Case Description. A 74-year-old farmer presented with high-grade, unsettling fever for a week. Muddy water exposure and suggestive symptoms prompted investigation and management in the line of leptospirosis  became positive). Subsequently, he developed severe acute hypoxemia requiring mechanical ventilation and acute renal failure requiring renal replacement therapy. Bone marrow biopsy and markedly elevated serum ferritin and triglyceride levels done on day 10  confirmed the diagnosis of HLH. The routine cultures, retroviral studies, CMV, dengue, hanta and mycoplasma antibodies, tuberculosis and COVID-19 PCR, and malaria screening were all normal. There was no improvement of hypoxemia following intravenous methylprednisolone. He died on day 15 despite escalating organ support.  Secondary haemophagocytic lymphohistiocytosis (sHLH), often associated with an array of infections, malignancies, and autoimmune diseases, is rarely seen with leptospirosis, which carries a relatively poor prognosis even with modern state-of-the-art medical care. We describe a patient with leptospirosis complicated by sHLH who succumbed to illness following multiorgan dysfunction.  Leptospirosis is a common zoonotic disease in the tropics with significant morbidity and mortality. In the case of severe leptospirosis, overlapping clinical features with sHLH make the diagnosis of the latter challenging. No assessment tools are available to date to predict the risk of developing sHLH in a patient having leptospirosis. Outcome following sHLH due to leptospirosis still remains majorly ominous. A high index of suspicion and low threshold for specific investigations could possibly alter the outcome following such an occurrence. First described in 1939  and carrLeptospira antibody test . He was started on intravenous ceftriaxone and methylprednisolone empirically with the suspicion of severe leptospirosis and pulmonary haemorrhages. Urgent haemodialysis was arranged every other day. Two days into the hospital stay, the patient acutely became drowsy and restless and electively intubated. Noncontrast CT brain excluded cerebral oedema or acute vascular events. Arterial blood gas analysis revealed a severe hypoxemia (P/F < 120). Lung-protective ventilation was continued although a fraction of inspired oxygen continued to escalate. Prone ventilation was opted approximately 14 hours a day. Pancytopaenia with hepatosplenomegaly was evident in the complete blood count and ultrasound abdomen, respectively. A multidisciplinary discussion with the general medical and haematological units was held. Persistent high fever with negative routine cultures and the rapid deterioration despite optimal supportive therapy pointed towards possible sHLH. Subsequent serum ferritin levels and trephine biopsy confirmed the diagnosis . Renal functions were markedly elevated, and bilateral moderate pleural effusions were noted in the ultrasound chest. Glycemic control was poor with initial random blood sugar of 350 mg/dl requiring an insulin infusion. Chest X-ray showed additional features of pulmonary haemorrhages. There were no skin rashes or lymphadenopathy. Blood was collected for routine cultures and  Figures , aided bMycobacterium tuberculosis are found in the literature, the leading bacterial infection of sHLH [Leptospira-induced sHLH are remarkably few. The reported cases are tabulated in comparison to our patient in Haemophagocytic lymphohistiocytosis (HLH) is a rare but possibly fatal, rather underdiagnosed disease state, affecting both adults and children . Amidst  of sHLH . Interesα), interleukin 1 or interleukin 6, and gamma interferon [Leptospira, being commonly extracellular, a genetic predisposition of the organism to utilize and internalize host haem proteins, could possibly contribute to its pathophysiology and HLH [Leptospira might induce a TNF-α-associated apoptosis of macrophages, excessive secretion of interleukins, and ultimately cytokine storm and multiorgan failure [The proposed mechanisms and models of HLH led organ dysfunction describe dysregulated T cell activation (mainly CD8+ cytotoxic T cells) which is propagated with macrophages, leading to a ‘cytokine storm.' It is postulated that the pathophysiology of infection-associated HLH includes excessive stimulation of Th1-mediated immune response resulting in the overproduction of tumour necrosis factor alpha , at least five out of eight criteria should be met to establish the diagnosis . While HThe management of sHLH is mainly in the aspects of controlling the trigger, chemotherapy, and immunotherapy with supportive care, frequently comprising the specialties of haematologists, oncologists, rheumatologists, microbiologists, and intensivists. In the case of leptospirosis-induced sHLH, early intravenous antibacterial therapy  should be instituted as patients are severely ill. According to the literature review , out of The diagnosis of sHLH in intensive care units poses a significant challenge to physicians due to unspecific symptoms which may overlap between conditions such as sepsis, multiorgan dysfunction syndrome, or other cytokine storm syndromes and coexistence of these conditions , 21. MorLeptospira, the response to which was assessed by means of serial CRP as procalcitonin was not available in our center. Even though there was a rise in CRP, which could have been due to the generalized inflammatory process, we opted for intravenous etoposide following hematology consultation due to poor response to steroid therapy. In comparison to the HLH-94 treatment protocol which is utilized primarily for paediatric HLH, modified, individualized HLH-94-like treatment protocols have been suggested for adult sHLH, although caution is warranted for immunosuppression following intracellular organism-related infections as resolution of HLH has been associated with specific antimicrobials alone [In the case of our patient, who presented late with acute kidney injury and probable setting severe adult respiratory distress syndrome, sHLH was suspected immediately after admission to the intensive care unit with pancytopaenia and rapid deterioration and pulsed corticosteroid therapy as intravenous methylprednisolone was commenced with other organ supports. The steroid therapy was continued with dexamethasone. The patient had already been started on third-generation cephalosporin on admission targeted for ls alone .By this review, it is evident that the outcome of leptospirosis-induced sHLH is poorer in adults in comparison to the paediatric age group. Associated comorbidities might play a role in accentuating or altering the immune response and accelerating the organ dysfunction; however, late diagnosis and nonadherence to the current guidelines during management are crucial factors contributing to these figures. It is likely that sHLH in association with leptospirosis may be misdiagnosed as severe leptospirosis due to the overlapping constellation of symptoms and signs, persistent fever, organomegaly, and cytopenia, making the diagnosis of sHLH underreported.Leptospirosis is considered as a neglected but reemerging disease throughout the world . A systeLeptospirosis will continue to burden the developing nations with increased morbidity and mortality. Even though being a rare association, most probably due to underdiagnosis and/or misdiagnosis, sHLH, due to severe leptospirosis, may also contribute to these figures. No assessment tools are available to date to predict the risk of developing sHLH in a patient having leptospirosis. When dealing with leptospirosis-induced sHLH, a high index of suspicion, especially in cases of abrupt deterioration of the clinical condition and poor response to standard therapy, is decisive to escalate the treatment protocol. Nonetheless, early diagnosis of sHLH by devising proposed scoring systems, early ICU care, and timely institution of corticosteroid therapy and immunosuppressant therapy, whenever appropriate, may dramatically improve the patient outcome as evident by this work.


