID1A). These mutations were found in the contiguous atypical endometriosis but not in distant endometriosis, further supporting local transformation of endometriosis tissue. Immunohistochemical staining for BAF250a was also lost in both OCCA and the contiguous atypical endometriosis but not in distant endometriosis ARID1A during malignant transformation of endometriosis.More recently, specific mutations and copy number abnormalities have been demonstrated in tumour and co‐existent endometriosis. Wiegand PIK3CA and found synchronous mutations in 17 of 42 (40%) of these tumours, most of which (71%) were also ARID1A‐deficient PIK3CA mutations also occur at a very early stage in the development of OCCA, before the appearance of the atypical precancerous lesions.These results were confirmed using immunohistochemistry for BAF250a MET proto‐oncogene is frequently overexpressed in OCCA and is frequently amplified. Copy number alterations and MET protein levels were studied in 13 tumours and precursor lesions [including 11 endometriosis and two clear cell adenofibromas (CCAFs)]. MET copy number gain and overexpression were demonstrated in the atypical precursor lesions. Non‐atypical endometriosis and benign CCAFs did not demonstrate MET gain MET gain is an early event in tumourigenesis in a subset of OCCAs and that it is a driver mutation.The To further identify the pattern of somatic mutations in OCCA and investigate whether there was a clonal relationship between benign gynaecological lesions, including endometriosis, and OCCA, whole‐genome sequencing of seven OCCAs was performed and targeted sequencing of other co‐existent adjacent or metastatic tumours, endometriosis, and other benign conditions The studies described above, considered collectively, make a compelling case for endometriosis being the precursor lesion of OCCA and OEA – although this progression is rare. The major clinical challenge is now to determine which individuals are at higher risk of the rare event of transformation of endometriosis to OCCA or OEA. However, our understanding of the molecular basis of this progression is far from complete. Effective patient screening and therapeutic intervention will only be possible once we have a fuller picture of the molecular pathways involved. Consequently, in the absence of any effective screening for any ovarian cancer type, current guidance is that no action be taken to monitor this potential progression in women with endometriosis For any system to be a useful model of a human disease, it needs to recapitulate the typical features of that condition but to be manipulable in a way that humans are not. Thus, there may be a naturally occurring veterinary condition that parallels the human disease; if so, this could be used to investigate the molecular mechanisms underlying the disease. If this is not the case, then a more interventional approach is needed. The simplest and the most commonly accepted pathological mechanism underlying endometriosis is the retrograde menstruation proposed by Sampson Macaca mulatta) develop endometriosis that “anatomically and clinically, appears to be identical to the human disease” Papio anubis), the frequency is broadly similar – 11% or 32% in animals held in captivity for less than 1 year or greater than 2 years, respectively. This difference may be due to an increase in the number of menstrual cycles (due to a reduction in the number of pregnancies) Unlike other model organisms, some primates menstruate and develop spontaneous endometriosis and this is very similar to the human condition. For example, rhesus monkeys . After 3 weeks, 76% of the subjects had endometriosis Iatrogenic endometriosis as a complication of surgery was seen to be so common that Ridley in 1968 wrote “this fact is now growing so commonplace that it is accepted and usually not reported in the literature” KRAS, FOS, and NODAL was altered The effects of the presence of endometriosis on the eutopic endometrium have been studied in humans In many ways, primate studies are attractive because these animals are closely related to humans, anatomically and physiologically, and spontaneously develop the disease, suggesting that the peritoneal environment is similar. These studies exemplify the usefulness of the baboon as a model animal for the study of endometriosis – the endocrine environment is similar to that in humans and can be well controlled; the induction of endometriosis is uniform and under tight temporal control; it is possible to visualize the lesions and collect samples over a prolonged time; and, lastly, the animal cycles naturally. This is counterbalanced by more complex handling and housing requirements; thus, experimental work with primates is costly and ethically challenging.Laboratory animals do not menstruate and never develop spontaneous endometriosis. However, hamsters, rabbits, and particularly rats and mice are widely used as scientific models for medical research, due to their short generation times and their ease of physiological and of particularly genetic manipulation. Rabbit and rodent models of endometriosis have been established based on transplanting endometrium or uterine fragments, either from the same species (homologous models) or from humans (heterologous models), to ectopic sites.A rabbit model of endometriosis was generated to study the effects of endometriosis on fertility Both rat and mouse models have been generated by autotransplantation of fragments of the uterus to the mesentery A model in which only the endometrium is transplanted has been developed. The endometrium was collected from ovariectomized and oestrogen‐treated mice. It was minced and injected into the peritoneum of syngeneic recipient animals An enhancement of this model which improved the quantitative assessment of lesion growth made use of donor mice in which green fluorescent protein (GFP) was ubiquitously expressed UbC‐Luc+/+ mouse was collected and the horns were opened to allow numerous 2‐mm tissue samples to be punched out. Four of these were then sutured into separate peritoneal locations in albino recipient mice. The luciferase‐derived signal level was measured 2 weeks post‐surgery and showed a clear signal in the location of suture sites. The mice were culled 2 weeks later and the lesions examined. All showed similarity to human endometriosis lesions, with evidence of angiogenesis. This approach is a significant advance as lesion growth can be serially monitored non‐invasively for up to 35 days While it is possible to image GFP in the internal organs of a mouse K‐ras in loxP‐Stop‐loxP‐G12D/+K‐ras mice (LSL‐K‐rasG12D/+) by injecting Cre‐adenovirus into the ovarian bursa resulted in the development of endometriotic‐like lesions  in the ovarian surface epithelium in all the mice, and endometriotic lesions with both glands and stroma in the peritoneum in 47% of the animals. When similarly transduced ovaries were transplanted, the endometriotic‐like lesions were limited to the ovarian surface epithelium. This suggests that the endometriotic‐like lesions on the ovary in this mouse model most likely arose from the ovarian surface epithelium itself. However, the peritoneal lesions probably arose from uterotubal cells in which K‐ras was activated along the needle track. Injection of AdCre virus directly into the peritoneal cavity of the LSL‐K‐rasG12D/+ mice did not lead to the development of any endometriotic‐like lesions, suggesting (in mice at least) that endometriosis does not arise through metaplastic transformation of the peritoneum LSL‐K‐rasG12D/+;PtenloxP/loxP mice leads to signs of invasive ovarian endometrioid adenocarcinoma between 7 and 12 weeks later. However, the unintended transduction of tubular cells (which was clearly demonstrated in this study) may in fact be an important characteristic of this method of delivering the Cre. While the method provides clear information about the function of the genes manipulated, conclusions about the precise cell of origin need to be drawn cautiously. Nonetheless, the presence of the K‐ras mutation in this model overcame the need for additional hormone stimulation and provides a useful model to study the histomorphology and biological behaviour of endometriosis.In 2005, the first mouse model in which endometriosis developed without any form of transplant was described Endometriosis is a condition in which normal menstrual material persists in an abnormal environment. Thus, an animal model of this condition would ideally mimic the processes that allow this material to adhere within the peritoneum and develop into a lesion. Indeed, this was the basis of the initial models in primates. Clearly, rodent models have many attractions but they do not menstruate naturally.A mouse model has been developed to mimic the endometrial breakdown seen in human menstruation. In this model, ovariectomized mice are treated with oestradiol and progesterone and the endometrium is stimulated to decidualize by injecting a small amount of oil directly into the uterine lumen. Examination 48 h after cessation of the hormone injections  revealed blood vessels filled with swollen erythrocytes, apoptotic changes, and invasion of leukocytes. By 79 h post‐withdrawal, blood cells and degenerating decidual cells had been shed into the uterine lumen This model has been refined: C56J/Bl6 ovariectomized mice were exposed to a programme of oestrogen injections to mimic the levels seen in the human cycle. However, progesterone was delivered via an implant inserted prior to the injection of oil into the uterus. The removal of the implant 49 h after the decidualizing stimulus allowed a more dramatic drop in serum progesterone levels, more