Tendon tissues have limited healing capacity. The incidence of tendon injuries and the unsatisfactory functional outcomes of tendon repair are driving the search for alternative therapeutic approaches envisioning tendon regeneration. Cellular therapies aim at delivering adequate, regeneration-competent cell types to the injured tendon and toward ultimately promoting its reconstruction and recovery of functionality. Mesenchymal stem cells (MSCs) either obtained from tendons or from non-tendon sources, like bone marrow (BM-MSCs) or adipose tissue (ASCs), have been receiving increasing attention over the years toward enhancing tendon healing. Evidences from in vitro and in vivo studies suggest MSCs can contribute to accelerate and improve the quality of tendon healing. Nonetheless, the exact mechanisms underlying these repair events are yet to be fully elucidated. This review provides an overview of the main challenges in the field of cell-based regenerative therapies, discussing the role of MSCs in boosting tendon regeneration, particularly through their capacity to enhance the tenogenic properties of tendon resident cells. Cells are Nature’s tissue engineers, intervening, through a spatiotemporal coordinated manner, in tissue homeostasis, disease development, tissue repair/healing, and ultimately regeneration. Within the Metazoa kingdom, regenerative abilities are unequally distributed . RegenerRegenerative medicine, including tissue engineering, envisions the generation of adequate therapeutic strategies to restore the functionality of an injured tissue or organ. Central to these approaches, cells are key players in orchestrating regenerative responses and thus, are the main targets of any strategy envisioning tissue regeneration. Thus, despite the fact that different approaches can be considered, all rely on controlling to some extent cellular responses, which implies knowing the biology of the target tissue. Within the human body, tendons are highly prone to fibrotic healing through excessive and disorganized deposition of the extracellular matrix (ECM) . This reOver the years, regenerative tendon approaches have been exploiting (i) cellular therapies ,9; (ii) Tendons are highly organized connective tissues comprising bottom-up assemblies of collagen molecules . This hiTendons are relatively hypocellular tissues. Nonetheless, tendon cell populations assure ECM dynamics during tissue homeostasis. As a result of the stretching mechanical forces, tendon cells exhibit a characteristic elongated, spindle-shape morphology. Traditionally, tendons were believed to comprise a homogenous population of tenocytes, which are differentiated cells expressing common tendon markers, such as scleraxis (Scx), a basic loop-helix-loop transcription factor, tenomodulin (Tnmd), a transmembrane glycoprotein, as well as the tendon ECM component collagen type I (Col1) ,19. NoneAlthough the cellular and molecular mechanisms intervening in tendon development, homeostasis and repair are still to be fully uncovered, it is evident that an interplay between biophysical and biochemical signals coordinates cellular function and impacts tenogenic differentiation of stem cells.Musculoskeletal conditions are the second largest contributor to disability, affecting people across the life-course worldwide. The Global Burden of Disease (GBD) study has estimated that 20–33% of people globally live with a painful musculoskeletal condition ,28. AmonTendinopathies are commonly divided into two major classes: acute and chronic (degenerative) damage . Acute tIn general, the quality of repaired tendon tissue rarely returns to pre-injury levels, leading to high re-injury risk and the onset of degenerative changes related to the development of chronic tendinopathies ,35. GiveCellular therapies envision the delivery of regeneration-competent cell populations, i.e., cells that are able to repopulate the injured tissue and/or of empowering resident cell populations with the ultimate goal of promoting tissue reconstruction and functional recovery. Tendon healing frequently encompasses the differentiation of resident TSPCs toward a phenotype resembling that of activated fibroblasts, leading to scar tissue formation, exacerbated inflammatory response and impaired functionality ,36. In tMSCs have been long described to possess multilineage differentiation ability in vitro and have been widely used for applications in Tissue Engineering and Regenerative Medicine, including in the field of musculoskeletal repair . NotwithAdult stem cells of mesenchymal origin hold great interest in the field of tendon tissue engineering and regeneration as promising alternatives to the scarce population of tendon cells. Herein, we will discuss the main outcomes of strategies using MSCs from different sources for tendon repair. Nonetheless, pluripotent stem cells have also been explored in the field, namely embryonic stem cells and induced pluripotent stem cells (iPSCs) ,43. In pKey issues to be considered when attempting to translate bioengineering strategies to tendon therapeutic approaches include (i) the lack of standardized isolation protocols, (ii) the inexistence of tendon-specific molecular markers and (iii) the definition of adequate differentiation protocols. Consequently, given the lack of well-established/standardized differentiation protocols and characterization markers for tenogenic differentiation, the selection of an ideal cell source represents a major challenge while trying to establish an effective cellular therapy.• Isolation protocolsSeveral problems remain unsolved concerning the isolation of tendon cell populations. Isolation of TSPCs and tenocytes still lacks the tune of enrichment and selection approaches. Tendon cells were initially isolated following collagenase digestion of tendon explants ,47. Howe• Molecular signatureTendon cells are highly-specialized fibroblasts, being the main producers of tendon ECM components. Given their mesenchymal origin and the limitations of characterizing fibroblasts and distinguishing them from MSCs , the defStrikingly, several factors such as age, donor variability, tendon type, and anatomic location, alongside with culture conditions, have also been reported to influence markers expression in these cell populations .• Differentiation protocolsThe establishment of adequate differentiation methods relies on the optimization of inductive protocols to commit stem cells toward a certain lineage. In opposition to chondrogenic and osteogenic differentiation, there is no standard induction protocol for tenogenesis. Generally, different growth factors can be added to culture medium to induce the desired phenotype, as these biomolecules are powerful tools in regulating biological responses. These regulatory effects have been demonstrated, for instance, after culturing human adipose-derived stem cells (ASCs) or human amniotic fluid stem cells (AFSCs) in the presence of growth factors associated with tendon development and healing, namely endothelial growth factor (EGF), PDGF-BB, bFGF, and TGF- β1 . An upreEven though several attempts have been made over the years to commit stem cells towards the tenogenic lineage, the identification of several major tenogenic biomarkers is of major importance. As the molecular signature of tendon cells is still to be uncovered, it is still difficult to clearly understand and clarify the differentiation process occurring in several cultures and in the proper tissue, posing a strong challenge to the development of effective cell-based therapies.MSCs can be applied in either autologous or allogeneic settings and can be obtained from tendon or non-tendon tissues. In the following sections, the contribution of MSCs from different sources to tendon healing, and ultimately regeneration, is discussed.Tendon stem cells are frequently obtained as a heterogeneous population of stem and progenitor cells, as highlighted before. The purity of tendon cell populations is highly debatable, but these cells are believed to hold potential for improving tendon repair mechanisms. Autologous tenocyte implantation is currently under clinical studies , but it is not clear whether the transplanted cells include only differentiated cells or a mixed population comprising also TDSCs or TSPCs.Tendon stem cells represent 1–4% of the total number of nucleated cells in tendon tissues  and haveNonetheless, the limited cell number obtained upon isolation from tendon tissues requires an additional step of in vitro expansion, which leads to phenotypic drift  and consBone marrow-derived MSCs are the most commonly explored source of stem cells in tendon tissue engineering and regeneration approaches ,78,79,80• Bone marrow-derived MSCsBM-MSCs are commonly harvested through a minimally invasive aspiration procedure from the iliac crest and then isolated from the mononuclear cell fraction upon density centrifugation. BM-MSCs have been frequently used as a comparison in tendon stem cell characterization studies and are known to express several tendon-related markers, including scleraxis, tenomodulin, collagen types I, II and III, decorin, biglycan, although to a lower extent than TSPCs .In vitro co-culture studies have been demonstrating the role of bi-directional crosstalk between tendon cells and BM-MSCs on the induction of a possible tenogenic phenotype through an up-regulation of tendon-related genes, including scleraxis and tenomodulin, and tendon ECM markers, like collagen type I, decorin and tenascin, together with significant ECM deposition ,80. OverInterestingly, BM-MSCs have been reported to generate embryonic tendon-like tissue in vitro through a process that is mediated by TGF-β3 signaling and requires a 3D environment . In thisFurthermore, BM-MSCs are being explored for their role in supporting tendon cells, particularly through the release of paracrine factors. Pre-conditioning tendon cells by in-vitro culture with BM-MSCs secretome and further combining those cells with an electrospun keratin-based scaffold resulted in improved biomechanical performance in an in-vivo rat model of chronic massive rotator cuff tear . These oBesides in-vitro tendon cell priming, other bioengineering approaches include the resort to scaffold-free cell sheet engineering. Cell sheets derived from co-culturing TDSCs and BM-MSCs have been reported to support tendon healing in a rat patellar tendon window injury model . The comIn summary, BM-MSCs are paving their way in tendon regenerative therapies owing to their contribution to new tendon-like tissue formation and to boost the tenogenic properties of tendon cells populations.• Adipose tissue-derived MSCs (ASCs)ASCs constitute an interesting candidate cell source for tissue engineering and regenerative medicine applications. These cells can be isolated from subcutaneous adipose tissue and, more recently, from liposuction aspirates ,85 and hAdditional concerns that must be undoubtedly considered arise from the hypocellular nature of tendon tissues. ASCs exhibit a high proliferation rate, whereas tendons possess relatively low cell numbers and an overproliferative phase may lead to fibrotic tissue formation and scarring. Strikingly, co-culturing ASCs with tendon-derived cells enabled a control over the proliferation rate; indeed, lower cell numbers have been reported for co-culture systems in comparison to ASCs cultured alone .Altogether, these results support a potential therapeutic effect for ASCs as they have been able to modulate the microenvironment of tendon niche in vitro, as well as to improve tendon healing in vivo, but more studies at the molecular level will be useful to help clarify the exact mechanisms behind these responses.Evidences from both in vitro and in vivo studies suggest that tendon resident cells, including tendon MSCs (TSPCs or TDSCs), may be the main orchestrators directing tendon-regenerative processes. Nonetheless, such biological response upon injury may be further boosted by the administration of non-tendon MSCs through two distinct but synergistic mechanisms—“cell replacement” and “cell empowerment”. Indeed, MSCs include cellular populations with potential therapeutic contributions by homing to the target site and exhibiting the ability to reconstitute/repopulate the injured tissue, simultaneously exerting an immunomodulatory effect that may shift the inflammatory environment. These events are crucial in switching tendon repair from pro-fibrotic to tissue regeneration, but functional clinical outcomes are still to be investigated.Future research in the field of cellular therapies for tendon regeneration must address several issues that still pose a huge obstacle to clinical translation, including the lack of standardized methods and the inexistence of an optimal panel of markers for characterizing tenogenic differentiation steps.Furthermore, there is clear influence from the bi-directional crosstalk between tendon MSCs and BM-MSCs or ASCs in enhancing the tenogenic properties of native cell populations. Nonetheless, the cellular and molecular mechanisms involved in these processes need to be investigated in more detail. Notwithstanding, tendon tissue engineering and regeneration can evolve from the combination of MSCs of different origins with biomaterials’ support to improve tendon healing and simultaneously provide adequate mechanical performance at the tissue level.