The advent of PSCs has also opened up a possibility of developing regenerative medicine therapies, either through PSC differentiation in vitro or by creating interspecies chimeras for organ replacement. Here, we discuss the emerging evidence of cell selection in human PSC populations in vivo and in vitro and we highlight the implications of understanding this phenomenon for human development and regenerative medicine.A small number of pluripotent cells within early embryo gives rise to all cells in the adult body, including germ cells. Hence, any mutations occurring in the pluripotent cell population are at risk of being propagated to their daughter cells and could lead to congenital defects or embryonic lethality and pose a risk of being transmitted to future generations. The observation that genetic errors are relatively common in preimplantation embryos, but their levels reduce as development progresses, suggests the existence of mechanisms for clearance of aberrant, unfit or damaged cells. Although early human embryogenesis is largely experimentally inaccessible, pluripotent stem cell (PSC) lines can be derived either from the inner cell mass (ICM) of a blastocyst or by reprogramming somatic cells into an embryonic stem cell-like state. PSCs retain the ability to differentiate into any cell type  •Cell selection might play a role in human embryo development.•Pluripotent stem cells provide a platform for studying human development.•Cell competition occurs in human pluripotent stem cell cultures.•Cell competition is a barrier to interspecies chimerism. Mediated by membrane receptors and signals, soluble factors or mechanical cues, cell-cell communication orchestrates development of nascent tissues and regulates homeostasis of mature ones. Remarkably, cell-cell interactions are not always cooperative, but rather, they can also be competitive in nature. A fascinating aspect of cellular interaction is cell competition, a quality control mechanism whereby cells utilise soluble signals, mechanical cues or perform direct fitness comparisons in order to weed out less fit, albeit otherwise viable, cells from their populations (reviewed in ). In thiViewed through the prism of quality assurance, it is difficult to imagine a more important context of competitive cellular interactions than that of the early embryo. Upon fusion of gametes and a zygote formation, development progresses through a sequence of cleavage divisions, leading to the rapid and exponential growth in cell numbers. Once the embryo reaches the blastocyst stage, at 5–7 days post-fertilisation, a small number of pluripotent epiblast cells are segregated and destined to produce all of the cell types in the human body. It is therefore reasonable to postulate that selection against unfit cells within the pluripotent population is pivotal for ensuring elimination of mis-patterned or genetically aberrant cells that could otherwise instigate developmental defects or pregnancy loss. Indeed, experimental evidence for the role of cell competition in development has been gathered from many model organisms, including mammalian species (reviewed in ). Nonethin vitro  from human blastocysts  and latein vitro . Whilst in vitro . Nonethein vitro . With sein vivo modelling of early developmental disorders, providing readouts of disease onset and progression, with relevant clinical value  serves as a novel platform for al value . MoreoveHere, we discuss the role of cell competition in human PSC populations within several different contexts. First, we speculate on the existence of cell fitness selection during early human embryogenesis, mainly inferred from descriptive studies of human embryos and mechanistic studies from other species. We then discuss the role of cell competition in establishing and expanding PSC cultures, relevant to applications of PSCs in basic biology research and regenerative medicine. Finally, we delve into the recent observations of cell competition in PSCs mixed from different species and consider the implications of understanding cell competition for applications from assisted reproduction to regenerative medicine.2The need to understand the exact timings and sequence of early embryonic processes in human development extends beyond academic interest into the area of substantial clinical significance. Indeed, estimates that as many as 70% of human pregnancies do not result in live births speak to the clinical need for unlocking the reasons behind a low fecundity rate in humans . The preSeveral observations in human embryos may help explain the apparent paradox. First is that the embryos harbouring mosaic aneuploidy can nonetheless result in live births  and secoApart from the elimination of genetically aberrant cells, cell competition in mouse embryos also selectively ablates cells that in some manner display an inappropriate or less-fit phenotype relative to the rest of their population. The undesired phenotypic features depend on the stage of the embryo development, with different features being selected for in mouse pre-implantation versus post-implantation development. For example, during mouse epiblast differentiation, cell competition eliminates cells that have a relatively low expression of pluripotency factors . The expThe quality surveillance for cell fitness also continues into the post-implantation stage of the mouse epiblast. At this stage, cell competition was shown to remove mis-specified cells, such as the ones with the defective Bone Morphogenetic Protein (BMP) signalling, when they are found in a milieu with wild-type cells . Moreovein vitro cultured embryos are being complemented by recently developed synthetic embryos fabricated from PSCs or by assembling multiple cultured stem cell types into embryo-like structures. A stem cell-based human embryo model based on the confinement of PSCs to 2D circular micropatterns mimics some features of post-implantation development, as cells self-organise to generate trophectoderm-like, mesendoderm-like and ectoderm-like layers upon exposure to BMP4 in vitro in vitro. Due to a lack of gold standard functional test of naïve pluripotency in humans, a flurry of papers reported the generation of human PSCs showing some degree of resemblance to mouse ESCs in vitro.use ESCs . Future in vitro and germline chimera formation in vivo. The same culture condition also supported the derivation of ESCs from horse blastocysts and generated transgene-free iPSCs from both horse and human fibroblasts. Horse FTW-ESCs/iPSCs transcriptionally resembled their mouse counterpart, could be directly induced into PGC-like cells (PGC-LCs) by BMP treatment, and contributed to chimera formation in mouse embryos. Human FTW-iPSCs also harboured intermediate pluripotency features and demonstrated PGC responsiveness in vitro. Interestingly, in another study, loXR-PSCs here). AloXR-PSCs share similar properties with FTW-PSCs, which include competence for germline specification, chimera formation, and transcriptome similarity to E5-6 epiblasts, among others. There are, however, noticeable differences between AloXR-PSCs and FTW-PSCs. For example, female FTW-PSCs retain two active X-chromosomes (XaXa) while one of the X-chromosomes in female AloXR-PSCs is inactive (XaXi). As random X-chromosome inactivation (XCI) is not fully established by E6.5 , AloXR-PSCs and FTW-PSCs may represent different types of formative cells between E5-6, or FTW-PSCs and AloXR-PSCs may resemble an early and late formative states, respectively. Overall, these findings suggest that formative pluripotency is a continuum rather than a singular state and demonstrate that different types of PSCs with formative features can be recapitulated in vitro under different culture conditions.A number of recent studies have reported that human and mouse PSCs putatively reside in intermediate states between naïve and primed pluripotency . Severalin vitro adaptations of human pluripotency continuum in vivo and provide us with readily accessible, inexhaustible, scalable and manipulatable resources for modelling human development and diseases, and for generating cells and tissues for regenerative medicine. Choice of which PSC type to use will likely depend on the application. For example, formative human PSCs are ideal for studying molecular mechanisms underlying human germ cell development and infertility. Naïve human PSCs are