closely representing the human cycle. Mice were sacrificed and the uterine response was examined 16, 24, 36, and 48 h post‐progesterone withdrawal. After 16 h, breakdown of the endometrium was observed; by 24 h, the decidual zone had separated from the rest of the endometrium. Tissue debris was fully cleared by 36 h after progesterone withdrawal and by 48 h, the endometrium was restored to its pre‐decidual state et al do not give progesterone after the injection of the oil into the uterine lumen et al leave a progesterone implant in place for 4 days after the interuterine oil injection These two studies form the basis of related but distinct models of endometriosis et al (described above) showed that activation of K‐ras was sufficient to generate an endometriosis‐like lesion in the peritoneum of mice LSL‐K‐rasG12D/+ mice. Thus, a small number of cells in which the LSL‐K‐rasG12D/+ had been activated were sufficient to lead to endometriosis.The work of Dinulescu et al used a LSL‐K‐rasG12V/+/Ah‐Cre+/+/ROSA26R‐LacZ+/+ mouse in which Cre was induced in the uterus by β‐naphthoflavone dissolved in the oil used as the decidualizing stimulus K‐ras and the LacZ reporter. After progesterone withdrawal, this endometrium was transferred to a subcutaneous pocket in wild‐type intact animals. No exogenous steroids were administered to the recipient animals but lesion growth could be inhibited by treatment with an oestrogen receptor antagonist . In the absence of the genetic modification, the ‘menstrual’ endometrium did not form lesions. Somewhat surprisingly, only a small proportion of the cells in the endometriotic lesions were LacZ‐positive, suggesting that Cre‐mediated recombination did not occur in all the endometrial cells, but that this low level of K‐ras activation was sufficient for lesion establishment Cheng et al is more efficient and yields a greater mass of ‘menstrual’ material for transplant. However, a significant difference in this model is that the recipient animal is primed with and continues to receive oestradiol; thus there is a strong oestrogenic drive which promotes endometriotic lesion growth et al as there is no need for complex animal breeding. This is a considerable advantage as it facilitates the inclusion of other genes of interest . Indeed, this approach has been effectively used to show that both host‐ and shed tissue‐derived macrophages are present in the growing lesions The extended progesterone treatment during decidualization used by Greaves An alternative to auto‐ or syngeneic‐transplant is to use human endometrium and immunodeficient mice as recipients. Collectively, these have the advantage that human tissue is used, so drugs or antibodies with species‐specific effects can be used. However, they have the disadvantage that the host immune system is severely compromised. Given that endometriosis is recognized as an inflammatory condition, this is a significant weakness in these models Multiple studies have described the transplantation of proliferative, secretory, and menstrual phase human endometrium into athymic nude mice Studies using this model have shown that angiogenesis is required for lesion growth, murine endothelial cells grow into the lesion, and that it is VEGF‐A derived from the human tissue that drives this et al prepared a single‐cell suspension of the glandular and stromal portions of human endometrium and transplanted this under the kidney capsule of ovariectomized NOD/SCID/γcnull mice. The mice were treated with oestrogen pellets with or without cyclic injections of progesterone. All mice transplanted were found to have endometrium‐like tissue in the kidneys when collected 10 weeks later, compared with only one of the 12 non‐hormone‐treated mice. Mice that received oestrogen and progesterone produced larger cysts than those treated with oestrogen alone. Introduction of a YFP or luciferase gene into the human cells prior to transplantation allowed lesion growth to be followed for 8 weeks using non‐invasive imaging. A cyclic increase and decrease in signal could be detected in the mice when progesterone was given or withdrawn, showing that the cyclic events of the menstrual cycle could be recapitulated in this model These studies broadly agree on the importance of oestradiol, angiogenesis, and invasion for lesion growth. However, the models are not well suited to investigating the underlying mechanisms. For example, there is considerable heterogeneity in the tissue source – human endometrium or endometriotic lesions et al is a significant disadvantage As summarized above, recent animal models of endometriosis have become more refined and now are able to recapitulate the presumed pathogenesis of the human condition. As might be expected, primate models are the closest to human disease but are ethically difficult and very expensive. Rodent models, which mimic the deposition of menstrual tissue, are now available and have the advantage of being easily genetically manipulable and immunocompetent. However, the need for exogenous oestradiol in rodents may be a limitation – for example, if studying a process that is directly oestrogen‐dependent. However, the complex breeding required to use the model described by Cheng in vivo imaging. Histological examination showed labelled cells close to site of the corpus luteum and disrupted epithelium. After 10 days, the cancer cells had invaded the ovarian stroma, leaving an intact ovarian epithelium. While this experiment used OTICs derived from high‐grade serous carcinoma, it demonstrates that these cells are attracted to the ovary. This is likely to be the case for the other types of epithelial ovarian cancers whose cells of origin are different but which lodge in the ‘fertile soil’ of the ovary.It is now understood that ovarian cancer is a heterogeneous disease originating from different epithelial cells in the Müllerian tract, which show marked tropism for spread and growth on the ovary. As early as 1925, Sampson recognized this and said of ovarian endometriotic lesions: “The ovarian implants are usually more active than the latter , thus suggesting that ovarian tissue is the most fertile soil” et al described above was a significant advance in the development of mouse models of ovarian disease because of the innovative targeting of genetic modifications to a specific site Arid1a loss and Pik3ca activation after intrabursal injection of AdCre in fl/flArid1a(GT)Rosa26Pik3caH1047R* mice leads to rapid development of ovarian cancer with OCCA‐like histology et alArid1a and Pten  using intrabursal AdCre in fl/fl;Ptenfl/flArid1a animals led to the development of endometrioid ovarian tumours The work of Dinulescu ApcloxP/loxPPtenloxP/loxP mice only develop tumours  after intrabursal AdCre. The relationship of these pathways to other pathways implicated in ovarian cancer can elegantly be studied using enhancements of this model: for example, the role of Tp53 (which is infrequently mutated in OCAA but nearly ubiquitous in HGS) The Wnt/β‐catenin pathway has been implicated in OEA by mutational profiling studies and functionally tested using models similar to those outlined above These mouse models replicate many but not all of the features of human OEAs or OCCA, but can now be extended to study in more detail the genetic alterations required for tumour development and progression. This will improve the use of these models to develop new therapies and to study treatment response. However, other models are needed to study the progression of endometriosis into OEA. If in the model the cancer arises in the OSE , then this is probably the wrong cell type. On the other hand, as the mode of delivery of the Cre  also targets a modest number of uterotubal cells, the possibility remains that these cells make a contribution to the pathology observed. The principal features of these models are summarised in Table When designing and using animal models of human disease, and specifically endometriosis, it is important to replicate the disease as closely as possible by taking into account the presumed tissue or cell of origin. The models of endometriosis all presume that the underlying mechanism depends on dissemination of endometrium by retrograde menstruation The epidemiological and the molecular data reviewed above very strongly suggest that the genesis of OCCA and OEA is not in the ovary – although the ovary is clearly a favoured seeding site. Thus, when developing improved models to study cancer progression, new approaches for targeting endometriosis cells in an appropriate environmental context are now required. The current models very elegantly demonstrate the interplay between genes and do cast light on the processes important for uncontrolled growth in the peritoneal environment. However, they do not fully address the nature of the presumed progression in humans. The nature of somatic changes necessary for the progression from eutopic endometrium to endometriosis, and then (rarely) to ovarian clear cell or endometrioid carcinoma, remains to be defined. As indicated in Figure Over the last 5 years, animal models of endometriosis and those developed to study epithelial ovarian cancer have improved significantly. There is now the prospect of combining genetic modification of murine endometrium with the ‘menstrual mouse’ models of endometriosis to define the role of single or combinations of particular genes in endometriosis and OCCA or OEA. Potential risk alleles identified from human genetic studies can be functionally tested in a model of the presumed precursor lesion. An outline of this approach is shown in Figure All authors contributed to the writing of the manuscript and approved the final version.