Alternate causes for such pleural pathology were absent. There is merit in physicians and pathologists having an awareness of these new manifestations when considering claimed asbestos related changes during life and at postmortem.Yellow nail syndrome is a rare acquired condition of unknown aetiology associated with distinct nail discolouration/xanthonychia, pulmonary manifestations, and lymphoedema. Pleural plaques and diffuse pleural thickening are typically, although not exclusively, recognised as markers of prior commercial asbestos exposure. The presence of such biomarkers may assist an asbestos personal injury evaluation. A postmortem examination performed on a 72-year-old man with known long-standing yellow nail syndrome identified pleural plaques and diffuse pleural thickening. An evaluation of the occupational history identified no known asbestos exposure. Electron microscopic mineral fibre analysis detected no asbestos fibres. To the best of our knowledge, this is the only case of yellow nail syndrome in which these benign pleural changes are reported This article describes the new manifestations of bilateral parietal pleural plaques and diffuse visceral pleural thickening/fibrosis in a subject with yellow nail syndrome with no discernible asbestos exposure. Careful patient evaluation revealed no known occupational history and no asbestos bodies or asbestos fibres were detected by light microscopy or transmission electron microscopy, respectively. We describe the patient's ante-mortem history, post-mortem findings including histology, together with fibrous and non-fibrous mineral analysis. The extent of the pleural changes in this case was such to mimic those seen in asbestos related disease. In the setting of potential asbestos related personal injury claims and in the investigation of asbestos related deaths at post mortem, there should be an awareness that the spectrum of manifestations present in yellow nail syndrome may include pleural plaques and diffuse pleural fibrosis.A 62 year-old male was diagnosed with yellow nail syndrome in 2000. He had a long history of recurrent sinusitis from 1983 and had developed numerous respiratory tract infections since 1996. Primary lower limb lymphoedema was diagnosed in 2000. Shortly thereafter he developed recurrent, initially right pleural effusions. Repeat thoracocenteses had revealed cloudy, thick fluid, exudative in nature. A right video-assisted thoracoscopic pleural biopsy was performed which showed chronic inflammation and reactive mesothelial changes but no malignancy. The diagnosis of yellow nail syndrome was made when xanthonychia developed in 2000. The clinical course continued with the development of bronchiectasis in 2003, complicated by recurrent chest infections and bilateral effusions.The subject had a history of heavy prior tobacco smoking and had worked as a general manager, policeman, clothing design director, and dark room technician.He died in 2010 following an infective exacerbation of his bronchiectasis.A CT scan performed shortly before his death showed extensive right pleuroparenchymal disease, including right diffuse pleural thickening, bronchiectasis and right airspace shadowing .A postmortem examination was performed. External examination revealed the presence of yellow discolouration affecting the finger- and toenails , along wMineral analysis performed on tissue from the left lung by transmission electron microscopy and energy dispersive X-ray spectrometry detected: no commercial or noncommercial amphibole asbestos fibres or serpentine chrysotile asbestos. Fibrous mullite, a nonasbestos aluminium silicate, fibrous silica and muscovite were detected, confirming a technically successful analysis. Non-fibrous mineral analysis detected silicon, talc, aluminium, titanium, and iron.Cause of death was recorded as yellow nail syndrome complicated by infection (lobar pneumonia) and diffuse pleural fibrosis (immediate cause).Yellow nail syndrome is a rare medical condition associated with yellow discolouration of the nails, lower limb lymphoedema and respiratory tract symptoms, including bronchiectasis and pleural effusions. The first series of patients were reported in 1964, by Samman & White, and since then less than 400 cases have been reported in the literature .The diagnosis of the syndrome requires at least 2 of the 3 features to be present simultaneously.The aetiology of the disease is largely unknown, lymphatic impairment, autoimmune disorders, paraneoplastic manifestations, and exposure to titanium from implants and medications have been postulated to play a role in the development of the syndrome , 3.Respiratory involvement was reported in 56-71% of the patients, with changes including pleural effusion, bronchiectasis, recurrent pneumonias, bilateral apical fibrosis, patchy alveolar infiltrates, and cystic lesions .Histopathological examination of the pleura either is noncontributive or shows features of chronic fibrosing pleuritis .We report a case of yellow nail syndrome in which the extent of pleural pathology was so marked as to mimic asbestos-related disease in a subject with no proven asbestos exposure.Diffuse visceral pleural thickening/fibrosis and parietal pleural plaques are an exceptional finding in yellow nail syndrome.Diffuse pleural thickening is defined as a uni- or bilateral thickening of the visceral pleura, at least 5 mm in thickness and involving at least one third of the affected lung surface.Histologically it is characterised by a basket-weave pattern of pleural fibrosis with subpleural parenchymal interstitial fibrosis superficially extending into the underlying lung .While diffuse pleural thickening is commonly associated with exposure to commercial forms of asbestos, it can also be caused by collagen vascular diseases , drugs (methysergide and chemotherapy), infection , and sarcoidosis and can also be idiopathic in nature.Pleural plaques are well-circumscribed lesions of the parietal pleura, composed of hyalinised, acellular collagen with a basket-weave pattern. Their presence is commonly associated with prior exposure to commercial forms of asbestos; however they can also be caused by local trauma and focal inflammation or even appear idiopathic in nature.In most cases of pleural thickening and pleural plaques, the clinician and pathologist will consider asbestos-related disease; however this paper highlights that in the right clinical context, yellow nail syndrome can also mimic these changes and must be ruled out.Mineral fibre analysis has an important role in determining an individual's inhaled, deposited and retained mineral content. Transmission electron microscopy and energy dispersive X-ray spectrometry was performed in this case and identified no asbestos fibres. A variety of nonfibrous minerals were identified including various metals and talc. The role of titanium in yellow nail syndrome has been suggested. To this end, it is of note that metal analysis detected low level titanium although the significance of this in isolation is uncertain. Talc was histologically observed and mineralogically identified in contralateral lung to the site of talc pleurodesis, supporting the notion that talc instillation may circulate remote from the pleural space. It is important for the clinicians to be aware that yellow nail syndrome may induce diffuse pleural fibrosis and pleural plaques without a history of asbestos exposure. It is also of equal importance that the pathologist performing the postmortem examination be aware of these new manifestations of yellow nail syndrome when investigating potential asbestos related deaths.