supposedly chimera competent, and therefore were believed to be the cells to use for interspecies blastocyst complementation for generating human tissues in animals. However, low chimeric contribution of naïve human PSCs in animals, even at the embryonic stage, has been reported . This shortage extends worldwide, and researchers are currently working on a variety of ways to increase the number of organs available. Classical developmental studies conventionally used to assess developmental plasticity of cells have inadvertently paved the way to achieving this feat. Specifically, an in vivo approach known as interspecies blastocyst complementation involves the injection of donor PSCs from one species into an organogenesis-disabled blastocyst of a different species. The growing mutant host embryo provides an emptied developmental organ niche exclusively for donor cells to fill , which has the main role of activating NF-κB. We found that MYD88 deficiency could also rescue human PSCs from being outcompeted by mouse EpiSCs. Suppression of human-mouse primed PSC competition conferred primed human PSCs with the ability to contribute to chimera formation in early mouse embryos.To gain insight into the molecular mechanisms underlying human-mouse primed PSC competition, we performed RNA-sequencing of human PSCs either separately cultured or cultured with mouse PSCs. Comparative analysis identified NF-κB signalling pathway was activated in co-cultured human PSCs. Consistent with this finding, knockout of MYD88 and P65 deficiency also prevented human PSCs from being eliminated by rat EpiSCs during co-culture. It remains to be determined if TLR/NFκB pathway plays a conserved role in primed PSC competition between other species.In addition to human and mouse, we also performed co-culture experiments using primed PSCs from several other species including rat, rhesus macaque and cow. Similar to human-mouse, cell competition could be observed in any co-culture combinations between a primate (human or rhesus) and a rodent (mouse or rat), but not between human and rhesus, or mouse and rat. Interestingly, we found primed bovine ESCs were outcompeted by both rodent and primate PSCs. These results suggest primed PSC competition is a general phenomenon among species with differential cell fitness. Similar to mouse EpiSCs, Drosophila wing disc development  to activate NFκB-dependent apoptosis in “aberrant” or “unfit” cells, and thereby preventing them from further participation in development. TLRs/NFκB is the key signaling pathway mediating the ancient and conserved innate immune recognition system activated in response to altered-self cells , which melopment , and thu6in vitro and in vivo. Therefore, recent findings implicating cell competition in determining the sub-clonal composition of human PSC populations came as no surprise to developmental biologists. Nonetheless, surprising was the diversity of cell competition mechanisms operating in different PSC contexts. The variety of these mechanisms likely reflects the exceptionally dynamic nature of pluripotency, with each pluripotent state favouring selection of specific cellular features. The dynamic states of pluripotency during development, which have also been captured in equivalent naïve, formative and primed states in vitro, therefore provide an exceptional model for exploring molecular rules of cell fitness selection. The use of complementation experiments, either through mixing with xenogeneic cells in vitro or by introducing PSCs into host embryos of different species, offers an additional route into the understanding of the molecular machinery of PSC cell competition. Ultimately, deciphering how cell fitness is sensed within a population and how elimination of unfit cells is executed, will have a significant impact on our ability to control stem cell fates in applications ranging from basic research and disease modelling through to regenerative medicine. Finally, a recent upsurge in PSC-based models of human conceptus offers an opportunity to delineate how cell selection contributes to upholding the cellular fitness in what is undoubtedly one of the most enigmatic contexts in biology – the human embryo.Cell competition, a quality control mechanism that scrutinises cellular fitness and removes less-fit cells from heterogeneous populations, has been demonstrated in many different cellular populations


Transformative and mutually beneficial solutions require decision-makers to reconcile present- and future interests  and to align them with those of other decision-makers . Despite the natural co-occurrence of intrapersonal and interpersonal conflicts in the transformation toward sustainability, both types of conflicts have been studied predominantly in isolation. In this conceptual article, we breathe new life into the traditional dialog between individual decision-making and negotiation research and address critical psychological barriers to the transformation toward sustainability. In particular, we argue that research on intrapersonal and interpersonal conflicts should be tightly integrated to provide a richer understanding of the interplay between these conflicts. We propose a novel, unifying framework of interdependent conflicts that systematically structures this interplay, and we analyze how complex interdependencies between the social  and temporal  dimensions pose fundamental psychological barriers to mutually beneficial solutions. Since challenges to conflict resolution in the transformation toward sustainability emerge not only between individual decision-makers but also frequently between groups of decision-makers, we scale the framework up to the level of social groups and thereby provide an interdependent-conflicts perspective on the interplay between intra- and intergenerational conflicts. Overall, we propose simple, testable propositions, identify intervention approaches, and apply them to transition management. By analyzing the challenges faced by negotiating parties during interdependent conflicts and highlighting potential intervention approaches, we contribute to the transformation toward sustainability. Finally, we discuss implications of the framework and point to avenues for future research. Third, from a multi-level perspective, we offer a systematic link between psychological negotiation research and transition management and highlight negotiation processes at different societal levels. Fourth, from an applied perspective, we aim to provide a more-comprehensive understanding of psychological conflicts in the transformation toward sustainability and to offer potential leverage points with hands-on tools for interventions that foster sustainable solutions. In essence, we seek to encourage future research to further examine human decision-making processes in the context of interdependent conflicts with the goal of fostering the transformation toward sustainability.present interpersonal conflict between their own and their counterparts’ present interests. This type of conflict has traditionally been investigated by social-conflict- and negotiation research . This type of conflict has predominantly been studied by individual decision-making research  ch e.g., . Simultach e.g., . Finallyct e.g., . The parOur paper is structured as follows: To establish our framework, we first introduce interdependent conflicts at the individual level. In so doing, we review the existing literature, outline characteristic psychological processes, derive propositions, and conclude with an intervention approach to addressing the proposed problems at the individual level. Second, we scale up our framework from the individual-group to the social-group level to establish interdependent conflicts as an interplay between inter- and intragenerational conflicts. We then follow the same structure as at the individual level.via negotiations.Interpersonal conflicts emerge whenever two or more parties perceive their views or interests as being incompatible , and negIn interpersonal conflicts, negotiators typically display the detrimental psychological tendency to devalue their counterparts’ interests . PartiesDecision-makers who experience intrapersonal conflict must make a choice between different alternatives that entail consequences that occur at different times e.g., . People Research has demonstrated that people tend to temporally devalue