Natural populations often show variation in traits that can affect the strength of interspecific interactions. Interaction strengths in turn influence the fate of pairwise interacting populations and the stability of food webs. Understanding the mechanisms relating individual phenotypic variation to interaction strengths is thus central to assess how trait variation affects population and community dynamics. We incorporated nonheritable variation in attack rates and handling times into a classical consumer–resource model to investigate how variation may alter interaction strengths, population dynamics, species persistence, and invasiveness. We found that individual variation influences species persistence through its effect on interaction strengths. In many scenarios, interaction strengths decrease with variation, which in turn affects species coexistence and stability. Because environmental change alters the direction and strength of selection acting upon phenotypic traits, our results have implications for species coexistence in a context of habitat fragmentation, climate change, and the arrival of exotic species to native ecosystems. Individuals of the same population often show extensive variation in morphology  structures population-level consumer–resource networks in predictable ways  increases over long time spans with increasing individual variation in life-history traits through a portfolio effect  and g(C) are the mortality rates for resource and consumers, respectively, and r(R) and εf are the reproductive rate of resource and consumers, respectively. The functional form of f is typically assumed to be the same for both consumers and resources, but its magnitude is scaled in the consumer equation by an efficiency parameter, ε, that can take any non-negative real value. May defined interaction strengths  in systems like wherewhere we x, of a quantitative trait , to be maximal at a given optimal trait value x = θα, and to then decrease away from that maximum in a Gaussian way:αmax is the maximal attack rate and τ2 determines how steeply the attack rate declines away from θα , to be minimal at a given optimal value x = θη, and to increase away from that minimum in a Gaussian way:ηmax and ηmin are the maximal and minimal handling times, respectively, and 2ν determines how steeply the handling time increases away from θη 10) can b2) as well as phenotypic mismatch (d2) and can be estimated numerically either at equilibrium (when C and R are constant), or instantaneously (for any given time t).Equations  and 12)12) depenK is the carrying capacity for the resource, m is the mortality rate of the consumer and all other parameters are as explained before. Our main objective is to tie the dynamic effect of phenotypic variation on attack rate and handling time through their effect on interaction strengths.To explore the effect of individual variation on consumer–resource interactions and species persistence through interaction strengths, we explored the dynamics of a Rosenzweig–MacArthur consumer–resource model Appendix .dα ~ 0 and dη ~ 0), interaction strengths decay in both consumers and resources with increasing individual variation  such that equation dα ~ 0 and dη ~ 0), the consumer is less likely to persist since Hence, if phenotypic mismatch is small , and variation is small enough , the carrying capacity (K) and the digestive efficiency (ε). Notice that equation (These numerical results are in accordance with our analytic predictions, where the condition for stability can be approximated as:whenever equation , where tCombined, these results suggest that the effect of variation in attack rates is dominant over that of handling times Appendix , which lIndividual variation in demographic parameters is pervasive in most systems (Bolnick et al. Although individual variation can increase species persistence in the eco-evolutionary dynamics of an apparent competition system (Schreiber et al. However, our results also suggest that the effect of individual variation on interaction strengths depends on the levels of phenotypic mismatch between consumers and resources, and these are ultimately controlled by existing and past selective pressures (e.g., Fellowes et al. Our results could have important implications for food-web theory. For example, interaction strengths have also long been known to drive the stability of large, complex networks of interacting species such as food webs (e.g., May We showed that variation can affect interaction strengths and species persistence. In what follows, we argue that this could have important consequences for the establishment of biological invaders. For small phenotypic mismatch between consumers and resources, interaction strength decreases monotonically with variation Fig. , which rInvasive species can enter a new environment with a single or a few individuals and could therefore have low individual variation during the establishment phase (Facon et al. Because of Jensen’s inequality it has been previously suggested that attack rates and handling times could have opposite effects on interaction strengths when individual trait variation was taken into account in each attribute independently Fig. . AlthougOne possible way of doing so is to use controlled microcosm experiments of consumer and resource protists (e.g., DeLong and Vasseur Our results are in accordance with previous theoretical studies that have shown that increased behavioral variation Okuyama  and variOther researchers have explored consumer–resource dynamics in the case where there is behavioral variation in foraging rates Okuyama ; howeverOverall our study shows that individual variation can affect species persistence and coexistence between consumer and resource through its effect on interaction strengths. Moreover, the effect of individual variation on interaction strengths depends on phenotypic mismatch and thus, on current and past selective pressures. This has important implications for species persistence embedded in food webs or the arrival of invasive species to native ecosystems. Finally, this study underlines the need for accurately estimating the distribution of ecologically relevant phenotypic traits, as well as their functional relationship with ecological attributes, in order to test our predictions of how individual variation affects the ecology and persistence of interacting populations.


Little attention has been given to how age affects the neural processing of movement within the brainstem. Since the brainstem plays a critical role in motor control throughout the whole body, having a clear understanding of deficits in brainstem function could provide important insights into movement deficits in older adults. A unique property of the startle reflex is its ability to involuntarily elicit planned movements, a phenomenon referred to as startReact. The noninvasive startReact response has previously been used to probe both brainstem utilization and motor planning. Our objective was to evaluate deficits in startReact hand extension movements in older adults. We hypothesized that startReact hand extension will be intact but delayed. Electromyography was recorded from the sternocleidomastoid (SCM) muscle to detect startle and the extensor digitorum communis (EDC) to quantify movement onset in both young (24 ± 1) and older adults (70 ± 11). Subjects were exposed to a startling loud sound when prepared to extend their hand. Trials were split into those where a startle did (SCM+) and did not (SCM−) occur. We found that startReact was intact but delayed in older adults. SCM+ onset latencies were faster than SCM− trials in both the populations, however, SCM+ onset latencies were slower in older adults compared to young (Δ = 8 msec). We conclude that the observed age‐related delay in the startReact response most likely arises from central processing delays within the brainstem. Our objective was to utilize the noninvasive startReact phenomenon, which is mediated through the brainstem, to gain insight into brainstem processing in older adults. We found that startReact hand extension was intact but delayed in older adults. The observed age‐related delay in the startReact response most likely arises from central processing delays within the brainstem. Our result that the startReact response is delayed in older individuals highlights that movements  and reflexes  that are coordinated by the brainstem may have similar deficits in older adults. Older adults face muscle weakness  but delayed in older adults when compared to younger individuals. We chose hand extension for two important reasons. First, the classic startle reflex results in hand flexion movement; thus by choosing hand extension, we can differentiate movements from the classic startle response allowing a clean method for identifying an intact startReact. Second, reticulospinal connections to the hand are weak and few in number  and 24 older subjects . Subjects were interviewed to ensure that they had no recent upper body injuries , hearing sensitivity, or heart conditions. All participants gave informed written consent prior to participating in the study in accordance with the provisions set forth by the Northwestern University Institutional Review Board IRB (STU9204).Bipolar electromyography (EMG) electrodes  were used to record muscle activity from the extensor digitorum communis (EDC) on the right arm and from the left and right sternocleidomastoid muscle (SCM). For grounding, a unipolar ground electrode  was positioned on the right styloid process. EMG signals were preamplified  and filtered with a band‐pass filter of 10–1000 Hz. Next, the signals were anti‐alias filtered by fifth order Bessel filters with a 500 Hz cutoff frequency and sampled at 2500 Hz .Subjects were comfortably seated in a chair with armrests supporting hands and arms against gravity. To minimize motion and to ensure a similar position for every subject, participants were strapped across the chest to the chair. Further, the right elbow joint was in a flexed position (90°) and placed in line with the right shoulder to ensure a comparable neutral posture.Visual feedback was delivered via a monitor placed approximately 3 feet in front of the subject. The screen displayed reaction times (milliseconds) and a visual representation of the relative success of the trial. Specifically, a happy face was displaced to indicate an appropriate onset latency and an unhappy face displayed when subjects initiated movement too slowly. The inclusion of visual feedback was important because the level of arousal and attention is known to improve the size and probability of startle responses . The first sound was a WARNING sound, which signaled the subject to get ready and plan the extension task. The second sound was a GO signal, where the subject was asked to perform the extension movement as fast as possible. To avoid anticipation of the GO, the time between the WARNING and GO was randomized between 1.5 and 3.5 sec . For classic startle trials, the WARNING signal was replaced with a startling acoustic stimulus (128 dB). In these trials subjects experienced the startling stimulus when they were not prepared to move (classic startle). For high‐intensity trials, the GO signal was replaced by a startling acoustic stimulus. Under these conditions, the subject had already planned the hand extension movement. The startling acoustic stimulus was emitted from a loudspeaker fixed to the chair directly behind the head of the subject. There was no instruction provided on how to react to this sound.Onset latency of the EDC and SCM muscles were determined for each trial. The EMG signal was first rectified and filtered with a 10‐point moving average filter. Then, onsets were identified with the help of an automatic detection file in Matlab , which automatically tagged the time point when the EMG signal rose significantly over background. All trials were then visually inspected for accuracy and to exclude trials when the subjects did not move , moved before the GO signal, or moved too late . Trial type and task were blinded to the reviewer.Next we assessed the presence of a startle during all trial types . SCM muscle onset latency within 120 msec after the GO signal indicated a startle occurred  was calculated by dividing the number of HI SCM+ and Clst SCM+ by the total number of all high‐intensity trials and classic startle trials, respectively.Our hypothesis was tested using an analysis of variance (ANOVA) of a linear mixed‐effect model with age , trial type  as the independent factors. Onset latency and probability were considered dependent factors. Subjects were treated as a random effect. Equal variance was not assumed in the statistical model. All individual trials were included in the analysis to decrease the probability of statistical errors by capturing all the variability in the data set Hedeker . Tukey'sF2,3087 = 1859.95, P < 0.0001) and age . Posthoc comparisons confirmed that the EDC onset latencies were significantly faster during HI SCM+ trials compared to HI SCM− trials for both young and old subjects (P ≈ 0). LI SCM− trials were significantly slower than HI SCM+ (P ≈ 0) and HI SCM− (P ≈ 0) in both young and old subjects.Hand extension movements were susceptible to startReact in both young and old subjects as demonstrated by HI SCM+ trials showing faster EDC onset latencies than HI SCM− trials Figs.  