Growing evidence suggests that endolysosomal and autophagic defects are key pathogenic processes in various neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The causal relationship between these defects and neurodegeneration is supported by human genetic studies identifying disease mutations in genes controlling endolysosomal function and autophagy. The canonical view is that defects in these processes lead to impaired lysosomal clearance of proteins prone to form toxic oligomeric assemblies and/or aggregates, ultimately resulting in cellular pathologies that define these disorders. Because lysosomes mediate the clearance of a large number of lipids, lipid storage is frequently associated with compromised endolysosomal and autophagic function. However, an emerging notion, supported by our recent study on class III phosphatidylinositol 3-kinase (PI3K) Vps34, is that neuronal endolysosomal and autophagic dysfunction can manifest itself with the occurrence of physically damaged endomembranes and with the release of exosomes enriched for Amyloid Precursor Protein COOH-terminal fragments (APP-CTFs) as well as atypical phospholipid bis(monoacylglycero)phosphate (BMP). Here, we summarize our recent findings and their potential implications in the context of lysosomal biology, lipid signaling and neurodegenerative diseases. Accordingly, genetic disruption of the Vps34-encoding gene (Pik3c3) in mice leads to major neuronal loss, suggesting that ablation and pharmacological inhibition of Vps34 are appropriate models to study the causal relationship between endolysosomal/autophagic defects and neurodegeneration.A master regulator of both endolysosomal function and autophagy is Vps34 and its lipid product phosphatidylinositol-3-phosphate (PI3P) through which the lipid kinase exerts most of its biological actions in eukaryotic cells. A host of PI3P effectors have been characterized and shown to mediate processes as diverse as endosomal fusion, retrograde transport, sorting of cargoes into intraluminal vesicles (ILVs) of multivesicular endosomes as well as autophagosome biogenesis and maturation. Interfering with the signaling of PI3P and its metabolite, PIPPrompted by our findings showing that PI3P is deficient in the brain of AD patients and mouse models thereof, we investigated the consequences of Vps34 kinase inhibition or genetic ablation on autophagy, endolysosomal function and APP processing. We observed that reducing PI3P levels in primary cortical neurons or the neuronal cell line N2a blocks autophagy initiation, causes an increase in autophagy receptor/substrate p62 and polyubiquitinated protein levels and slows degradation of APP-CTFs, including APP-CTF. However, those puncta were also positive for cholesterol/sphingolipid-rich membrane marker flotillin, yet, negative for LAMP-1 , suggesting that they were organelles of endocytic origin lacking standard markers for those compartments. APP did not appear to concentrate on these organelles either. We speculated that their permeabilization causes loss of specific endolysosomal markers through excessive proteolysis. Importantly, accumulation of p62, ubiquitin and galectin-3 on those physically compromised organelles suggested that they are targeted to the autophagy pathway but not properly cleared by lysophagy , as Vps34 inhibition blocks autophagy initiation. What mechanisms account for endomembrane damage and what endolysosomal compartments are permeabilized in response to Vps34 inhibition? While we have not answered this question, we hypothesized that altered trafficking along the endolysosomal pathway causes secondary accumulation of sphingolipids and possibly sterols, which, in turn, leads to destabilization and permeabilization of endolysosomal membranes. Alternate explanations include increased oxidative stress or loss of glycocalyx coating of their luminal leaflets through missorting of glycoproteins. It will thus be fundamental to better characterize the identity of these damaged structures, perhaps via immunoisolation combined with proteomic and lipidomic analyses. Future work will address whether endolysosomal membrane permeabilization is a common pathogenic process in neurodegenerative disorders associated with LSDs and whether blocking it confers therapeutic benefits.Given the impact of Vps34 inhibition on endolysosomal function, we hypothesized it may also impair the metabolism of lipids and specifically, those degraded in lysosomes. Using lipidomics, we found that Vps34 inhibition increases levels of specific sphingolipids, namely ceramides and dihydrosphingolipids, somewhat reminiscent of some lysosome storage disorders (LSDs). While dysregulated ceramide metabolism can trigger cytotoxic signaling cascades, including apoptosis and necroptosis, missorting and accumulation of these sphingolipids in membrane subdomains may destabilize lipid bilayers and cause their permeabilization. Thus, we examined levels and subcellular localization of galectin-3, which, similar to other galectins, is a cytosolic lectin with high affinity for glycan . Exosomes are small vesicles secreted upon fusion of multivesicular endolysosomal compartments with the plasma membrane and correspond to the ILVs of those organelles. There is significant heterogeneity in the types of internal vesicles based on their differential protein and lipid composition, and logically, this heterogeneity is carried over in exosomes when internal vesicles are secreted. For instance, it has been reported that internal vesicles can be either PI3P- or BMP-positive, although no studies had reported the presence of either of those lipids in exosomes. Also, whether various subpopulations of exosomes derive from common or distinct endolysosomal compartments remains to be elucidated. We found that by reducing levels of PI3P with the Vps34 inhibitor, the co-secretion of BMP- and APP-CTF-positive exosomes is enhanced, although these molecules may not necessarily reside on the same exosomes . Pharmacological experiments have begun to delineate the mechanisms responsible for this atypical exosome release. First, it is also triggered by inhibition of the V-type ATPase with Bafilomycin A1 (BafA1), suggesting that elevation of endolysosomal pH may be the underlying mechanism, although neither chloroquine nor NH4Cl phenocopied these findings, arguing against a simple pH effect . This may rather reflect an important role of the V-type ATPase in exosome release, as suggested by recent studies. Second, inhibitors of serine palmitoyl transferase and neutral sphingomyelinase 2 both blocked exosomal release of APP-CTFs, suggesting that de novo sphingolipid synthesis and ceramide generation are critical. This is consistent with the fact that Vps34 inhibition causes a profound alteration of sphingolipid metabolism, including ceramides. We also note that previous studies from our group have found that the ESCRT pathway, which relies on PI3P primarily due to its ability to recruit ESCRT0/Hrs to endosomal membranes, mediates APP sorting into ILVs and exosome. However, Vps34 inhibition and the ensuing drop in PI3P levels shunt APP/APP-CTFs into an ESCRT-independent pathway, leading to robust exosomal secretion of APP-CTFs. This highlights the plasticity of ILV sorting and exosomal secretion routes for specific cargoes. It remains unclear if BMP is required for secretion of APP-CTF and other endolysosomal cargo in exosomes, a question that may be addressed by identification of the BMP-synthesizing enzymes and their manipulation.Another key finding reported in our study is that Vps34 inhibition causes robust secretion of exosomes enriched for APP-CTFs , and TDP-43, which accumulate primarily in the neurons of patients with AD, PD and ALS/FTD, respectively. In fact, if exosome release is robust enough to eliminate these toxic intracellular proteins, blocking exosomal release may cause their intracellular accumulation and increase their cellular toxicity, perhaps exacerbating intracellular pathologies.What could be the purpose of exosome release triggered by endolysosomal dysfunction? Both cell-autonomous and non-autonomous aspects must be considered. Growing evidence indicates that exosomes can serve as vectors for eliminating DNA, proteins or lipids. Our work, along with studies from others, suggests that exosome release is a Regarding potential cell non-autonomous functions, growing evidence suggests that exosomes participate in cell-cell communication. In contrast to diffusible, soluble molecules that ensure long-range signaling within a tissue, an organ or an organism, exosomes are more likely to play paracrine roles with limited spreading abilities, unless they reach blood circulation or the cerebrospinal fluid (CSF) or undergo long-range transcytosis along neuronal processes upon internalization. Because exosomes harbor intracellular molecules such as proteins and lipids, the very presence of such molecules on the outer leaflets of their lipid bilayers likely plays a signaling role, driving specificity in cell-cell communication. For instance, the molecules exposed to the surface of exosome could determine what cell types ultimately captures them and transmits their signals, through specific cell surface receptors. In the case of exosomes released upon inducing lysosomal dysfunction in brain tissue, they may carry "eat me signals" destined for immune cells of the brain, such as microglia, which could help eliminating the toxic waste neurons cannot handle. They could also carry danger-associated molecular patterns (DAMPs) stimulating innate immune receptors on astrocytes or microglia, favoring chemotaxis and deleterious gliosis. Future studies will address whether neuronal exosomes triggered by lysosomal dysfunction are endowed with signaling abilities towards glial cells and what the physiological and pathophysiological consequences of this communication are.In the realm of pathology, exosomes may also contribute to the spreading of neuronal aggregates within the central nervous system, including tau and  of patients and preclinical models for disorders associated with lysosomal dysfunction, including AD, PD, ALS, FTD and obviously LSDs, may be informative, and may also provide novel opportunities in clinical settings for identification of pharmacodynamic biomarkers in relation to treatments that improve or correct lysosomal dysfunction.Finally, our work has implications for biomarker discovery. Since lysosomal dysfunction causes robust secretion of APP-CTFs and BMP, measuring those molecules in bodily fluids (