their own future interests relative to their immediate ones  may only be resolvable via joint control. Due to these differences across conflicts, parties may experience more constraints in resolving conflicts of interests with their counterparts  compared with resolving conflicts of interests with themselves . We therefore conclude that negotiators tend to prioritize the resolution of inter- over intrapersonal conflicts because solutions between people require interpersonal coordination, whereas solutions over time are less constrained by coordination with other parties.Based on the distinction between full actor and joint control, parties could perceive of having different degrees of freedom in resolving their conflicts of interests over time and between people. Specifically, conflicts over time  may be resolvable Building on the above-mentioned research, our framework of interdependent conflicts postulates how parties cognitively process the interplay between different psychological conflicts. In contrast to a rational approach in which parties cognitively process interdependent conflicts in a comprehensive, unbiased way , we hypothesize that parties systematically prioritize the consideration of certain conflicts in a biased way.Proposition 1: In interdependent conflicts, parties prioritize the consideration of present interpersonal conflicts (first priority) over intrapersonal conflicts (second priority) and future interpersonal conflicts (third priority).These priorities are derived both from parties’ tendency to discount their future interests  and to dIn line with this reasoning, prioritizing the consideration of conflicts should also determine which conflict is resolved at the cost of another.Initial support for our assumptions can be found in a survey study  that invProposition 2: Prioritizing the consideration of conflicts determines the extent to which parties can exploit integrative potential and reach integrative agreements.To resolve interdependent conflicts in an integrative way, decision-makers must consider their interests in a comprehensive rather than in an isolated, prioritized way. From a rational perspective, parties can maximize the utility of a solution  by makinWe assume that prioritizing the present interpersonal conflict is caused – in part – by constraints in decisional control. Resolving interpersonal conflicts requires negotiating between parties to overcome divergent interests, whereas resolving intrapersonal conflict does not require negotiating to overcome divergent interests in the present or future. To balance the consideration of interdependent conflicts, we propose also applying a negotiation strategy to intrapersonal conflicts over time . NegotiaSocial-conflict research has revealed that integrative solutions are particularly likely when each negotiator (1) has a strong concern for his or her own outcomes ; (2) intergenerational conflict ; and finally, (3) future intragenerational conflict .This example can be systematically structured using the framework of interdependent conflicts. Conflicts in the transformation toward sustainability include a social dimension between groups  and a temporal dimension between generations over time  resources as the root of competition in intergroup conflict . When covia different strategies, including social competition, for instance, by discriminating the out-group.However, another line of research suggests that merely categorizing oneself and others as members of an in- and out-group, respectively, is sufficient to induce intergroup conflict , 1986. SOverall, greed and fear as well as the need for positive distinctiveness all contribute to intergroup devaluation. Greed and fear are particularly pronounced when outcome interdependence exists. However, the need for positive distinctiveness can be explained by the psychological process of self-categorization as an in- or out-group member.In contrast to intergroup conflicts within a generation e.g., , much-leSpecifically, outcomes are not reciprocally interdependent in intergenerational conflicts. Instead, the outcomes of the future generation are fully determined by the present generation. Present generations therefore have complete actor control without the need to coordinate their interests with future others. Consequently, future generations have no voice in intergenerational conflicts .Proposition 3: In interdependent conflicts at the social-group level , parties prioritize the consideration of present intragenerational conflicts (first priority) over intergenerational conflicts (second priority) and future intragenerational conflicts (third priority).Social groups have a tendency to prioritize present intragenerational conflicts because joint control with the other group within the present generation places constraints on the decision-making process and requires coordination between groups. This joint control stands in contrast to intergenerational conflicts over time, which should be given second priority because the present generation has full actor control when it comes to resolving these conflicts. In line with this reasoning, future intragenerational conflicts should be given third priority because in addition to intergenerational devaluation, the need for positive distinctiveness from the other group  also contributes to the prioritization of these conflicts.These priorities also determine which conflict will be resolved at the cost of another. Conflicts of higher priority may be resolved at the cost of lower-priority conflicts because present intragenerational conflicts should receive more consideration than intergenerational conflicts or future intragenerational conflicts. Prioritizing the consideration of interdependent conflicts thus has important implications for the transformation toward sustainability.Recent research has found initial support for Proposition 3  by showiProposition 4: A prioritized consideration of conflicts determines the extent to which social groups  can exploit the integrative potential and reach integrative agreements.To achieve mutually beneficial, transformative solutions at the group level, a balanced and unbiased consideration of all conflicts (rather than a prioritized consideration) is necessary. However, we assume that the involved groups prioritize conflicts with detrimental consequences. Specifically, parties consider the coordination of diverging interests in higher-priority conflicts to a greater extent than in lower-priority conflicts. Integrative potential and the trade-off opportunities embedded within lower-priority conflicts are therefore less-likely to be discovered. A prioritized, biased consideration of conflicts should thus result in suboptimal solutions for involved groups. In other words, resolving interdependent conflicts should be transformative and mutually beneficial if future generations’ interests are considered in an unbiased and balanced way.via interventions (via negotiations requires that (1) the two subgroups consider their common concerns by creating a new superordinate, common in-group identity and (2) that each subgroup maintain its distinct group membership and consider its dual concerns . If the groups consider their superordinate, common in-group identity and common concerns, while simultaneously considering their dual identities and dual concerns, intragenerational conflicts can be resolved in an integrative, unbiased way  create a common in-group identity with their succeeding future generation that includes common concerns shared by the present- and future generations and (2) acknowledge their distinct dual identities over time – including dual concerns of the present- and future generations – in order to stimulate negotiations with future others.via negotiations. Contemporary representatives of the future generation may take responsibility for speaking up for their generations’ interests , increasing affinity with future generations , and empWe developed and introduced a framework of interdependent conflicts for stimulating novel research that examines individual- and joint decision-making processes in the transformation toward sustainability. The critical relevance that negotiations entail in this transformation is undisputed ; howevervia negotiations are often not in favor of our own or others’ long-term interests. One of the key contributions of our novel framework