EDC muscP = 0.002). Though a delay in HI SCM− trials between young and old was observed, it did not reach significance  indicating it was not consistent across all subjects. Finally, older subjects were not significantly slower during LI SCM− trials  suggesting that old and young subjects could perform hand extension equally efficiently.Older subjects showed delayed muscle activation during trials when a startle was present  and age . Posthoc comparisons confirmed that the right SCM muscle onset latency was slower in older adults during both classic startle  and startReact  conditions. The left SCM muscle showed a similar, but less robust, trend. Onset latency was influenced by trial type  though not age . The interaction showed a trend toward significance ; therefore posthoc comparisons were completed that demonstrated that the left SCM muscle onset latency was slower in older adults during both classic startle  and startReact  conditions.Sternocleidomastoid muscle activation was slower in older adults during both classic startle and high‐intensity SCM+ trials Figs. , Table 1P ≈ 0) and old  subjects. The left SCM muscle showed the same result in both young  and old  subjects.The onset latency of both right and left SCM muscles were faster during classic startle than during startReact in both young and old adults. The onset of the right SCM muscle was faster during classic startle compared to startReact in both young  but not trial type  with no interaction effect . The probability of eliciting a SCM+ was decreased in older adults during classic startle , but did not reach statistical significance during high‐intensity trials  though there was a trend toward significance. There was no difference in the probability of eliciting a SCM+ between classic startle and high‐intensity trials within each population  indicating that each population's sensitivity to SCM+ was the same during both classic startle and high‐intensity trials.The probability of detecting a SCM+ did not reach statistical significance during high‐intensity trials but was lower during classic startle trials in older adults. The probability of eliciting a SCM+ was influenced by age  resulted in faster reaction times during high‐intensity trials compared to high‐intensity trials when startle was not present (SCM−) indicating that older individuals possess an intact startReact response. Therefore, despite evidence that older individuals rely more heavily on the cortex for movement planning (Wild‐Wall et al. The observed age‐related delay in the startReact response likely arises from central processing delays within the brainstem. Although the specific neural mechanism remains debated, there are several key papers that indicate that the cortex and corticospinal tract is not the primary neural tract utilized during startReact. First, individuated movements of the hand that are expressed predominately through the corticospinal tract (Lawrence and Kuypers An alternative mechanism that could explain the observed delay in the startReact and classic startle response is an age‐dependent slowing in central and peripheral motor conduction. However, voluntarily initiated extension movements were not different in latency between old and young indicating that motor conduction is not impaired during voluntary tasks. Further, previous reports indicate that central and peripheral motor conduction deficits in older adults likely can only account for 3–5 msec of delay in distal muscle (Dorfman and Bosley Our results showing an intact startReact response indicate that older adults still can utilize the brainstem for planning movement – at least in simple ballistic tasks. Movement planning is a global process that requires all layers of the nervous system (Rushworth et al. The startReact reflex has been previously used as a probe of both brainstem utilization (Valls‐Sole et al. The results of this paper highlight that the classic startle reflex and the startReact response are closely linked but still appear to be somewhat distinctive phenomena. It has been previously suggested in literature that startle and startReact are unique phenomena (Kumru et al. Our result that the startReact response is delayed in older individuals highlights that movements and reflexes that are coordinated by the brainstem may have deficits in older adults. Since the brainstem is known to be highly involved in postural control and locomotion (Lyalka et al. This is the first report to demonstrate that hand extension is susceptible to startReact. It was recently demonstrated that startReact elbow movements were improved in terms of onset latency and muscle activation patterns in stroke survivors compared to voluntary initiated movements (Honeycutt and Perreault The authors would like to thank T. Goetz‐Haswell his technical and scientific expertise.The authors have nothing to disclose.


A new group of arsenolipids based on cell‐membrane phosphatidylcholines has been discovered in herring caviar (fish roe). A combination of HPLC with elemental and molecular mass spectrometry was used to identify five arsenic‐containing phosphatidylcholines; the same technique applied to salmon caviar identified an arsenic‐containing phosphatidylethanolamine. The arsenic group in these membrane lipids might impart particular properties to the molecules not displayed by their non‐arsenic analogues. Additionally, the new compounds have human health implications according to recent results showing high cytotoxicity for some arsenolipids. Arsenic‐containing lipids, so‐called arsenolipids, are natural constituents of marine organisms thought to be involved in arsenic detoxification processes.Research activity over the last eight years, however, has revealed several new groups of naturally occurring arsenolipids, including arsenic‐containing fatty acidsA recent study of a fish oil showed the presence of an unidentified group of arsenolipids thought to be conjugates of arsenic fatty acids because they could be base‐hydrolyzed to known arsenic fatty acids.Clupea harengus; ≈0.8 μg As g−1 dry mass, originating from the Norwegian Sea) with DCM/MeOH and washing the extract with water yielded an organic layer containing approximately 80 % of the initial arsenic. After all traces of DCM were removed, the residue was re‐dissolved in absolute ethanol and analyzed by reversed‐phase HPLC coupled to an elemental mass spectrometer . The HPLC/ICPMS measurements performed in selected‐ion monitoring mode with m/z 75 (As+) indicated the presence of several known arsenic fatty acids and arsenic hydrocarbons, in addition to a cluster of later‐eluting arsenolipids  and one with m/z=123 (C2H8OAs+), when subjected to collision‐induced‐dissociation. Owing to their mass defects, these fragment ions can be uniquely identified by a high‐resolution mass spectrometer even from a complex matrix. Searching the fragmentation spectra for these two fragment ions exposed a multitude of precursor ions, from which six known arsenic fatty acids  and five known arsenic hydrocarbons  could be expressly identified . This procedure similarly implied an ion with [M+H]+ at m/z=529. The accurate mass and fragmentation data of this ion were consistent with a hitherto unknown arsenic fatty acid with eight ethylene groups MexHyO9NAsP, and inspection of the MS/MS (fragmentation) spectra of these ions confirmed similarity as well as a set of prominent fragment ions characteristically observed in the MS/MS spectra of phosphatidylcholines.In the later part of the HPLC/ESIMS chromatogram  did not reveal the presence of AsPCs, but again AsFA 528  and also an arsenic‐containing phosphatidylethanolamine (AsPE) incorporating AsFA 528 were identified , and both contained the five AsPCs described above. The same approach applied to an extract of salmon caviar  PEs and PCs.m/z=585.2919, corresponding to the arsenic fatty acid with part of the glycerol molecule after loss of phosphorylethanolamine and docosahexaenoic acid as the corresponding ketene.The fragmentation behavior of the protonated AsPE 1035  parallels the behavior observed for the protonated AsPCs with the exception that protonated phosphorylethanolamine at The origin of the arsenic phosphatidylcholines in the membrane‐rich fish eggs remains an open question. Phosphatidylcholines are key components of biological membranes. It is possible that infidelity in their biosynthesis results in the arsenic analogues, although a more fundamental role for the arsenic compounds cannot be dismissed. These compounds are likely to be widespread in fatty fish, based on the ubiquity of free arsenic fatty acids and the recent report of unidentified arsenic fatty acid conjugates in fish oil,As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