So far, the large and expanding body of research on meditation has mostly focussed on the putative benefits of meditation on health and well-being. However, a growing number of reports indicate that psychologically unpleasant experiences can occur in the context of meditation practice. Very little is known about the prevalence and potential causes of these experiences. The aim of this study was to report the prevalence of particularly unpleasant meditation-related experiences in a large international sample of regular meditators, and to explore the association of these experiences with demographic characteristics, meditation practice, repetitive negative thinking, mindfulness, and self-compassion. Using a cross-sectional online survey, 1,232 regular meditators with at least two months of meditation experience  responded to one question about particularly unpleasant meditation-related experiences. A total of 315 participants  reported having had particularly unpleasant meditation-related experiences, which they thought may have been caused by their meditation practice. Logistic regression models indicated that unpleasant meditation-related experiences were less likely to occur in female participants and religious participants. Participants with higher levels of repetitive negative thinking, those who only engaged in deconstructive types of meditation , and those who had attended a meditation retreat at any point in their life were more likely to report unpleasant meditation-related experiences. The high prevalence of particularly unpleasant meditation-related experiences reported here points to the importance of expanding the scientific conception of meditation beyond that of a  health-promoting and self-regulating technique. We propose that understanding when these experiences are constitutive elements of meditative practice rather than merely negative effects could advance the field and, to that end, we conclude with an overview of methodological and conceptual considerations that could be used to inform future research. In fact, a careful study of the literature reveals that most studies on meditation have not explored experiences that meditators would describe as particularly unpleasant or difficult  practice of meditation”  developeRelated research on mindfulness-based interventions has generally not included measurement or evaluation of potential meditation-related difficulties. A recent systematic review of randomised controlled trials of mindfulness-based stress reduction and mindfulness-based cognitive therapy found that only 1% of the 4031 participants included in 36 randomised controlled trials reported “adverse events” . ImportaEmpirical evidence regarding the relationship between particularly unpleasant meditation-related experiences and meditation types is sparse. A recently proposed taxonomy grouped meditation practices into attentional, constructive, and deconstructive practices based on their underlying cognitive mechanisms . BrieflyIn brief, research on meditation and mindfulness-based interventions seldom include measures of particularly unpleasant meditation-related experiences, and standardised assessment methods for such experiences have not yet been developed and implemented. Subsequently, prevalence estimates of particularly unpleasant meditation-related experiences across secular and contemplative contexts remain inconclusive, while preliminary findings suggest that their occurrence is highly dependent on a complex interaction of contextual factors. In the context of 18 million meditators in the United States alone , 39, 40,Individuals were informed that the study aimed to advance the scientific understanding of meditation practices and their relationship to cognitive and emotional processes, and that by submitting their completed questionnaire they consented to participate. This study and consent procedure was approved by the University College London ethics committee (ref no: 10043/001) and conducted in line with the Declaration of Helsinki. This study used a cross-sectional design.An anonymous online survey that took approximately 20 minutes to complete was used between April and August 2017 to recruit regular meditators (practicing at least once a week) with a minimum age of 18 years and a good understanding of the English language. The survey link was shared on social media  and sent to Buddhist communities, meditation centres, and mindfulness associations. A total of 2,599 individuals started the survey, and 1,706 participants completed it. We excluded a total of 474 participants: 383 indicated only a yoga practice; 42 did not indicate a meditation practice; 34 had less than two months of meditation experience; another 15 did not respond to the question about particularly unpleasant meditation-related experiences and were also excluded. Therefore, 1,232 participants were included in the subsequent analyses. Individuals did not receive a financial reward for participation.Participants were asked to report demographic information and to answer the following question regarding particularly unpleasant meditation-related experiences: “Have you ever had any particularly unpleasant experiences , which you think may have been caused by your meditation practice?”. The term “particularly unpleasant” was chosen over other potential labels , which we evaluated as more likely to predetermine the value and significance (or lack thereof) of the experience and nudge participants towards theorising about their experiences. Meditators also reported their total years of regular practice (lifetime meditation experience), weekly number of meditation sessions (frequency), average length of their meditation sessions (session length), whether they had ever attended a meditation retreat (retreat experience), and the types of meditation they practiced. Meditation types were based on a taxonomy categorising meditation practices into attentional, constructive, and deconstructive types . The folRepetitive negative thinking was measured using the Perseverative Thinking Questionnaire (PTQ) . The PTQtypically think about negative experiences or problems. Total PTQ scores are computed by summing all item scores (possible range: 0 to 60), with higher scores indicating higher levels of repetitive negative thinking. PTQ scores are associated with symptoms of anxiety and depression, and the PTQ has displayed excellent psychometric properties across samples: Cronbach’s alpha ranged from 0.94 to 0.95 , 25underestimation of the actual rate of particularly unpleasant meditation-related experiences, coupled with an exaggeration of their intensity, given that only extreme cases would have come to the attention of researchers and clinicians. In other words, passive monitoring in meditation research could have led to an underreporting of particularly unpleasant meditation-related experiences. This proposal is reflected in the published literature containing a host of case reports of severe meditation-related experiences, yet not including any long-term randomised controlled trials of regular meditation that have quantitatively or qualitatively captured particularly unpleasant meditation-related experiences. Fourth, we did not assess possible pre-existing mental health problems, which could have confounded our prevalence estimate of particularly unpleasant meditation-related experiences. The reported prevalence estimate could therefore be more a reflection of the lifetime or 12-month prevalence of mental health problems in the general population ),Until recently, only a few overview and opinion papers –76 on thFirstly, it is unclear how to properly evaluate particularly unpleasant meditation-related experiences. One important issue demanding special attention in meditation research relates to the lack of semantic and conceptual clarity pertaining to these experiences. Characterisations that have been applied to describe these experiences have ranged widely (even within a single article) and have included the following labels: aberrant, adverse, challenging, difficult, distressing, extreme, harmful, impairing, negative, pathological, terrifying, unexpected, and unwanted. This is not a trivial point: operational definitions and conceptualisations are not simply a passive reflection of the researcher’s conceptual framework(s) and epistemological stance ; rather, they are also likely to influence meditators, other researchers, study methodology, study outcomes, and their interpretation. This, in turn, will influence the trajectory of this nascent research field along with the perception of funders, policy makers, and the public. To illustrate this point further, research conducted within, for instance, a biomedically informed framework has previously operationalised these experiences as adverse events, side effects, or contraindications (see ). While Secondly, current discussions on unpleasant meditation-related experiences have not elaborated sufficiently enough on how the empirical research on psychotherapy’s side effects—including the methodological and theoretical challenges encountered in defining, finding, classifying, and assessing side effects—can inform meditation research. Strikingly paralleling meditation research, there is extensive evidence for the positive effects of psychotherapy but a surprising paucity of research on side effects . AnotherEudaimonia, or existential flourishing). Davidson and Dahl (2018) [a priori.Thirdly, previous research on unpleasant meditation-related experiences has not clearly distinguished meditation as a means of treating various pathologies from meditation as a means of cultivating well-being   point oul (2018) ). We sugl (2018) , 83 or aIn conclusion, we believe that in order to be able to successfully address the issues briefly outlined here, an open-ended exploration is required, one that would enable us to explicate, broaden, and—if need be—modify the epistemic and methodological frameworks  into which both researchers and meditators are embedded, without giving way to an epistemic asymmetry in which one framework  explains away another . We propose that a constructive interdisciplinary discussion of these topics would advance the field and help construct a unified theoretical framework that would do justice to the plurality of traditional and scientific approaches.S1 Appendix(PDF)Click here for additional data file.


Cancer is a complex disease in which cells progressively accumulate mutations disrupting their cellular processes. A fraction of these mutations drive tumourigenesis by affecting oncogenes or tumour suppressor genes, but many mutations are passengers with no clear contribution to tumour development. The advancement of DNA and RNA sequencing technologies has enabled in-depth analysis of thousands of human tumours from various tissues to perform systematic characterization of their (epi)genomes and transcriptomes in order to identify (epi)genetic changes associated with cancer. Combined with considerable progress in algorithmic development, this expansion in scale has resulted in the identification of many cancer-associated mutations, genes and pathways that are considered to be potential drivers of tumour development. However, it remains challenging to systematically identify drivers affected by complex genomic rearrangements and drivers residing in non-coding regions of the genome or in complex amplicons or deletions of copy-number driven tumours. Furthermore, functional characterization is challenging in the human context due to the lack of genetically tractable experimental model systems in which the effects of mutations can be studied in the context of their tumour microenvironment. In this respect, mouse models of human cancer provide unique opportunities for pinpointing novel driver genes and their detailed characterization. In this review, we provide an overview of approaches for complementing human studies with data from mouse models. We also discuss state-of-the-art technological developments for cancer gene discovery and validation in mice. This disruption is generally attributed to mutations in so-called driver genes, which provide cells with a selective growth advantage and drive their malignant transformation. Broadly speaking, driver genes can be divided into two classes of genes: oncogenes and tumour suppressor genes (TSGs) . OncogenTumours are however not the product of single mutations, but develop progressively over time through the accumulation of multiple mutations. Depending on the affected genes, these mutations can increase the fitness or tumourigenic potential of cells , or have no clear contribution towards tumourigenesis (passenger mutations). Over time, this accumulation gives rise to subpopulations of cells (subclones) harbouring distinct sets of mutations, which are subject to Darwinian competition  within the tumour lesion  figure . This coEffective treatment of cancer patients is increasingly based on precision medicine, i.e. tailored treatments designed to target specific mutation-associated dependencies and/or vulnerabilities of a patient's tumour. It is therefore crucial to identify exactly which mutations contribute to tumourigenesis and how they do so. Although human sequencing studies have identified many genes contributing to cancer development, they do not provide evidence for causality or detailed insight into the biological mechanisms by which these genes drive tumour development. These studies also do not reveal whether drivers are essential for tumour maintenance and may therefore be of limited use for designing effective therapeutic strategies. In contrast, preclinical model systems such as genetically engineered mouse models (GEMMs) provide an experimentally tractable approach in which the biological effects of specific mutations can be studied in detail in a controlled genetic background. In this review, we describe several aspects of mouse models and how these can ultimately be applied to improve treatment of cancer patients. To this end, we first highlight several