is that it enables an analysis of decision-making settings in the transformation toward sustainability in a more-comprehensive, unifying, and systematic way. Moreover, our framework provides a parsimonious structure for disentangling these complex conflict situations, analyzing the arising psychological phenomena, and designing interventions that tap into the psychological barriers that impede transformative solutions. At best, agreements create integrative solutions for all parties involved – not only in the present, but also over longer timespans. Our framework offers a systematic integration of the social and temporal dimensions and thereby helps in reaching these transformative and mutually beneficial solutions.Typically, decision-makers must simultaneously consider their own interests and those of other decision-makers in addition to long-term future consequences for themselves and future others. We aimed to provide a novel perspective on why agreements reached Sustainability challenges represent the largest collective-action problem ever faced by humanity . Joint dThe described tensions may lead to a rather skeptical view of the transformative potential of negotiations. Indeed, the challenges for parties in creating transformative solutions are difficult. However, we hope that our framework and the proposed intervention approaches might help negotiators navigate toward more-transformative solutions across different societal levels and contexts. In grassroots initiatives, small groups of societal frontrunners may initiate negotiations over innovations and, in the management of the transition, representatives of larger societal-interest groups, institutions, or organizations may negotiate their interests in contributing to the transition pathway. Thereby, negotiations may also help to bridge structural changes across societal levels. We believe that existing and potential future tools for implementing intervention approaches should be tested, adapted, and refined depending on the interdependent-conflict situation. Nevertheless, we wish to emphasize the idea that interdependent conflicts are negotiable not only between individual actors and societal groups but also within ourselves and across generations. Making use of the transformative potential of these negotiation processes may open new transition pathways toward sustainability. We, therefore, remain optimistic that negotiations as collaborative decision-making approaches are most promising for reaching transformative solutions and are our only true alternative to collaboratively achieving long-term societal prosperity . In acknThe original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.JM developed the theory, created the figures, and drafted the manuscript. MB, HZ, MT, and RT contributed to theory development, structuring, and revising the manuscript. All authors contributed to the article and approved the submitted version.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.


Review of US medical and recreational marijuana laws (MML and RML), their effects on cannabis potency, prevalence of non-medical cannabis use and cannabis use disorder (CUD) in adolescents and adults, and implications for neuroscience research, given what is known about the relationship of cannabis to neurocognitive impairments and underlying brain functioning.Cannabis potency may be increasing faster in states with MML or RML than in other states. MML and RML have not impacted prevalence in adolescents but have consistently been shown to increase rates of adult non-medical use and CUD.Recent neurocognitive or neuroimaging studies may be more impacted by cannabis than studies conducted when MML and RML were less common. Neurocognitive or neuroimaging studies conducted in MML or RML states should carefully test potential participants for recent cannabis use. More research is needed on cannabis and cognition in medical marijuana patients. Cannabis has been used in the United States since the 1800s, with public attitudes towards its acceptability and potential harmfulness varying considerably over time , and theLegalization of cannabis would not be relevant to considerations in behavioral neuroscience research if cannabis were unrelated to neurocognitive functioning. However, extensive research over the past 50 years, including hundreds of individual studies and many reviews and meta-analyses suggests that cannabis use is related to cognitive impairments and underlying brain functioning. Many debates remain in this area, including the causal nature of the relationships and reasons for some of the inconsistences in the literature. Nevertheless, broad agreement exists that acute, persistent, and heavy cannabis use affects cognition in many domains, e.g., memory, attention, and executive functioning.Impaired attention is a key feature of acute cannabis intoxication . WhetherMemory is the cognitive domain most consistently reported as impaired by cannabis, with such effects found in adolescents, adults, and older adults , 14–16. Executive functioning involves tasks of planning, reasoning, interference control, decision-making, and problem solving. In the US adult general population, frequency of cannabis use in the past year predicted lower scores on an executive functioning self-report scale , and amo1 receptors are downregulated in people who were cannabis users, especially shortly after use. These changes are mainly located in the neocortex and limbic cortices, which regulate cognition, and in the ventral striatum, which is involved in reward and goal-directed behavior. Reduction of CB1 receptors in cannabis-dependent subjects returns to normal ~2 to 28 days after abstinence, although among those with chronic heavy use, the reduction in CB1 receptors may impact downstream systems that maintain changes underlying later behavioral characteristics of cannabis use disorder. Although evidence is somewhat conflicting, acute THC administration appears to cause increased dopamine release and neuronal activity, whereas long-term cannabis use is associated with blunting of the dopamine system [1 receptors that reduce glutamate release and are sensitive to THC. In the limited research in humans, chronic cannabis use appears to reduce levels of glutamate-derived metabolites in cortical and subcortical brain areas, while animal studies indicate that THC depresses glutamate synaptic transmission via CB1 receptor activation [A recent review of structural and functional neuroimaging studies compiled considerable information from positron emission tomography (PET) and magnetic resonance imaging (MRI) studies about brain structure and functioning related to cannabis use . PET stue system , which itivation .Human structural magnetic resonance imaging (MRI) studies show alterations in corticolimbic structures in those with CUD, e.g., prefrontal cortex (PFC), hippocampus and amygdala. The integrity of the orbitofrontal cortex (OFC within the PFC), which contributes to cognitive flexibility and decision-making, is often impaired in SUD and related to problem use. A similar sensitivity is evident in the hippocampus, a region central to learning and memory. An additional recent review and meta-analysis also showed smaller volume of the hippocampus and orbitofrontal cortex in people who were regular cannabis users compared with controls .Functional magnetic resonance imaging (fMRI) studies of differences in brain functional alterations in people who were cannabis users and non-users while performing memory tasks showed that functional brain activation during the tasks was altered in the people who were cannabis users. The results suggested that the altered brain activation drove the deficits in memory performance . More spIn 1996, California became the first U.S. state to pass a medical marijuana law (MML) legalizing the use of cannabis for medical purposes. As of this writing, 34 states have MML, covering 67% of the US population . State mIn 2012, Washington and Colorado became the first states to pass laws permitting legal use of marijuana for recreational purposes (RML). Eleven states now have such laws , covering 28% of the US population, and several additional states are considering the passage of such laws . Recreat1 receptors, which mediate the action of THC, are particularly concentrated in brain regions such as the hippocampus and amygdala, basal