VZV) is a neurotropic α‐herpesvirus that causes chickenpox and establishes life‐long latency in the cranial nerve and dorsal root ganglia of the host. To date, VZV is the only virus consistently reported to have an inverse association with glioma. The Glioma International Case‐Control Study (GICC) is a large, multisite consortium with data on 4533 cases and 4171 controls collected across five countries. Here, we utilized the GICC data to confirm the previously reported associations between history of chickenpox and glioma risk in one of the largest studies to date on this topic. Using two‐stage random‐effects restricted maximum likelihood modeling, we found that a positive history of chickenpox was associated with a 21% lower glioma risk, adjusting for age and sex : 0.65–0.96). Furthermore, the protective effect of chickenpox was stronger for high‐grade gliomas. Our study provides additional evidence that the observed protective effect of chickenpox against glioma is unlikely to be coincidental. Future studies, including meta‐analyses of the literature and investigations of the potential biological mechanism, are warranted.Varicella zoster virus ( All cases were recruited within a year of diagnosis and consented at their clinic visits. All sites started recruiting participants in April 2010.Controls were 18–80 years of age. Because not all sites were able to recruit controls using the same methods (due to issues related to existing infrastructure and resources), four sites recruited clinic‐based controls, three sites recruited population‐based controls, and seven sites recruited visitors of cancer patients as controls All 14 sites used a common study protocol and the same risk factor questionnaire. Study coordinators were trained to ensure site‐to‐site homogeneity in data collection practices. Data were stored in a centralized database, and were managed by the lead statistician. More details on our data collection methods have previously been published The GICC risk factor questionnaire included demographic characteristics, past medical history, and occupational exposure history. Questionnaires were administered through phone and/or in‐person interviews, or through mailed self‐administered forms. Specifically with regard to VZV‐related conditions, participants were asked whether they had ever had any of a list of viral infections, which included chickenpox and shingles. If they answered yes, they were asked their age or what year it was when they had chickenpox or shingles.The overall GICC analysis plan, as well as details of key sensitivity analyses, are available elsewhere I2 statistic was used for each meta‐regression model to evaluate the proportion of variability in the effect estimates due to heterogeneity, and the τ2 statistic was calculated to assess the intersite variance. In some stratified analyses, the numbers became too sparse to calculate mORs, and thus pooled ORs (pORs) had to be provided instead.Site‐specific unadjusted and adjusted odds ratios (ORs), and their corresponding 95% Wald confidence intervals (CIs), were calculated, using unconditional logistic regression. Sites with less than five cases or controls in the exposed or unexposed groups were excluded from the meta‐analyses. To calculate the meta‐analysis ORs (mOR), we utilized both two‐stage random‐effects maximum likelihood and two‐stage random‐effects restricted maximum likelihood (REML) modeling Age and sex were considered potential confounders (determined a priori) and were adjusted for in all multivariable models, though adjustment for these factors did not meaningfully alter effect estimates. Education, race/ethnicity, allergy status, and cigarette smoking history were evaluated as potential data‐based confounders and were not found to be such, based on a 10% change‐in‐estimate criterion. These variables were therefore not included in the final models. We also stratified our models by age at chickenpox development , and separately by glioma diagnosis/study enrollment age groups .http://www.R-project.org).Sensitivity analyses were conducted in which we included and excluded proxy respondents in the final models and compared the results to ensure that there were no meaningful differences between the ORs. Possible patterns or discrepancies in effect estimates between sites with different control types  or different questionnaire administration methods  were also evaluated Table Approximately, 79% of cases and 83% of controls reported a positive history of chickenpox. Overall, a positive history of chickenpox was associated with a 21% lower glioma risk, controlling for age and sex , between the ages of six and nine , or above age nine .In the overall study population, 10.3% of cases and 9.2% of controls reported having at least one episode of shingles. About 28% of participants reported having their first episodes of shingles before age 30 . A positive history of shingles was not significantly associated with glioma risk . The mORs were similar stratified by tumor grade.In our study, a positive history of chickenpox was associated with a 21% lower glioma risk, adjusting for age and sex. The protective effect of chickenpox was stronger for high‐grade glioma, particularly among those under age 40. Our findings, which represent the results of the largest study to date on this topic, confirm the inverse associations previously reported in the literature on VZV and glioma.The majority of published studies on VZV infection and glioma risk are from the San Francisco Bay Area Adult Glioma Study (SFBAGS) series Like our study, the SFBAGS analyses have implied that the inverse association with prior VZV infection may be stronger for high‐grade glioma P for trend = 0.03). Because of the use of prediagnostic specimens, this study provided further evidence that VZV antibody‐glioma associations reported in the literature are unlikely to be a result of postdiagnostic or treatment‐related factors . Furthermore, such serologic studies also suggest that the associations observed between self‐reported history of chickenpox and glioma risk are unlikely to be completely attributable to memory problems or cognitive deficits in glioma patients.Besides the SFBAGS series, a few other epidemiologic studies have found similar associations between chickenpox and glioma risk A particularly interesting finding of our study is that the protective effect of chickenpox against high‐grade glioma was strongest among the youngest (<40) age group. Median age at glioma development is 55 years In our study, shingles was not associated with glioma risk. Some previous studies have found an inverse association with glioma risk, though shingles has not been studied as frequently as chickenpox and has often been combined with chickenpox, rather than examined separately The biological mechanism through which chickenpox may confer protection against glioma is currently unclear. One proposed mechanism is that VZV antibodies may demonstrate some cross reactivity to tumor cells (or other oncogenic viruses), and are thus capable of helping mount a protective immune response against existing tumor cells A limitation of our study is the amount of intersite heterogeneity between our 14 international sites. Accordingly, we have provided site‐specific odds ratios and have used random‐effects meta‐regression in an effort to account for some of this heterogeneity. Because questionnaire administration methods and control types differed between sites, we have also conducted a number of sensitivity analyses (methods described in reference 15) to ensure that these differences did not detectably bias the results of our analyses.Findings from the previous literature, bolstered by those of our study, provide strong epidemiologic rationale for continued investigation of the potential role of chickenpox (or other manifestations of VZV infection) in glioma development The authors declare no conflicts of interest.


PTMs), such as phosphorylation, acetylation and ubiquitination, has highlighted their importance in the regulation of a myriad of signaling events. While high‐throughput technologies have tremendously helped cataloguing the proteins modified by these PTMs, the identification of lysine‐methylated proteins, a PTM involving the transfer of one, two or three methyl groups to the ε‐amine of a lysine side chain, has lagged behind. While the initial findings were focused on the methylation of histone proteins, several studies have recently identified novel non‐histone lysine‐methylated proteins. This review provides a compilation of all lysine methylation sites reported to date. We also present key examples showing the impact of lysine methylation and discuss the circuitries wired by this important PTM.Large‐scale characterization of post‐translational modifications ( The convergence of high‐throughput proteomics efforts with targeted studies of site‐specific PTM and protein‐modifying enzymes has shed light on the scope of these modifications across a wide variety of organisms. Among the 20 amino acids, lysine is one of the most heavily modified. To this day, lysine residues are known to be covalently modified by acetyl  to chromatin  as a methyl donor, catalyze the addition of a methyl group to the ε‐amine group of a lysine side chain , belongs to an extended superfamily of methyltransferases found throughout eukaryotes, prokaryotes and archaea. Members of this family methylate DNA, RNA or amino acids such as arginine, glutamine, aspartate and histidine  cannot be separated using low‐resolution mass spectrometers. Fortunately, the precision of recent instruments such as Orbitrap and triple TOF simplifies their respective identification  on both K37   will mark which lysine will be methylated during a given cellular process.Methyllysine residues have also been mapped on other pioneer transcription factors. Methylation of GATA4 by EZH2 regulates association of the activator to p300, regulating the expression of GATA4 target genes  recognizes trimethylated K3 of the ribosomal protein L12. This association, mediated by the chromodomain of Corto, recruits the RNA polymerase III and activates transcription of the heat‐shock responsive gene hsp70   demethylates RelA on K218Me and K221Me, opposing the activation of this transcription factor. Interestingly, given that RelA regulates et al, In comparison with other post‐translational modifications, methylation appears to present only limited ways to affect the chemistry of a residue. For example, acetylation of lysine ε‐amine neutralizes its positive charge and the addition of a carbonyl's dipole makes possible new types of interactions. Phosphorylation drastically modifies the charge of a protein (‐3 per phosphate group) and adds a relatively important mass to an amino acid side chain . The addition of ubiquitin and ubiquitin‐like molecules, which increase the size of the targeted proteins by at least 10 kDa, is linked to cell trafficking, transcriptional regulation and endocytosis , allowing association with PAF receptor and strongly contributing to bacterial invasion and pneumonia onset (Barbier et al, In addition to cancer, lysine methylation plays key roles in bacterial pathogenicity. Vaccination efforts against typhus' agent Since its discovery over half a century ago, lysine methylation has been found in all domains of life. It is a dynamic modification, as it can involve the addition of one, two or three methyl groups, and it can be reversed by dedicated demethylases. Although histone lysine methylation is held as a canonical example of the importance of this PTM, it still remains unclear whether it acts as repository of epigenetic instructions or whether it is a consequence of transcriptional and replicative DNA‐based processes. Importantly, methylation of lysine residues influences protein function beyond the context of chromatin, predominantly by modulating the deposition of other PTMs such as phosphorylation, acetylation and ubiquitination or by regulating protein–protein interactions. The versatility of lysine methylation is highlighted by the fact that the same mark, mediated by different methyltransferases, can trigger distinct biological effects in different cellular contexts. Similarly, modification of different residues on a given protein by the same methyltransferase can elicit different biological responses. Future efforts involving the high‐throughput analysis of protein methylation and the identification of the specific subsets of substrates attributable to each PKMT will advance our understanding of the regulatory networks underlying the lysine methylome and will provide novel functional insights regarding this PTM. Moreover, considering the involvement of protein methylation in pathologies, such analyses would be beneficial for developing diagnostic biomarkers and for revealing mechanisms of pathogenicity.The authors declare that they have no conflict of interest.Supplementary Table S1Click here for additional data file.Review Process FileClick here for additional data file.