challenges in translating findings from human sequencing studies to the clinical setting, before explaining how some of these challenges can be addressed using complementary approaches in mouse model systems.2.Several major human sequencing studies have been undertaken over the past years, aiming to identify and catalogue potential driver mutations across many different cancer types –8. One oAlthough these approaches have proven successful in identifying many driver genes affected by hotspot mutations, other types of mutations have proven more challenging. This is especially the case for DNA copy-number driven diseases such as breast cancer, in which approaches aimed at identifying recurrent copy number alterations typically identify regions harbouring many genes ,17. Simiin vivo validation of candidate genes . O. OBcr-AbVhl) (c) , in whicVhl) (c) ,56.d). Using these approaches, oncogenes can be introduced by injecting  vectors expressing these gene(s) into the tissue of interest [A limitation of conditional GEMMs is that generating new models is still time-consuming and expensive. Recent developments in somatic gene-editing techniques provide incredible potential to speed-up this process by allowing mutations to be introduced somatically into existing mouse models d. Using interest . Similarinterest . Furtherinterest  and CRISinterest ,61 promiAnother approach for upscaling experiments is to orthotopically transplant mouse tumour fragments into syngeneic mice, allowing further stages of tumour development to be studied in parallel . A parti4.4.1.a). Following this approach, driver mutations can be detected using various computational approaches, in the same fashion as previously described for human tumours. This can be particularly powerful when combined with mouse models of spontaneous tumour development, such as radiation- or carcinogen-induced [Kras-mutant mouse skin squamous cell carcinoma [Egfr-, Myc- and Kras-driven lung cancers [Brca1- and Brca2-deficient breast cancer [Besides candidate cancer driver analysis, mouse models can also be used to identify additional driver mutations by sequencing mouse tumours and identifying additional genes that are frequently mutated across tumours a. Follow-induced –67 tumou-induced –70, whicarcinoma  and Egfr cancers ,73. Simit cancer ,75.FiguBrca1/Brca2-deficient breast cancer models [In general, mouse tumours tend to have lower mutational loads than their human equivalents. This reduced complexity offers easier interpretability, allowing for the extrapolation of critical genetic driver processes that may not be apparent in genetically complex human cancers , but mayr models ,75.4.2.ad hoc setting, by identifying drivers of mouse tumours and comparing these with known mutations in human datasets, or as a deliberate strategy using comparative oncogenomics. In the latter approach, sequencing data from mouse and human tumours are typically first analysed to identify candidate driver genes for both species individually. These species-specific candidates are then integrated by only selecting genes and/or networks that are aberrated in both species (b). Remaining candidates can optionally be filtered using additional criteria, such as correlation with gene expression or prior knowledge from literature.A more powerful approach for identifying driver genes using sequencing approaches is to combine insights from mouse and human datasets and prioritize genes that are mutated in both species, as these are most likely to represent true driver genes. This can be done in an  species b. Remainet al. [cIAP1 and Yap, which were shown to act synergistically in tumourigenesis. Similarly, copy-number sequencing of metastases from an inducible Hras model of a traditionally non-metastatic melanoma identified a focal amplification on mouse chromosome 16, which contained only eight candidate driver genes [RAS- and MET-driven melanomas identified a single gene, NEDD9, as the driver of these metastases. A recent study from Liu et al. [This comparative strategy has proven particularly effective for distinguishing driver genes from passengers in chromosomally unstable tumours. For example, in a mouse model of hepatocellular carcinoma (HCC), Zender et al.  identifier genes . Furtheru et al.  used who5.Although additional driver genes can be identified by mouse tumour sequencing, this approach is not always optimal as mouse models can have a long tumour latency and may be more prone to acquire other types of mutations than those of interest (i.e. copy-number aberrations rather than point mutations or vice versa). Forward genetic screening approaches can address these issues by using various mutagenesis strategies to induce additional mutations and accelerate tumour formation, after which any tumours that developed can be studied to identify new drivers. The type of mutations that occur depends strongly on the type of mutagenesis that is being employed, meaning that different mutagenesis strategies can be used to specifically induce different kinds of mutations.5.1.in vivo chemical mutagenesis screens, animals  are treated with a controlled dose of a chemical mutagen and subsequently monitored for tumour formation (a). Developed tumours can be sequenced using whole-genome or targeted sequencing strategies to identify mutations that may be driving tumourigenesis or metastasis [Chemical-based mutagenesis is one of the oldest mutagenesis strategies, in which cells or animals are treated with a chemical substance that damages the DNA and thereby induces mutations. The induced mutations are typically single nucleotide changes; however, the spectrum of these mutations depends on the used substance and can vary greatly between different chemicals. In ormation a. Develotastasis ,80.FiguKras-driven lung cancers [An advantage of chemical mutagenesis compared to other mutagenesis approaches is that its bias towards single nucleotide changes makes it suitable for modelling the effects of human variants, which are often single point mutations that result in changes in the levels of expression or activity of a gene product. For this reason, chemical mutagenesis approaches have been used to mimic human mutational processes and to characterize the genomic landscapes of the mutational landscapes of mouse skin squamous cell carcinoma  and Kras cancers . However cancers .5.2.b). In transposon-based insertional mutagenesis (TIM) strategies, this process is mediated by a transposase enzyme, which excises transposons from a concatemer introduced in the genome of the mice and reintegrates them stochastically elsewhere. By placing the expression of this transposase under a tissue-specific promotor, mutagenesis can be restricted to specific tissues in the mouse.The bias towards point mutations also limits the utility of chemical mutagenesis strategies for modelling other types of mutations, such as increased gene expression resulting from gene amplifications. Insertional mutagenesis (IM) strategies provide an alternative approach, in which viral  or transfigure 4T2Onc/2 transposon, which is frequently used in Sleeping Beauty IM screens [T2Onc/2 transposon also contains two splice acceptor sites (SA/En2SA) combined with a bi-directional polyA sequence, which allow the transposon to truncate transcripts if integrated within a gene. Depending on the gene and the relative location of the insertion, these truncations can inactivate genes by resulting in an unstable transcript or inactive protein, or activate genes by removing inhibitory protein domains [The effects of insertions depend on the used transposon, but typically involve the activation of oncogenes using promotor sequences and/or inactivation of tumour suppressors by truncating genes. For example, the  screens ,84, cont domains ,88.A considerable advantage of IM strategies is that insertions can be specifically captured via PCR amplification before sequencing, enabling cheap and efficient retrieval of the insertion sites compared to genome-wide sequencing. A drawback of transposon-based systems is that they generally show some bias in terms of their insertion patterns, due to sequence integration biases or biases towards specific gene features (e.g. gene bodies or promotors) . For thiDespite these drawbacks, TIM and other mutagenesis systems have been valuable for identifying cancer-associated driver genes in mouse models of a large variety of cancer types, including breast cancer –93, mela5.3.in vivo RNAi screen, (tumour) cells transduced with a lentiviral shRNA library can be injected orthotopically in animals [c). Once developed, tumours are harvested and sequenced to quantify the frequency of each shRNA in the tumour cell population. By comparing these frequencies to those of the initial starting population, this approach can identify which shRNAs are enriched in the tumour and are therefore likely targeting TSGs whose loss is beneficial for tumourigenesis. Conversely, depleted shRNAs may identify potential oncogenes and/or genes that are crucial for tumour maintenance.In contrast to the previously described non-targeted genome-wide screening approaches, library-based screening approaches, such as loss-of-function screens based on RNA interference (RNAi) technology, can be used to target specific sets of genes. In pooled RNAi screening approaches, cells are transduced with lentiviruses encoding short hairpin RNAs (shRNAs) targeting specific genes that, when integrated into the genome, result in stable and heritable suppression of the corresponding gene . To perf animals , which ain vitro approaches, in vivo shRNA screens provide the opportunity to expose the vulnerabilities of tumour cells in the context of their microenvironment and can be used to study drivers of metastasis and therapy resistance [in vivo shRNA screens have been reported for a variety of cancer types, including xenograft models of hepatocellular carcinoma [The scope of shRNA screens depends entirely on the used library, meaning that screens can be designed to target all genes in a genome-wide fashion or to test a small number of pre-selected candidate genes. As such, shRNA screens can not only be used to identify novel driver genes, but can also be used for narrowing down lists of potential drivers or to validate putative driver genes. Compared to sistance –106. Drasistance  and off-sistance . Successarcinoma , lymphomarcinoma ,111, leuarcinoma ,113 and arcinoma ,115.5.4.-mediated genome editing. CRISPR/Cas9-based loss-of-function screens are generally performed in the same fashion as shRNA screens by transducing cells with pools of single guide RNAs (sgRNAs) targeting different genes, injecting the transduced cells in vivo and contrasting the abundance of sgRNAs in tumours with their abundance in the starting population [With the development of CRISPR-based technologies, it has also become possible to perform pooled loss-of-function screens by inactivating genes using CRISPR/Cas9pulation –118. Howin vivo validation of candidates from a Sleeping Beauty IM screen [Compared to shRNA screens, CRISPR-based screens have been reported to be remarkably efficient and suffer less from off-target effects than shRNA screens ,120. CRIM screen . With thM screen , gene inM screen  and the M screen ,61.6.in vitro models suffer from several drawbacks, including the presence of additional mutations and the lack of a tumour microenvironment. An alternative approach is to introduce candidate driver mutations into an established GEMM of the cancer type in which the mutations were identified, in order to study their effect on tumourigenesis (a). These additional mutations can be introduced using either germline approaches  or somaOnce established, the resulting mouse models can be studied to determine how the additional mutations affect tumour incidence, latency and/or metastasis compared to the baseline mouse model. The new models can also be used to study the effects of the extra mutations on therapy response. Any developed tumours can be studied in detail to determine how the additional mutations affect tumour morphology and the tumour (immune) microenvironment. Additionally, by using sequencing strategies or screening approaches to identify additional driver genes in these more complex GEMMs, mutational landscapes can be compared