ganglia, anterior cingulate cortex, and prefrontal cortex [The primary psychoactive component in cannabis is delta-9-tetrahydrocannabinol (THC). THC directly targets the body’s natural endogenous endocannabinoid system, including the receptors that mediate the direct actions of cannabinoids . Cannabil cortex . These bIn samples of illegal cannabis seized by law enforcement between 1990 and 2010 , mean THConcerns that MML would increase adolescent cannabis use emerged about 10 years ago , 48, basTo our knowledge, only one study to date used national data to examine the effects of recreational marijuana laws (RML) on adolescent marijuana use that analyzed data appropriate for this purpose . This stIn contrast to the relatively large literature on adolescents and MML effects, fewer studies investigated the relationship of MML or RML to adult cannabis use or related outcomes. A cross-sectional analysis of national 2004–2005 data showed higher rates of adult cannabis use and cannabis disorders in MML than in non-MML states . HoweverA study of RML effects on adults in yearly national surveys from 2008 to 2016 found no effects in young adults age 18–25 . HoweverDiagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) consists of at least 3 of the following symptoms developing within 7 days of reduced cannabis use: (1) irritability, anger, or aggression; (2) nervousness or anxiety; (3) sleep disturbance; (4) appetite or weight disturbance; (5) restlessness; (6) depressed mood; and (7) somatic symptoms, such as headaches, sweating, nausea, vomiting, or abdominal pain. The duration of this syndrome post-abstinence is unclear, but about half of people who are regular cannabis users experience cannabis withdrawal syndrome [The findings on the post-MML and -RML increases in cannabis use and cannabis use disorders indicate that increasingly permissive state marijuana laws do have implications for behavioral neuroscience studies of adults, particularly those age 26 and older. Potential participants in studies conducted in states with MML and particularly RML are more likely to be people who are cannabis users and frequent users than participants in other states. For studies comparing people who are cannabis users or those with cannabis use disorders to cannabis-naïve controls, this may only affect feasibility since the pool of cannabis-naïve participants that can potentially be recruited will be smaller. However, the scientific findings of studies focused on other groups or conditions of interest may be impacted in a more serious way if participants are not carefully tested for recent cannabis use and excluded if they test positive. This may particularly affect studies of neurocognitive or brain functioning studies of depression or anxiety disorders. Cannabis withdrawal syndrome as defined in the syndrome . Becausesyndrome . In suchStudies of the relationship of marijuana to neurocognition and neurofunctioning have largely focused on samples of people who are recreational marijuana users. However, over two-thirds of US states now permit legal use of marijuana for medical purposes, raising issues about potential implications of medical marijuana use for neuroscience research. These include questions about whether people who are medical users are different from people who are recreational users, and whether cannabis may differentially affect people who are medical users. Several early studies from California, the first state to pass MML, showed that people who were medical and recreational users had similar characteristics and that people who were medical users often had histories of recreational use –76. ThesBehaviors such as use and misuse of illicit substances often cluster empirically within the externalizing domain of psychopathology –81, typiIn summary and conclusion, the population prevalence of people who are regular or heavy use of cannabis among US adults has increased over the last 20 years, medical marijuana use is now largely permitted across the USA, and adult residents of states that have legalized cannabis are more likely to be people who use cannabis recreationally and to have cannabis use disorders. Therefore, when evaluating participant eligibility for studies of neurocognitive or brain functioning, carefully evaluating a history of recent use  via interviewing and biological tests will be increasingly important to avoid misleading results or simply reduce error variance in such studies. Additionally, for clinical care, careful histories about marijuana use at the beginning of treatment could avoid diagnostic confusion and provide a better basis for treatment planning, monitored by periodic check-ins on marijuana use as treatment proceeds.


Despite strenuous studies since the last century, the precise cause and pathology of schizophrenia are still largely unclear and arguably controversial. Although many hypotheses have been proposed to explain the etiology of schizophrenia, the definitive genes or core pathological mechanism remains absent. Among these hypotheses, however, GABAergic dysfunction stands out as a common feature consistently reported in schizophrenia, albeit a satisfactory mechanism that could be exploited for therapeutic purpose has not been developed yet. This review is focusing on the progress made to date in the field in terms of understanding the mechanisms involving dysfunctional GABAergic system and loops identified in schizophrenia research. Schizophrenia (SCZ) is a debilitating neurological disorder with a worldwide prevalence of 1% . It is oUnfortunately, the neuropathological finding of schizophrenia is subtle. A slight loss of brain volume has been so far consistently confirmed in growing literature. Interestingly, this reduction largely results from loss of neuropil rather than neuron numbers, potentially highlighting abnormal synaptic plasticity and cortical micro-circuitry. A meta-analysis of neuroimaging studies on patients with SCZ also confirmed the enlargement of lateral and third ventricles and smaller cortical and gray matter volumes . AlteratThough separate but linked, hypotheses for schizophrenia pathology include glutamatergic and dopaminergic hypotheses are being highlighted here. Clinical research suggest that glutamate level may alter prior to the onset of psychosis , whereasThe hypo-functioning of NMDA type of glutamate receptor (NMDAR) has long been proposed as a key contributor to schizophrenia. It is believed that it may alter the excitability of GABAergic neurons and thus neural network oscillation, which promotes the occurrence of cognitive deficit. NMDAR hypofunction may directly reduce the tone of GABAergic interneurons, which leads to overexcitability of glutamatergic pyramidal neurons. The latter may result in an excessive stimulation by glutamate and promote neuronal damage or death . PostnatA reduced GABA level in patients with schizophrenia also associates with lower intelligence quotient (IQ) compared to healthy controls , which sCortical GABAergic interneuron is one of the key elements to coordinate cognitive processes and complex emotions via the release of γ-amino butyric acid (GABA). Glutamate serves as a precursor for GABA synthesis, and the reaction is mediated by glutamate decarboxylase enzymes (GAD65 and GAD67). GABA is stored in cytoplasmic vesicles via vesicular GABA transporters (VGAT1 and VGAT2). Upon release into synaptic cleft, the GABAergic signaling can be terminated in two ways: (1) GABA uptake by presynaptic terminals via plasma membrane GABA transporters (GAT1–4) and (2) GABA uptake by glial cells (via GAT1–4) . Further3A receptors expressing interneurons but infrequently contain more than one of these markers. To conclude, there are three distinct types of interneurons with non-overlapping markers . Interneurons are extended vertically across cortical layers (translaminar), horizontally (transcolumnar), or restricted locally to a single layer oriented horizontally, which has been extensively elaborated by GABAergic interneurons are categorized into various subtypes based on the following: (1) morphology (chandelier cells or basket cells) ; (2) eleIt is understood that by early adulthood, cortical circuits are refined through the pruning of excitatory synapses, elaboration of inhibitory circuits, and remodeling of pyramidal dendrites. The discovery of effective therapeutic strategies is