There are variations in the proportions of head and neck cancers caused by the human papillomavirus (HPV) between countries and regions. It is unclear if these are true variations or due to different study designs and assays.We tested formalin‐fixed paraffin‐embedded diagnostic biopsies for p16 immunohistochemistry and HPV‐DNA (by polymerase chain reaction [PCR] and in situ hybridization [ISH]) using validated protocols on samples from 801 patients with head and neck cancer recruited prospectively between 2006 and 2011 in 4 randomized controlled trials (RCTs).p < .0001) and between Western Europe and Asia . Other independent determinants of HPV positivity were tumor site and smoking.Twenty‐one percent of patients (170 of 801) showed both HPV‐DNA and p16‐positivity, detected almost exclusively in oropharyngeal cancer ; and only 1% of the patients (5 of 499) with nonoropharyngeal cancer were HPV positive. HPV‐positive oropharyngeal cancer differed between Western and Eastern Europe (37%, 155 of 422 vs 6%, 8 of 144; Head Neck38: E1863–E1869, 2016This is the first study to establish geographic variability as an independent risk factor in HPV‐positive oropharyngeal cancer prevalence, with higher prevalence in Western Europe. © 2016 The Authors Head & Neck Published by Wiley Periodicals, Inc.  The proportion of head and neck cancers caused by the human papillomavirus (HPV) seems to have increased considerably over the past decade in several countries.It has become important to ascertain the regional differences in HPV‐positive head and neck cancer to understand the current trends and relative burden of HPV‐positive and HPV‐negative head and neck cancer. This will enable local clinicians and health commissioners to plan for the provision of health care, rehabilitative, and social support services for patients who are HPV‐positive who enjoy higher survival rates but have to deal with the considerable sequelae of their intensive treatment.The variations in reported regional prevalence rates may be a reflection of regional differences in the profiles of established risk factors, such as primary site, smoking, or sexual habits,In this study, our purposes were to overcome these potential deficiencies, to provide a recent and more accurate representation of the proportion of HPV‐positive head and neck cancer in different geographic regions, and to assess the role of region as a risk factor for HPV‐positive head and neck cancer. We compared the proportion of patients with HPV‐positive head and neck cancers and analyzed possible risk factors in a multinational cohort of patients with head and neck cancer who were all recruited prospectively in randomized controlled trials (RCTs), within a recent time frame. All samples were assessed for high‐risk HPV‐DNA using the same validated protocols in 2 quality‐assured laboratories.We examined formalin‐fixed paraffin‐embedded diagnostic biopsies from a total of 1049 patients with head and neck cancer who gave informed consent and were recruited to 4 multicenter RCTs: PET NECK trial (187 subjects), GSK EGF104334 (107 subjects), GSK EGF105884 (67 subjects), and GSK EGF102988 (688 subjects). Most subjects included in our study were recruited between 2007 and 2010.Ethical approval for PET NECK trial (reference: 07/Q1604/35) was granted by the Oxfordshire Research Ethics Committee, May 2007. GSK EGF Ethical approval (reference: 06/MRE12/82) was granted by the Thames Valley Research Ethics Committee, March 2007.The procedures followed were in accordance with the ethical standards of the committees on human experimentation and in accord with the Helsinki Declaration of 1975, as revised in 1983.Analysis was undertaken on tissue sections (4 μm) or on tissue microarrays (TMAs) with up to four 0.6‐mm diameter cores from each case. We took 10‐μm thick tissue curls from available blocks for DNA extraction and downstream polymerase chain reaction (PCR) analysis. All samples were analyzed for p16 expression by immunohistochemistry using a proprietary kit  on a Benchmark Autostainer . Two pathologists (H.W. and M.R.) scored the test results independently, after undergoing calibration. Samples were assessed for high‐risk HPV‐DNA using a protocol based on that described and validated by Smeets et al.For all samples in this study, cases that showed evidence of HPV‐DNA (either by ISH or consensus PCR) and showed high p16 expression (HPV‐positive/p16‐positive) were defined as “harboring biologically relevant oncogenic HPV infection.”All subjects who were enrolled into the studies but who had not yet provided tumor tissue for biomarker testing were included in a summary of baseline and disease characteristics, but excluded from all other analyses. Subjects who had provided tumor tissue that was inadequate for testing were not included in the patient characteristics summaries. The latter are based on the subset of subjects who had valid results for both HPV and p16.Subjects with at least one positive result for HPV and one positive result for p16 for any of the cores and sections were considered to be positive overall for that biomarker. If the patient had no positive cores or sections, then they were considered negative, otherwise their result was treated as unknown, and they were not included in the analysis. Subjects who tested negative for p16 and who had an inconclusive result for HPV were considered as HPV‐negative overall.The analysis includes only subjects with valid results for both biomarkers (HPV‐DNA and p16). The demographic and baseline disease characteristics, including age, sex, primary tumor type, disease stage, smoking status, and geographic region, were summarized for subjects who had valid results for biomarkers, as well as those who did not provide tumor tissue for testing or who had insufficient tissue. The frequency and proportion of subjects testing HPV‐positive, p16‐positive, and those with combined positivity (HPV‐DNA‐positive and p16‐positive) were also reported by these characteristics. Where possible, odds ratios for combined positivity (HPV‐DNA‐positive and p16‐positive) were calculated for each characteristic, with suitable dichotomization where necessary. A logistic regression analysis was used to model the probability of testing positive for both HPV‐DNA and p16, adjusting for the risk factors listed above. Combined test results were grouped into 4 categories: (1) HPV‐positive/p16‐positive; (2) HPV‐positive/p16‐negative; (3) HPV‐negative/p16‐positive; and (4) p16‐negative/HPV‐negative, and summarized for patients with oropharyngeal cancer and nonoropharyngeal cancer tumors, as well as by geographic region.A total of 1049 patients submitted samples, constituting 81% of total subjects recruited in all 4 studies at the time of the analysis. Eight hundred one patients had valid results for both biomarkers. The primary tumors included were squamous cell carcinomas originating from the oral cavity in 241 patients (30%), the oropharynx in 302 patients (38%), the larynx in 159 patients (20%), and the hypopharynx in 99 patients (12%). Compared to those who were included in the analysis, the cases that were not included had very similar baseline characteristics  showed HPV‐DNA (by ISH or PCR), 218 (27%) tested positive for p16, and 170 (21%) showed both HPV‐DNA and p16‐positivity .There was a large difference in the distribution of HPV and p16‐positivity between tumor sites. Biologically relevant, oncogenic high‐risk HPV infection (HPV‐DNA‐positive/p16‐positive) was detected almost exclusively in oropharyngeal cancer , whereas 1% (5 of 499) of nonoropharyngeal head and neck cancers were positive  and Eastern Europe  compared to Western Europe  and 9% (2 of 23) in Asia  in oropharyngeal cancer also differed significantly by geographic location: 63% of the patients 152 of 242) in Western Europe, compared to 24% (8 of 33) in Eastern Europe , and laryngeal cancers  and for former smokers relative to nonsmokers . Subjects with stage IV disease were more likely to have HPV‐positive cancer  compared to those with stage II disease. Importantly, subjects from Western Europe had a significantly higher risk of being HPV‐positive compared to those from Eastern Europe  compared to Eastern Europe, but this was not statistically significant. There was no association with age or sex , for former smokers relative to nonsmokers  and significantly higher for subjects from Western Europe  compared to those from Eastern Europe , or sex , or disease stage , and second using stepwise selection. Only primary site, smoking status, and geographic region were found to have a significant influence on the probability of biologically relevant oncogenic HPV infection (HPV‐DNA and p16‐positivity) in head and neck cancer (Table p = .69).When examining risk factors for oropharyngeal cancer alone, multivariate analysis revealed only smoking status and geographic region to be significantly associated with combined HPV‐positivity and p16‐positivity . This was the same whether the factors were introduced together or in a stepwise fashion. There was no statistically significant interaction between the risk factors (This study demonstrated large, significant differences in the proportion of HPV‐positive oropharyngeal cancer among Western Europe, Eastern Europe, and Asia, with HPV‐associated head and neck cancer being predominantly a disease of the oropharynx in Western Europe. Geographic location seems to have a strong association with HPV‐positivity in oropharyngeal cancer, regardless of smoking status, which is a well‐established risk factor. To the best of our knowledge, this is the first time that a study firmly has established geographic variability in the prevalence of HPV‐positive oropharyngeal cancer. The study highlights the need for ascertainment of local rates before deciding on