between models with different drivers to determine how additional drivers affect the evolution of tumours initiated by the engineered mutations and if this provides clues to any driver-specific vulnerabilities.in vivo testing of (combinations of) candidate cancer genes and have been used to validate driver genes for a wide range of cancer types, including breast cancer [In particular, CRISPR/CAS9-based somatic cancer modelling approaches enable rapid t cancer , colorect cancer , pancreat cancer  and livet cancer . Additiot cancer ,127. Fint cancer .7.BRAF-mutant melanoma, which is targeted by inhibiting the mutant BRAF kinase and/or MEK, a protein downstream of BRAF in the RAS/MAPK signalling pathway. Alternatively, tumours can be targeted therapeutically by exploiting a synthetic lethality resulting from the driver mutation(s). A classic example of synthetic lethality is poly(ADP-ribose) polymerase (PARP) inhibition in BRCA-deficient tumours, which specifically targets cells with defects in their homologous recombination (HR) pathway due to loss of HR factors such as BRCA1 and BRCA2.Ultimately, knowledge of driver genes and their effects is used to develop novel therapeutic strategies that target specific vulnerabilities of the tumour, enabling effective treatments with minimal side effects. Following this premise, personalized therapies are generally designed by either targeting identified driver genes directly (if possible) or by targeting other genes in the same signalling pathway. A well-known example is in vitro models  or in vivo models . To identify which treatments are most effective in different cancer types or tumours with different genetic backgrounds, several efforts have been made to set up large biobanks of PDX models for high-throughput drug screening purposes [et al. [Before moving into the clinic, drugs are generally first tested for anti-cancer efficacy in a preclinical setting, either using purposes ,129,130. [et al. , who idein vivo resistance mechanisms by grafting cell lines, organoids or tumour fragments into multiple recipient mice, which are then subjected to different treatments (b). Upon relapse, resistant tumours can be sequenced and compared with tumours from vehicle-treated mice to identify possible resistance mechanisms. Using this approach, our laboratory identified several resistance mechanisms to PARP inhibitor treatment in mouse models of BRCA1- and BRCA2-deficient breast cancer [Besides intrinsic therapy resistance, many targeted therapies fail in the clinic due to the emergence of drug resistance which is acquired during treatment. As such, a key challenge for improving the efficacy of these therapies is to identify  (epi)genetic changes that underlie this acquired treatment resistance. Both GEMMs and PDXs can be used to identify potential eatments b. Upon rt cancer –133. Thit cancer ,99,134.Besides identifying potential resistance mechanisms, an important challenge is to determine how resistance actually arises and to design therapeutic strategies accordingly. For example, in cases where tumours acquire additional (epi)genetic changes during treatment (as described above), therapies should ideally be designed to pre-empt and prevent the most likely paths of resistance. On the other hand, cases where resistance is driven by pre-existing sub-populations of intrinsically resistant cells  will req8.in vitro and in vivo model systems provide crucial platforms for assessing sensitivity to different therapies across multiple cell lines or tumours, enabling the construction of correlative models that predict the efficacy of these treatments for new tumours. More detailed genetic modelling in (mouse) model systems can further refine these correlative models by providing causative evidence that combinations of mutations drive cancer development and/or affect therapy response. In addition, mouse modelling enables detailed characterization of the effects of drivers on other tumour phenotypes, such as tumour latency, morphology, mutational landscape and interactions with the (immune) microenvironment. Genetically engineered mouse models also provide powerful platforms to critically evaluate new candidate drug targets [The success of personalized anti-cancer therapies hinges on how accurately we can predict whether a given patient tumour will respond to a given treatment, allowing clinicians to select the most effective therapeutic strategy for treating a patient. Ideally such an approach would be implemented by feeding omics data and other data types  from patient tumours into  models that predict which therapies are most likely to be effective based on specific tumour biomarkers . Creatin targets  and ther targets .Figure Modelling of human cancer using genetically engineered mice is complicated by the observation that tumours generally contain multiple driver lesions, which can strongly influence their sensitivity to treatments targeting specific drivers. As a result, accurately assessing therapy response may require the generation of complex mouse models containing multiple driver genes that are frequently encountered together in a given type of cancer. Using germline engineering approaches, generating models with multiple driver lesions has been challenging due to the extensive breeding and animal husbandry involved. Somatic approaches using CRISPR/CAS9-based gene editing and over-expression vectors alleviate this bottleneck, by providing the technology to quickly create new mouse models by introducing different combinations of mutations into a pre-existing baseline mouse model. The rapidly ongoing refinement of these tools will further expand the repertoire of mutations that can be modelled in this manner, enabling the rapid creation of new models reflecting the mutations observed in patient tumours, which can be used to test the effects of novel therapeutic strategies targeting these mutations.in vitro models or xenograft models lacking an active immune system. As such, immune-competent mouse models such as GEMMs are better suited for studying interactions between tumours and the immune system, although care should be taken to ensure that used models reflect the variety of genetic and other factors that may influence an immune response. Additionally, differences in the mouse and human immune systems may limit the relevance of GEMMs, requiring the use of humanized mouse models to accurately model immune responses in human patients.Mouse models are of particular interest for studying immune-based therapies, which have recently garnered much interest due to their high efficacy in treating advanced stages of several cancer types –147. Howin vivo setting. Similarly, mouse models may also be used to model the effects of specific lifestyles  on cancer risk and development [Besides designing novel therapies, other important clinical challenges include identifying which patients are most at risk of developing cancer and should be screened for preventative treatments. Although factors such as genetic background and lifestyle have been shown to have a profound influence on cancer risk and survival, our insights into how these factors influence cancer development is still limited. Due to their tightly controlled genetics, mouse models are uniquely suited for examining the effects of genetic backgrounds and how these interact with specific driver genes in an elopment . CombineImproved screening imposes its own challenges, as population screening programmes identify many early lesions that will not necessarily progress to cancer and therefore do not actually require treatment. Unfortunately, in many cases it is currently not possible to distinguish which lesions are indolent and which will progress to invasive cancer, leading to overdiagnosis and overtreatment . IdentifTumour progression, metastasis and escape from therapy are phenomena which are driven by intra-tumour heterogeneity . Single-It is important to keep in mind that tumours arising in GEMMs of human cancer may not necessarily reflect all characteristics of human tumours. For example, GEMM tumours may contain lower numbers of somatic mutations compared to the cognate human tumours  and feweFinally, efforts to collect and catalogue mouse sequencing data have been relatively limited compared to efforts involving human sequencing studies. To fully exploit the large compendium of mouse sequencing and screening data, it will be important to collect these data in centralized repositories and create portals to query these data, allowing researchers to quickly explore existing datasets and compare tumour characteristics across different mouse models. Fortunately, several efforts are already underway to collect data in application-specific databases , to crea


Arsenic exposure is a public health concern of global proportions with a high degree of interindividual variability in pathologic outcomes. Arsenic metabolism is a key factor underlying toxicity, and the primary purpose of this review is to summarize recent discoveries concerning the influence of the human gut microbiome on the metabolism, bioavailability, and toxicity of ingested arsenic. We review and discuss the current state of knowledge along with relevant methodologies for studying these phenomena.Bacteria in the human gut can biochemically transform arsenic-containing compounds . Recent publications utilizing culture-based approaches combined with analytical biochemistry and molecular genetics have helped identify several arsenical transformations by bacteria that are at least possible in the human gut and are likely to mediate arsenic toxicity to the host. Other studies that directly incubate stool samples in vitro also demonstrate the gut microbiome’s potential to alter arsenic speciation and bioavailability. In vivo disruption or elimination of the microbiome has been shown to influence toxicity and body burden of arsenic through altered excretion and biotransformation of arsenicals. Currently, few clinical or epidemiological studies have investigated relationships between the gut microbiome and arsenic-related health outcomes in humans, although current evidence provides strong rationale for this research in the future.The human gut microbiome can metabolize arsenic and influence arsenical oxidation state, methylation status, thiolation status, bioavailability, and excretion. We discuss the strength of current evidence and propose that the microbiome be considered in future epidemiologic and toxicologic studies of human arsenic exposure. Arsenic is among the most widespread and dangerous environmental toxicants in the world. It is ubiquitous in the environment and primarily originates from the natural weathering of the earth’s crust. As such, it inevitably becomes associated with human food and water as well as surface soils and particulates in the air . AnthropA microbiome can be defined as the community of microorganisms occupying a defined ecosystem and the sum total of their physical, biochemical, and ecological activities . The humIn this review, we overview human exposures to arsenicals, relevant pathways of human arsenical metabolism and excretion, and the influence of arsenic-microbiome interactions on host physiology, arsenical metabolism, bioavailability, and toxicity. We also discuss evidence of arsenic-induced compositional and functional changes in the microbiome and potential contributions to host health associated with those changes.Inorganic arsenic (iAs) leeches from mineral deposits in the earth’s crust and into underground aquifers, leading to human exposure when wells are drilled or dug to meet water needs. In fact, the most common route of arsenic exposure in humans is contaminated drinking water . ArsenicV) exists as an oxy-anion , while reduced trivalent arsenite (iAsIII) is typically uncharged (H3AsO30) and more mobile in the subsurface [V is most stable, but a shift to even mildly reducing conditions can favor the formation of the more mobile iAsIII. iAsIII is also considered more toxic and bioavailable to human cells compared with iAsV. Thus, by altering the redox conditions and pH as part of normal metabolism, microorganisms both directly and indirectly influence iAs cycling in soil, subsurface, and aquatic ecosystems [The rate of iAs leeching into the water table