yet awaited to rescue postnatal maturation hindrances and over-pruning and to maintain a healthy GABAergic system to alleviate the risk of developing and also the developed schizophrenia.via gap junctions  interneurons are fast-spiking neurons containing a sufficient number of mitochondria to compensate for high demand of energy  and are unctions , which iunctions . Whereasunctions . Howeverunctions .Each PV interneuron innervates hundreds of pyramidal neurons and regulates their output and synchrony , energizPV interneurons regulate and maintain excitatory and inhibitory balance in the cortex to control the targeted neuron’s activity in a homeostatic range. Spiking outputs from pyramidal neurons to targeted brain areas are regulated by PV interneurons . The tha3a receptor expressing) interneurons inhibit both the PV and SST interneurons and are implicated as an important disinhibitory player in cortical circuits (Somatostatin (SST)-expressing Martinotti cells are low-threshold spiking interneurons that project expansively to distal dendrites of pyramidal cells to control incoming cortical signals . Increascircuits . SST celcircuits . On the circuits . Furthercircuits . It shoucircuits . GlutamaA reduced expression of SST has also been reported within the dorsolateral prefrontal cortex in patients with depression , which iMedium spiny neurons are GABAergic interneurons abundantly presented in the striatum and basal ganglia. Recently, the association of medium spiny neurons with schizophrenia has been evidenced, which is independent from cortical interneurons and pyramidal neurons. The genetic risk associated with medium spiny neurons does not overlap with that of glutamatergic pyramidal cells and cortical interneurons, indicating that different cell types have biologically distinct roles in schizophrenia . FurtherNotably, schizophrenia has shared molecular neuropathology with bipolar and autism spectrum disorders, and bipolar presents a similar synaptic dysfunction with schizophrenia , which iOrchestrated neural oscillations are fundamental to establish precise temporal relationships between neuronal responses important for memory, perception, and consciousness. In patients with schizophrenia, the beta- and gamma-band activity is dis-synchronized, suggesting a crucial role for dysfunctional oscillations in the generation of the cognitive deficits and other relevant symptoms. Dysfunctional oscillations may arise owing to anomalies in the brain’s rhythm-generating networks of GABAergic interneurons and in cortico-cortical connections . Neural Reduced synaptic connectivity is sufficient to produce a large deficit in gamma oscillations, and gamma (γ) deficit in schizophrenia patients could be a marker of reduced synaptic connectivity underlying cortical network. Though gamma deficit would not be indicative of the type of the underlying synaptic connectivity abnormality, it has been suggested that gamma oscillations may be a more sensitive indicator of circuit integrity than MRI measurement, as a 10% decrease in synaptic connectivity is enough to produce a substantial gamma deficit . The bloSource analyses revealed that in schizophrenia patients, the gamma-band oscillations triggered by transcranial magnetic stimulation (TMS) did not propagate beyond the area of stimulation, whereas in controls, it can be found in multiple motor and sensorimotor regions. Patients with schizophrenia also demonstrate reduced amplitude and less synchronization of self-generated, rhythmic activity in several cortical regions. The absence of theta coherence between left frontal and temporal electrodes suggests a failure in the preparation of temporal areas for speech production, which could lead to the misattribution of self-generated speech to an external source . ModificPV interneurons display a fast-spiking pattern of firing, which is not only crucial for temporal control of cortical inhibition but also generates synchronous membrane potential gamma band oscillations (30–80 Hz)  relatingThe EEG results also demonstrate that synchronized oscillatory activity, particularly in the gamma range (30–80 Hz), is abnormal in schizophrenia patients . These oin vivo that, as a result, contributes to cognitive deficits (in vivo brain imaging and post-mortem examination (Myelination of fast-spiking interneurons (FSIs) has crucial roles in regulating the function of FSI. Hence, the defect in FSI myelination likely alters local cortical circuit oscillations deficits  in schizdeficits . Therefodeficits . Moreovedeficits , which ideficits . The latdeficits , which imination . HoweverImaging technique is a non-invasive approach to investigate different brain regions providing an activity-based picture of the brain. Various dysfunctional or hyperactive/hypoactive brain regions serve as the cause of disease, which can be differentiated easily via imaging. 1H-MRS) is a non-invasive approach to identify and quantify certain biochemical compounds in brain tissues (1H-MRS). It is mostly likely that the elevated GABA level in the mPFC could be the overcompensation of a subclass of GABAergic neurons rather than PV interneurons A+/− model with 22q11.2 microdeletions (a highly penetrant genetic variant for schizophrenia) and the chronic ketamine administration model both produced deficits in stimulus-elicited gamma power analogous to those reported in sensory cortices of human SZ patients . InteresTo date, a couple of rodent models have been created to mimic altered GABA levels in human patients. For example, a global GAD1 knockout rat model was recently developed using CRISPR/Cas9 genome editing technology. The rats successfully re-capitulated cognitive impairment and behavioral alterations relevant to negative and positive symptoms of schizophrenia. However, the mutants cannot mimic the physical GAD67 level in patients due to the complete depletion of GAD67 . As oppoDisorder-specific human-induced pluripotent stem cells (hiPSCs), which bear the genetic makeup of donor patient and can differentiate into specific cell types of interest, are one of the tools to study or even to compensate the damages leading to schizophrenia . A compain vivo multicellular calcium imaging, for cells integrated in circuit, in an animal model would help to identify the interaction between GABAergic, glutamatergic, and dopaminergic systems to delineate the landscape of interconnecting mechanistic pathways.To conclude, the reduced neural connectivity, brain volume, and spine density have been reported as commonly agreed endophenotypes of schizophrenia, but the reduction in synaptic function has not yet been studied in detail. In addition, the perturbed information flow among different brain regions is common for many psychiatric disorders, but schizophrenia can be assessed more precisely via transcriptome analysis. Moreover, demyelination of PV interneurons, which in turn reduces gamma oscillations, also promotes the onset of schizophrenia. It remains unclear how to regulate the postnatal maturation and migration of inhibitory neurons, over-pruning of excitatory synapses, pyramidal dendritic remodeling, and deciphering the underpinning mechanisms, which may alleviate the process of or even the onset of schizophrenia. Importantly, studies need to be warranted in the future to target cell type-specific remyelination, synaptogenesis and neurite outgrowth. The literature regarding GABA alterations is heterogeneous in terms of GABA level and also the affected brain areas. Therefore, so far, a commonly agreed mechanism is lacking. Apart from genetic predisposition, perhaps, each type of environmental insult is hitting different areas in the brain. It is speculated that each cell type of GABAergic system probably responds differently to environmental stress. It is also assumed that possibly each type of environmental stress (nature of stress) has different effects on different cell types. Designing animal studies regarding exposure to various environmental insults with various intensities on different times would help to answer the speculations. Moreover, there is a scope for developing schizophrenia animal model with cell-specific demyelination to probe that either cell-specific sensitivity occurs. Furthermore, BL and X-PW conceived the idea. All authors contributed, read and approved the final 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.