local health policies and strategies related to oropharyngeal cancer. The results also demonstrate the very low, relative prevalences of HPV in nonoropharyngeal cancer tumors, and emphasize that, despite the current interest in HPV‐related disease, the prevalence of HPV‐negative head and neck cancer in many countries remains considerably higher than that of HPV‐positive disease.By collating one of the largest cohorts ever collected, using prospective multinational trials, and analyzing them using the same assay and methodology, this study has been able to overcome some of the main limitations of previous studies. After accounting for tumor site and smoking status, geographic region has a significant influence on the probability of testing positive for HPV. The reasons for these geographic variations remain speculative. A likely factor may be related to differences in the prevalence rates of oral HPV infection.Some hypothesize that smoking may protect against the development of HPV‐positive oropharyngeal cancer.This study had some limitations. Only 76% of eligible subjects had evaluable samples. Although this is high when compared to similar studies, it may have still introduced bias to our results. The subjects included had been recruited within trials with eligibility criteria, including having treatment for curative intent. This introduces the possibility of bias compared to population‐based estimates, which may also include subjects with very early disease or those undergoing palliative treatment, who are more likely to have HPV‐negative oropharyngeal cancer.Our study also enabled the quantification of the relative prevalence of HPV in nonoropharyngeal head and neck cancer compared to oropharyngeal cancer, reports of which have varied widely in the literature.Our findings have implications for the planning of future clinical trials. It is clear that HPV‐positive and HPV‐negative head and neck cancers have different characteristics and survival rates, and are thus different disease entities.The findings of this study also have significant implications for local health care policies. In regions where HPV‐positive disease is prevalent, it will be necessary to consider and plan for the long‐term support and survivorship needs of these younger subjects, who are much more likely to survive their cancer, but who will also have to deal with the long‐term effects on them and their families of their disease and treatment.Over three fourths of the subjects recruited in our study had HPV‐negative head and neck cancer Table . AlthougFinally, the significant differences in the prevalence of HPV‐positive head and neck cancer have implications for preventative public health measures, especially in resource‐limited settings. The cost‐effectiveness of the HPV vaccination for prepubescent boys is highly dependent on the prevalence of the disease, among other factors. In areas of low HPV prevalence, especially in countries with low health care expenditure, resource and efforts may be best directed toward smoking cessation policies.Supporting InformationClick here for additional data file.


Cooperation is essential for the success of societies and there is an ongoing debate whether individuals have therefore developed a general spontaneous tendency to cooperate or not. Findings that cooperative behavior is related to shorter decision times provide support for the spontaneous cooperation effect, although contrary results have also been reported. We show that cooperative behavior is better described as person × situation interaction, in that there is a spontaneous cooperation effect for prosocial but not for proself persons. In three studies, one involving population representative samples from the US and Germany, we found that cooperation in a public good game is dependent on an interaction between individuals’ social value orientation and decision time. Increasing deliberation about the dilemma situation does not affect persons that are selfish to begin with, but it is related to decreasing cooperation for prosocial persons that gain positive utility from outcomes of others and score high on the related general personality trait honesty/humility. Our results demonstrate that the spontaneous cooperation hypothesis has to be qualified in that it is limited to persons with a specific personality and social values. Furthermore, they allow reconciling conflicting previous findings by identifying an important moderator for the effect. Cooperation is important for societies to achieve collective goals2343427Previous experience with the experimental setting of social dilemma games has been found to reduce the effect10We guide the perspective towards social value orientation (SVO), which is a continuous measure of social preferences and can be expected to be an important moderator for the spontaneous cooperation hypothesis on theoretical grounds31214We conducted three studies including one lab and two online experiments. The studies were approved by the local ethics committee of Göttingen University’s Psychological Department and conducted in accordance with the approved guidelines. Before conducting the studies, we obtained informed consent from all subjects. With the methods and instructions used, we strictly replicated the previous core investigation by Rand and colleaguesThe basic procedure is described in Decision time was measured via the programs used for data collection (i.e.‘Unipark’ for the online studies and ‘Bonn Experiment System’ for the lab study). In the first study we analyzed data of 134 Amazon Mechanical Turk (MTurk) workers  half of them from the United States and the remaining from other western countries and India. Second, we elaborated these results in a more controlled setting and conducted a laboratory study with mostly German students . Finally, as a third experiment, we conducted a high-power online study with representative samples concerning age and gender for the US (N = 249) and German populations (N = 255), resulting in a total sample of N = 504 . Data for the third experiment was gathered via a professional panel provider. The endowment for the PGG varied between studies  in the lab experiment and USD 1.50 in the third study (Panel)) but the multiplication factor of two for contributed money remained constant.In line with previous studies we additionally gathered data about beliefs (the expectations about the other players’ cooperation behavior) as well as cooperativeness of interaction partners in daily life and previous experience with the experimental setting of social dilemma games. As an important extension, we additionally measured social value orientation using the SVO Slider Measure0.5 = 3.16) to 31.6 sec (101.5 = 31.6) was accompanied by a decrease of cooperation by 54.52% according to a Tobit regression and by 19.08% according to an OLS regression  = 17.21, p < 0.001). This difference was paralleled by a difference in social value orientation  = 30.68, p < 0.001) with around 5° of SVO angle differences between studies. The parallel development of contributions and SVO is–as expected–reflected in a strong correlation between both measures . Hence, differences in social values  among the different samples used in our studies have most likely driven the level differences in contributions. To correct for these differences, we included study dummies as control variables in the overall analysis of contributions.In an overall analysis of the three experiments , we findsion see . ConsideMost importantly, in this overall analysis we find the expected interaction of decision time and SVO . In the p < 0.001) but does not hold for proselfs . This also holds true in separate analyses for the three studies  and proselfs (SVO angle < 22.45°) reveal that the spontaneous cooperation effect is only observed for prosocials , but the interaction effect persists on a marginal significance level also when controlling for SVO and its interaction with decision time (p = 0.064). The observed interaction concerning the specific construct social values therefore also generalizes to the related but broader personality construct Honesty-Humility.To validate our results also on the basis of a broader personality construct, we measured Honesty-Humility as one of the six factor personality scale HEXACO1719In a set of studies we show that the controversially debated spontaneous cooperation effect replicates in population representative samples and beyond. Given its small effect size, large samples are needed in order to find the effect on a conventional significance level. Crucially, however, we show that the spontaneous cooperation effect is also strongly dependent on personal characteristics. For persons that are prosocial and high on the personality factor Honesty-Humility cooperation decreased with increasing decision time. These persons drive the spontaneous cooperation effect reported in some of the previous studies. No relation between decision time and cooperation exists for selfish persons. The demonstration of this moderator allows reconciling previous diverging results.The finding that only prosocials show the spontaneous cooperation effect is consistent with the explanation that cooperation might be their default response, which is sometimes overwritten by increased deliberation. Given that prosocials are mainly conditional cooperators that are particularly sensitive to changes in beliefs concerning the behavior of other group membersData Availability: Data and materials are also available online at Open Science Framework (https://osf.io/w7hsk/).How to cite this article: Mischkowski, D. and Glöckner, A. Spontaneous cooperation for prosocials, but not for proselfs: Social value orientation moderates spontaneous cooperation behavior. Sci. Rep.6, 21555; doi: 10.1038/srep21555 (2016).