depends on arsenical redox state and geological factors, such as soil type. Unlike other toxic metal(oid)s in the environment, which may form inorganic cations in solution, dissolved iAs is found as oxy-acid compounds . Under nbsurface . In oxygosystems .Collectiosystems , 15.5OH(AsO4)3) became the most prominent agricultural pesticide of the time and was not officially prohibited in the USA until 1988 [Contaminated soils and dust have also been considered as potential sources of arsenic exposure, especially among children because of common hand-to-mouth behaviors , 17. Alttil 1988 . This witil 1988 , and eletil 1988 .Despite the phasing out of most inorganic arsenicals from use, organic arsenical compounds (oAs) are still widely used in agriculture and landscape management. Monosodium monomethyl arsenate (MSMA) is a commercially available arsenical herbicide used for residential lawn care and commercial weed control in golf courses, sod farms, and highway rights-of-way . SynthetV; monomethylarsonic acid (MMAV); and dimethylarsinic acid (DMAV)), compared to other grains and vegetables [Detectible arsenic in foods raises concerns about the potential contribution of dietary exposures. Arsenic can accumulate in cereal grains, vegetables, and fruit crops from contaminated irrigation water or surface soil environments . Rice stgetables . These pgetables . Arsenicgetables –34. As wgetables . More re10) can be inhaled, while only particles smaller than 2.5 μm (PM2.5) penetrate into the lungs where they can be deposited onto the pulmonary epithelium [10 and PM2.5 [A number of different processes contribute to atmospheric arsenic levels, including volcanic activity, mining and industrial processes, combustion of fossil fuels, use of agricultural pesticides, and volatilization of arsine compounds. Compared with drinking water exposure, inhalation is considered a minor source of arsenic exposure for the general population ; howeverithelium . Arsenicnd PM2.5 . Atmosphnd PM2.5 .Tobacco smoke is another significant source of inhaled arsenic, as arsenic-based pesticides in the soil can be taken up by tobacco plants. The risk of disease from arsenic contaminated tobacco is difficult to determine because smoking behavior carries an independent carcinogenic risk and has a synergistic effect with arsenic in food and water . ArsenicIn contrast with the health problems associated with chronic incidental exposures, arsenic compounds have also been used therapeutically throughout history. In the pre-antibiotic era, drugs like arsanilic acid (atoxyl) and arsphenamine  were commonly used to treat syphilis and trypanosomiasis, among other common ailments . Before Currently, arsenic trioxide (ATO) is still used as an anticancer treatment for patients with acute promylocytic leukemia (APL).The exact molecular mechanisms of its anti-cancer activity are still being studied, but it has been suggested that arsenic biotransformations in the body are important for ATO’s clinical efficacy . There iV), monomethylarsonous acid (MMAIII), dimethylarsinic acid (DMAV), and dimethylarsinous acid (DMAIII). Methylation is enzymatically catalyzed by arsenic (3+) methyltransferase (AS3MT), utilizing S-adenosylmethionine (SAM) as a methyl group donor in concert with endogenous reducing agents such as thioredoxin (Trx) and glutathione (GSH) [In humans and many of other animals, iAs entering the body in food and water is methylated as the primary means of systemic detoxification. Methylated arsenicals are more readily excreted, leading to enhanced body clearance , and effne (GSH) , 55. Recne (GSH) .3) and monomethyl arsenite di-glutathione (MMA(GS)2) have been detected in urine and bile of mammals [V) and dimethyl monothioarsinic acid (DMMTAV), have cytotoxicity more similar to trivalent arsenicals compared with their non-thiolated counterparts  [2S) is an important signaling molecule throughout the body and source of sulfide ions. The liver is perhaps the most well-known source of biogenic H2S, suggesting that hepatocytes play a significant role in thiolation. This hypothesis, however, has yet to be experimentally addressed.While GSH is an important reductant in the methylation pathway, arsenic-GSH-conjugates are also actively transported and effluxed from cells, and arsenic tri-glutathione (As(GS) mammals . These r mammals . For exa mammals , 60. Hum mammals , but theenicals) . A numbeenicals) . In geneenicals) . HydrogeV) or arsenite (iAsIII), even though most humans are naturally and chronically exposed to much lower levels. However, the intent of most acute studies is to test whether a certain factor  influences the onset and/or progression of an observable outcome. As such, acute studies in mice often utilize doses between 1 and 50 ppm  and somewhat lower than the immediate lethal dose (LD50). Unfortunately, there is considerable variability in LOAEL and LD50 estimates for humans, but according to the ATSDR and EPA’s toxicological profile on arsenic[−1 body weight day−1. Assuming  a 55-kgThird, and arguably the most important consideration, is allometric conversion. Such conversions take into account important physiological differences between humans and animals, such as body surface area. In other words, if mice have less surface area but the same uptake rate, then a higher dose is needed to reach the same exposure. For example, the FDA suggests  that a hKm = 37) . Using tKm = 37) . The midars operons, were first characterized in plasmids isolated from Escherichia coli and Staphylococcus aureus [ars genes have since been identified and characterized in a variety of clinically important pathogens, including Listeria monocytogenes, Campylobacter jejuni, and Yersinia strains [ars operon has also been found in human gut symbionts like Bacillus subtilis [Bacteroides vulgatus [Bacterial arsenic metabolism has largely been identified and studied by examining microbial “resistance” to arsenic-induced effects . Although metabolism and resistance often detoxify arsenicals, not all resistance pathways involve the biochemical transformation of arsenic (see below). Resistance gene clusters, dubbed s aureus , 84. Bac strains . The arssubtilis  and the vulgatus . The molvulgatus , 88–90. ars operon is conferred by the arsBC genes, encoding for an iAsIII-specific efflux pump (arsB) and a cytosolic iAsV oxidoreductase (arsC) [III can be as simple as a one-step process consisting of ArsB-mediated efflux, while resistance to iAsV involves at least a two-step process via ArsC-mediated reduction to iAsIII followed by ArsB-mediated efflux. Most ars operons also contain a regulatory gene, arsR, that encodes a trans-acting, iAsIII-responsive transcriptional repressor [III efflux with H+ ion exchange, and both can function as subunits in a heterodimeric, ATP-driven iAsIII pump in the presence of the catalytic subunit, ArsA [arsB and acr3 genes are found in bacterial ars operons in roughly equal frequency, suggesting that iAsIII resistance was very important in the evolution of arsenic resistance [The ‘core’ function of the e (arsC) , 92. Resepressor , 94. Effepressor , 96. Desit, ArsA . arsB ansistance .arsC genes are structurally and functionally similar to low molecular weight tyrosine phosphatases [V reduction is coupled with cellular thiol-disulfide exchange systems, which vary depending on the lineage of ArsC expressed [E. coli and other proteobacteria, iAsV reduction is commonly coupled with GSH and glutaredoxin, while thioredoxin-dependent ArsC enzymes are common in Firmicutes and Bacteroidetes phyla [Corynebacterium, utilizes the cellular mycothiol/mycoredoxin system [Oxidoreductase enzymes encoded by phatases . ArsC-mexpressed . In E. ces phyla . A thirdn system . This liars operons also carry extended ars genes that support this core functionality. Two of the most common are arsA and arsD. As mentioned above, arsA encodes for a catalytic ATPase that forms a heterodimeric complex with ArsB/Acr3 transporters capable of ATP-driven primary efflux of iAsIII [III to the ArsAB complex for efflux [arsA and arsD are not essential for arsenic resistance, their presence greatly improves the efflux efficiency and transcriptional regulation of this arsenate reduction/arsenite efflux resistance pathway.Many f iAsIII . ArsD isr efflux . While aV reduction first because they take place in the periplasm and second because they harness energy from the redox conversion between iAsIII and iAsV in the form of electrochemical gradients and electron transport. To date, there is little evidence of respiratory arsenic metabolism in the human gut environment. This may be due to the abundance of more favorable electron donor/acceptor couples in the intestine, although it is possible that they have simply not been identified yet. Several expert reviews are available that detail the structures, mechanisms, and distribution of these energy-harnessing arsenic systems [In addition to cytosolic arsenate reduction, many prokaryotes are capable of utilizing iAs for respiration. These systems differ from ArsC-mediated iAs systems , 104.III utilizing SAM as a methyl donor in a similar fashion to that of human AS3MT. Arsenic-methylating bacteria can generate significant amounts of mono-, di-, and trimethylated arsenicals from iAsIII [V and DMAV under oxidizing conditions, bacterial methylation has been shown to yield more toxic trivalent forms of mono and dimethyl arsenic in a simulated gut environment [III into their surroundings, thus inhibiting the growth of competitors. Interestingly, greater amounts of MMAIII are formed when arsenic-methylating bacteria are grown in co-culture, whereas the less toxic MMAV is the favored product of the same organisms grown in monoculture [III and other toxic trivalent organoarsenicals, including efflux mediated by ArsP [III to oAsV) by ArsH [III but can also confer resistance to other trivalent organoarsenicals. ArsK is more promiscuous, conferring resistance to a variety of organic and inorganic trivalent arsenicals. ArsI is a carbon-As bond lyase capable of demethylating MMAIII to the less toxic iAsIII [Human AS3MT, mentioned above, is a homolog of prokaryotic ArsM. The presence of this methylation enzyme in representative organisms from all three domains of life indicates that this metabolism was essential very early in earth’s biological history. Bacterial ArsM catalyzes the methylation of iAsm iAsIII . While aironment . Li et aironment . Similaroculture . Li et a by ArsP  and ArsK by ArsP , demethy by ArsP , and che by ArsH . ArsP mec iAsIII , 112.arsM has been found in the genomes of different bacteria that inhabit the human gut, it is not yet clear whether arsenic methylation occurs in this environment. Similarly, ArsP, ArsH, and/or ArsI activities have not been experimentally evaluated and so it is unknown whether these play a role in human arsenic toxicity. It is worth noting that arsI has only been identified in aerobic bacteria, suggesting that it may not be common amongst the abundant anaerobic bacteria of the human gut [Although uman gut . That saMuch of what is known about arsenic-microbe interactions comes from environmental microbiology and in ecosystems such as soil and the subsurface, where microbial metabolisms are the primary determinants of arsenic speciation, mobility, and toxicity. Many of the same principles used in these environmental microbiology studies of arsenic can be directly applied to understand arsenic interactions with the human microbiome. For example, arsenic-microbiome interactions can have three general and sometimes overlapping outcomes: no noticeable effect, perturbation of microbiome taxonomic structure and function, and alteration of the pharmacological and/or toxicological properties of the toxicants . More thScopulariopsis brevicaulis (formerly Penicillium brevicaule) as trimethyl arsine gas [E. coli isolated from the cecal contents of rats were shown to metabolize DMAsV, producing TMAVO and an unidentified arsenical [The notion that intestinal bacteria contribute to health outcomes following arsenic exposure is now more than a century old. In 1917, Puntoni reported the ability of spore-forming bacteria isolated from human stool to produce a potent garlic odor when cultivated in the presence of cacodyl arsenic . It was sine gas . More rersenical .ex vivo). Incubations of the intestinal contents of rodents demonstrated a high capacity for enzymatic reduction and methylation of iAs mediated by the microbiome [V in a simulated human gut environment, yielding both toxic (MMAIII and DMAIII) and comparatively less toxic methylated arsenicals (MMAV and DMAV) [V was reduced to iAsIII even in autoclave sterilized experimental controls, suggesting that non-enzymatic processes may contribute to this transformation.In addition to experiments in pure cultures of bacteria, arsenic metabolism has been studied in the context of microbiome members experimentally evaluated in the lab  , 120. InFaecalibacterium prausnitzii, but not Gram-negative E. coli, provided protection to gnotobiotic AS3MT-KO mice during exposure [While the above studies provide strong evidence that the microbiome has the potential to influence arsenic toxicity, none of them experimentally determined the overall impact that the microbiome has on host exposure. In Coryell et al., our lab showed that antibiotic treatment of mice prior to arsenic exposure significantly reduced fecal arsenic excretion and increased host accumulation of arsenic in the liver and lung tissues . We specexposure .III [Arsenic has been found to change the taxonomic structure of the microbiome in lab animals and human populations. In mice and rats, arsenic exposure induced shifts in microbiome community membership, metabolite profiles, the functional metagenome, and proteomic expression , 123–125III  , have reported arsenic-induced perturbations dependent on host sex, arsenic dose, exposure time, and dietary micronutrients. Arsenic may be an important factor in the enrichment, spread, and/or maintenance of antibiotic resistance genes, and further investigation is needed to determine whether arsenic and other metal toxicants have an important influence on the reservoir of antibiotic resistance genes currently in circulation. Both clinical and epidemiological studies examining arsenic-microbiome interactions should clarify these and other potential links and these studies should include arsenical speciation as well as proxies for microbiome function.Blautia genus as well as representatives of the Lachnospiraceae, Ruminococcus, and Faecalibacterium families were significantly associated with survival across humanized groups, but these are all very common and diverse groups of bacteria. It is possible that although these taxa were consistently associated across subjects, important strain level differences would weaken or even remove the statistical associations. In other words, taxa that are beneficial in one individual may not be beneficial in another person because bacterial taxonomy often does not reliably define function (Note: the first three E. coli genomes to be sequenced shared only 40% of their protein-coding loci) [In vivo, microbiome “phenotypes” have been linked to altered ratios of methylated and inorganic arsenicals in the host . Howeverng loci) . Regardlng loci) . Since tTerminology like “toxicomicrobiomics” and “pharmacomicrobiomics” have emerged in recent literature from increased interest in microbiome-mediated metabolism of xenobiotic compounds, and its influence on physiological outcomes . These eSo far, very few epidemiological studies have investigated arsenic-microbiome interactions and even fewer interrogated host health as a variable. While single-timepoint observational studies may identify potential associations with microbiome composition or activity, they are limited to correlative inference. Prospective cohort studies tracking oral or fecal communities over time could help clarify key interactions between microbiome arsenic-related diseases, and it is important to recognize that the microbiome is highly dynamic at the strain level . InformaThe science of arsenic toxicology underwent a paradigm shift with the development of technologies and methods allowing for rapid and accurate speciation of arsenical compounds. Similarly, advancements in DNA sequencing and meta-omics technologies have changed our understanding of microbial interactions in human health. We reviewed and summarized recent insights into the influence of the microbiome on arsenic metabolism, excretion, and toxicity and discussed the influence of microbiome perturbations on arsenic exposure in both animal models and humans. The microbiome clearly has the potential to alter host arsenic metabolism and disease outcomes in mice. However, more research is needed to quantify microbial metabolism of arsenic, in vivo, and to identify underlying mechanisms influencing host uptake, metabolism, and excretion. Animal research is currently limited by availability of adequate animal models for arsenic-induced disease, especially with regards to health effects from chronic arsenic exposures, which represent perhaps, one of the greatest threats to human health. Further exploration and application of germ free and gnotobiotic animal models may help identify causal arsenic-microbiome links relevant to clinical practices and interventions.


The rift between evolutionary psychology and the biosocial model of gender relations impedes a fuller understanding of gender roles and gender inequality. In a novel evolutionary account that complements both existing theories, we highlight life history strategies as intermediate mechanism linking distal environmental forces to variations in gender relations. Specifically, traditional versus modernized gender roles are seen as shaped by present-oriented versus future-oriented reproductive strategies, which are sensitive to uncontrollable morbidity-mortality risks. Gender inequality stems from a combination of present-oriented reproductive strategies adapted to high-risk environments and dominance hierarchies resulting from societal competition . By contrast, gender egalitarian values develop as people increasingly enact future-oriented reproductive strategies in a competitive but orderly and controllable environment, which is conducive to prestige hierarchies. The current account provides novel interpretations of phenomena ranging from sex differences in mate preference, sociosexuality, and sexism to cross-cultural variability in marital systems and cultural practices. All of these serve to support the view that gender relations are evolved, changeable, and influenced by the interaction between ecological and social environments in ways predicted by the life history mechanism. Both social constructionists e.g.,  and evolThe current paper seeks to combine life history theory  with sexA number of explanations of gender roles and gender inequality have been proposed, ranging from the invention of the plow for intensive agriculture  to the gEvolutionary psychologists argue that at least some sex differences in human behaviors and psychological dispositions, especially those related to mate seeking and selection, are attributable to selective pressures of intersexual selection and intrasexual competition imposed by a number of adaptive challenges . These cWhile different evolutionary psychology theories differ in their emphasis on various processes of sexual selection , they all maintain that sex differences are ultimately produced by selection of inheritable traits, instead of non-genetic processes such as social learning . This isContrary to this misguided impression, contemporary evolutionary psychologists actively reject genetic essentialism by acknowledging non-genetic, environmental inputs and phenotypic plasticity in human life history strategies . Despitevia the monopoly of “warfare, agriculture, and production activities,” which generate far more material wealth than domestic labor  extrinsic risks (often divided into harshness and unpredictability), which represent the morbidity and mortality threats that cannot be avoided through individual efforts , and (2)In human society, harshness and unpredictability , through their indirect effects on families , have been demonstrated to “accelerate” individuals’ life history, which manifests as earlier physiological maturation, earlier sexual debut, and earlier reproduction . As an eHowever, the mentioned strategies impose long-term costs on an individual’s health, relationships, and offspring competitiveness , 2002. INotably, drastically imbalanced parental investment is not a fixed nature of humanity. Humans are exceptional among mammals in terms of paternal investment in offspring . HoweverSince the ecological dominance of human species has overcome numerous extrinsic risks, including predators and cold climates, humans have become their own worst enemy because of societal competition . The hyp2) have higher population density than stable, prosperous countries  female partners . Indeed,Societal competition expressed as non-agonistic contests based on skills and altruism can also benefit high-prestige individuals reproductively . One resThe above analyses lead us to a series of predictions regarding gender roles and gender inequality . Research has shown that in such dangerous environments, women prefer men with indicators of good genes . In particular, the interaction between extrinsic risks and societal competition might shed light on an array of cultural phenomena (mostly documented in ethnographic studies) relevant to gender roles and gender inequality.Small-scale, nonagricultural societies, some of which probably resemble those inhabiting the human EEA , generalNevertheless, the proportion of polygynous mating systems, which is usually associated with constraints on women’s autonomy , differsPolygyny is also rare in agricultural societies, but this might be due to socially imposed monogamy, rather than indicating equal power between sexes in such societies . In sociIn contrast, these practices rapidly declined and gender egalitarian values replaced traditional values that suppress women’s freedom as Europe and East Asia went through industrialization and demographic transition. Two interrelated reasons might account for this cultural practice and cultural value transition. First, increased concentration of population in urban areas and in industrial jobs leads to large-scale cooperative societies comprising mostly strangers, instead of kin-groups . From thOur life history account complement existing theories about gender relations by: (1) emphasizing the fact that the evolutionary processes, including sexual selection, that shape traditional gender roles and gender inequality are flexible rather than fixed, and (2) providing specific predictions regarding how these processes are contingent on the interaction between extrinsic risks and societal competition. Future research is needed to improve the evidentiary status of environmental influences on gender relations, and it faces several challenges.First, identifying the sources of extrinsic risks in modern environments while ruling out confounding genetic effects can be difficult. Previous research has examined familial resource insecurity (e.g., income-to-needs ratio; Secondly, further theoretical and empirical works are needed to elaborate different evolutionary pressures for and different social developmental consequences of dominance versus prestige competition in a life history framework. As two forms of societal competition or status-seeking strategies, dominance and prestige are conceptually separable . HoweverWe propose that the relative importance of dominance and prestige in societal competition might have more to do with life history tradeoffs in the face of extrinsic risks. Specifically, dominance competition might prevail in high-risk environments, as present-oriented reproductive goals prompt male–male competition and agonistic confrontations over resources . PrestigA third challenge lies in recognizing individual differences in susceptibility to environmental influences at different levels . For insA fourth challenge is to distinguish gender inequality from gender roles—although they are occasionally intricately related to each other see —and to aFinally, our position should not be mistaken as yet another version of gender essentialism. We concur with the biosocial model , 2012 anNZ and LC conceptualized the manuscript. NZ prepared the first draft and LC revised and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.