Frequent evolutionary birth and death events have created a large quantity of biologically important, lineage‐specific DNA within mammalian genomes. The birth and death of DNA sequences is so frequent that the total number of these insertions and deletions in the human population remains unknown, although there are differences between these groups, e.g. transposable elements contribute predominantly to sequence insertion. Functional turnover – where the activity of a locus is specific to one lineage, but the underlying DNA remains conserved – can also drive birth and death. However, this does not appear to be a major driver of divergent transcriptional regulation. Both sequence and functional turnover have contributed to the birth and death of thousands of functional promoters in the human and mouse genomes. These findings reveal the pervasive nature of evolutionary birth and death and suggest that lineage‐specific regions may play an important but previously underappreciated role in human biology and disease. CAGEcap analysis of gene expressionChIPchromatin immunoprecipitationDHSDNaseI hypersensitive siteH3K27acacetylated histone H3 lysine 27H3K4me2dimethylated histone H3 lysine 4H3K4me3trimethylated histone H3 lysine 4lncRNAlong non‐coding RNALTRlong terminal repeatmiRNAmicroRNATFtranscription factorwww.ensembl.org) The large and varied diversity observed between individuals and across species is reflected in high levels of genetic diversity. The study of this diversity between mammalian species has been possible on a genome‐wide scale since the publication of the first complete drafts of the human and mouse genomes in 2001 This wealth of data has stimulated the field of comparative genomics, which investigates both the similarities and differences between genomes. Much early work focussed on identifying shared features between sequenced genomes and restricted itself to the small proportion of the genome which encodes for protein‐coding genes. Many of these genes have been deeply conserved throughout evolution from yeast to human, the lineages of which diverged approximately one billion years ago C20orf203 is found only in the human genome, and is absent from closely related primates. This gene is highly expressed in the brain, and is further upregulated in Alzheimer's disease, which suggests a potential role for this lineage‐specific gene in the development of the disease Not all protein‐coding genes, however, are evolutionarily ancient. For example, Comparative genomics has also been applied to studying the remaining, non‐coding, regions of the genome, which make up almost 99% of the genome Poldi. This lncRNA is restricted to the post‐meiotic cells of the testis, and promotes sperm motility and testis development miR‐941 – which was born in the human lineage between one and six million years ago from the expansion of an evolutionarily unstable tandem repeat sequence – was recently discovered to be important for neurotransmitter signalling in the brain As for protein‐coding genes, there are also corresponding examples of non‐coding RNA genes that have been emerged during evolution, such as the mouse‐specific lncRNA HACNS1 has evolved rapidly in humans and only the human sequence, but not the orthologous primate sequence, is able to function as a limb enhancer in mouse reporter experiments AR gene, which is conserved across most mammalian species has been deleted from the human genome. The activity of this enhancer is correlated with the formation of whiskers and penile spines in non‐human mammals, and it has been speculated that this loss in humans may be linked to increased monogamous reproductive strategies relative to other primates There are a handful of known examples of enhancer birth and death in the human genome. For example, the non‐coding element On a genome‐wide scale, non‐coding elements are less conserved between mammalian species than protein‐coding genes In this essay, I will first discuss sequence turnover, where sequence is either inserted (born) or deleted (dies) along a lineage. I will then describe analyses of functional elements that have been identified through experimental profiling Box  and wereBox 1Chromatin immunoprecipitation followed by sequencing (ChIP‐seq): Identifies the locations of histone modifications or the sites of a DNA‐binding protein, such as a transcription factor, by high‐throughput sequencing of DNA pulled down with an antibody specific to the modification or protein of interest.DNase 1 hypersensitivity sites sequencing (DNase‐seq): Discovers all classes of active regulatory elements by digesting accessible chromatin which is not packaged into nucleosomes, followed by high‐throughput DNA sequencing.Cap analysis of gene expression (CAGE): Clones the 5′ ends of transcribed mRNA molecules and then subjects them to high‐throughput sequencing to precisely identify the sites of transcription initiation at promoters and other transcribed elements, such as enhancers.The insertion or deletion of sequence along one of the lineages that separates two species (collectively known as ‘indels’) results in gaps in the sequence alignments which describe the relationship between orthologous sequences within the two genomes being compared. Insertions can be discriminated from deletions by comparing the sequences of three or more genomes simultaneously. The principle of parsimony makes the assumption that the most likely evolutionary history for a set of related sequences is the scenario that can be explained by the minimal number of mutations. In this way, a sequence is defined as having been deleted if it is present in the outgroup species, while an inserted sequence will be absent from this species Fig. . This tyWithin the human population, there are likely to be many millions of polymorphic indels which are found in some, but not all, individuals. A study of 79 diverse human genomes reported almost two million small indels Polymorphic indels disrupt the coding sequence of over 6% of annotated human genes, but 72% of genes contain an intronic indel The distribution of indels throughout the genome has been used to quantify the amount of functional, but lineage‐specific, sequence within the genome. This model assumes that indels occur randomly within the genome and that unexpectedly large distances between indels therefore contain sequence which is preserved by natural selection There are a number of molecular mutations that insert or delete sequence in the genome. Transposable elements, which are capable of jumping around the genome, make up approximately half of the human genome Sequence can also be inserted or deleted from the genome through the activity of normal cellular processes, such as recombination and replication. Unequal crossing‐over takes place when non‐homologous regions are paired during cell division. This can result in one chromosome gaining sequence and the other losing the same sequence Fig. C, but thDespite reports of sequence gain and loss within the human genome, their accurate discovery remains a difficult task, requiring the development of specialised computational pipelines Genome sequencing projects have been followed by a second wave of functional genomics studies, as exemplified by the work of the ENCODE consortium FOXA2, HNF1A, HNF4A and HNF6) in human and mouse revealed that 41–89% of their binding locations within aligning sequence were found in only one of these two species Large‐scale functional turnover of both transcription factor (TF) binding and promoter locations have been reported. A comparison of four liver‐specific TFs . The degree of overlap required to identify orthologous regions also affects the detection of functional turnover events. Some studies consider a single 1 bp overlap between regions as sufficient to define them as being conserved while others have required at least a 50% overlap in reciprocal comparisons between species While both mechanisms of birth and death in the genome – sequence and functional turnover – are clearly important contributors to lineage‐specific genomics, it is only recently that they have been explicitly investigated simultaneously in the same experimental system.DrosophilaThe FANTOM5 project, which identified promoter locations across a range of matched human and mouse cell lines and tissues, described the half‐life of promoters when aligned to increasingly divergent species The sequences of a large number of promoters have been born or died along the human lineage , since its divergence with mouse cis‐regulatory elements found at a constant distance from the promoter. This contrasts with the inverse correlation seen between expression and substitution rate divergence at promoters activated in lipopolysaccharide‐stimulated macrophages Many promoters whose sequence has been conserved between human and mouse have experienced functional turnover , as they show no detectable evidence of transcription in the opposing species. These species‐specific promoters are specifically associated with decreased evolutionary constraint at the promoter elements While sequence gain and loss are clearly important factors in promoter evolution along the mouse and human lineages, the lack of well‐matched data across more species remains the limiting factor for resolving the large number of functional turnovers into births and deaths at aligned sequence. As shown in Fig. The birth and death of both sequence and function is a common occurrence within the human genome, and represents an important contributor to genetic diversity. These turnover events have been observed at both distal regulatory elements and functional promoters and confirm that, while a useful predictor, evolutionary conservation is not required to identify functioning, lineage‐specific elements in the human genome.The availability of large amounts of functional genomics data in both human and mouse have already allowed evolutionary turnover to be investigated across a number of tissues. Extending the available datasets to more distantly related species will permit functional turnover events to be resolved into births and deaths along individual lineages Fig. . InvestiInvestigations into the combined effects of sequence and functional turnover in the birth and death of genetic elements have only recently been attempted. Further work will likely focus on the direct relationship between these, for example one might ask if functional deaths result in biologically unimportant sequence that is then a target for sequence deletion and the complete removal of the element from the genome.Despite this work, the biological relevance of these evolutionarily volatile elements still remains unclear. Do they drive the diversification of gene expression profiles, or do they simply represent the neutral churn of redundant genetic elements in the genome? By carefully matching current datasets to increasing amounts of functional genomics data from multiple species The author has declared no conflicts of interest.