Under physio-pathological conditions, cells release membrane-surrounded structures named Extracellular Vesicles (EVs), which convey their molecular cargo to neighboring or distant cells influencing their metabolism. Besides their involvement in the intercellular communication, EVs might represent a tool used by cells to eliminate unnecessary/toxic material. Here, we revised the literature exploring the link between EVs and redox biology. The first proof of this link derives from evidence demonstrating that EVs from healthy cells protect target cells from oxidative insults through the transfer of antioxidants. Oxidative stress conditions influence the release and the molecular cargo of EVs that, in turn, modulate the redox status of target cells. Oxidative stress-related EVs exert both beneficial or harmful effects, as they can carry antioxidants or ROS-generating enzymes and oxidized molecules. As mediators of cell-to-cell communication, EVs are also implicated in the pathophysiology of oxidative stress-related diseases. The review found evidence that numerous studies speculated on the role of EVs in redox signaling and oxidative stress-related pathologies, but few of them unraveled molecular mechanisms behind this complex link. Thus, the purpose of this review is to report and discuss this evidence, highlighting that the analysis of the molecular content of oxidative stress-released EVs (reminiscent of the redox status of originating cells), is a starting point for the use of EVs as diagnostic and therapeutic tools in oxidative stress-related diseases. Extracellular Vesicles (EVs) are a heterogenous group of membranous particles, originating from different cell compartments and processes that are released outside the cell and retrieved in body fluids, as well as in cell culture media. Originally identified during reticulocyte maturation , they weg), enriched in exosomes, and large/medium EVs (l/mEVs) for EVs isolated at lower centrifugal forces  have been recommended. Other separation methods are available, but all of them suffer drawbacks and pitfalls, and none of them assure more than an enrichment on specific EV subtypes , a key event that induces both MV and exosome release by promoting membrane blebbing as well as fusion of MVBs with plasma membrane [Several studies focused on the effect of cell redox status on EV biogenesis mechanisms. Yarana reports in a recent review  the diffmembrane . Doxorubmembrane ,129, a cmembrane . In factmembrane , whereasmembrane ,133. Doxmembrane . Thus, tmembrane ,136.Lipid membrane remodeling seems to be one of the key events responsible for the increased release of EVs from PAHs-treated hepatocytes . PAH treOther membrane rearrangements that modulate membrane fusion and EV release are changes in the level of cell surface protein thiol groups ,124,141.2O2 can induce tolerance to oxidative stress in target cells. Indeed, the pre-treatment with exosomes released under conditions of oxidative stress increases the viability of cells exposed to H2O2, compared to cells pre-treated with exosomes harvested from normal conditions. This protective role seems to be partly mediated by the mRNA exosomal cargo [2O2-treated cells was remarkably different from that of exosomes produced by cells cultured under normal conditions, whereas no differences were observed in the levels of carbonylated proteins [EVs released under oxidative stress can mediate either protective or harmful signals in target cells exposed to the same stress, depending on their specific biochemical cargo. Thus, the knowledge of the molecular cargo of oxidative stress-released EVs could be predictive of the effects that they exert on target cells. Exosomes released by mouse mast cell line (MC/9) exposed to Hal cargo . In factproteins .Interesting information regarding the biochemical composition of oxidative stress-related EVs derives from studies on airway epithelial cells exposed to CSE or cigarette smoke condensate (CSC). One of these studies examined the content of small noncoding RNAs (sncRNAs) in EVs released by human small airway epithelial (SAE) cells treated with CSC . SignifiProteomic analysis of EVs released by airway epithelial cells exposed to CSE confirmed the reliability to predict EV biological effects on the basis of their protein composition . EVs relBiasutto et al.  analyzedIn addition to RNA and proteins, EVs released under oxidative stress conditions convey oxidized lipids generated from peroxidation of cell membrane phospholipids. Lipid peroxidation products disseminated to other cells via EVs seems to mediate relevant biological effects. Endothelial cells exposed to peroxides release MVs containing oxidized phospholipids able to activate neutrophils and monocytes ,148. Con2O2 protect C-kit+ cardiac stem cells against oxidative stress-induced cell death by transferring miR-21 [2O2 present high levels of Nrf2 and its downstream antioxidants  mRNAs, which once internalized by target cells counteract oxidative stress [Another important issue is the capability of EVs to convey antioxidants, thus acting as ROS scavengers, as well as enzymes involved in ROS production . The capg miR-21 . Furthere stress . As preve stress . Exosomee stress . AltogetConversely, EVs can autonomously produce ROS, both in extracellular fluids, or in target cells, after their internalization . Indeed,In the previous section, we reported evidence derived from in-vitro studies performed in different cell lines, where oxidative stress was induced by different oxidants/stressors. To have a more reliable picture regarding the role of EVs in redox-mediated intercellular signaling, in this section we report evidence regarding the role of EVs in the pathogenesis and progression of oxidative stress-related diseases.Oxidative stress is known to be associated with various pathologies . Aging aEvidence points to the involvement of EVs in intercellular communications in senescence and aging . The indAs recently reviewed by Kahroba et al., exosomes containing Nrf2 and Nrf2-induced products can be transferred to recipient cells and activate anti-stress defense mechanisms in recipient cells . Nrf2, aFurther evidence indicates that EVs may be mediators of oxidative stress and inflammation in cardiovascular system, thus contributing to cardiovascular diseases and atherosclerosis. Circulating EVs have been shown to increase significantly in cardiovascular diseases and EVs released by leukocytes, erythrocytes, smooth muscle and endothelial cells strongly accumulate in atherosclerotic plaques, favoring local inflammation ,171. MorOxidative stress also plays a critical role in the pathogenesis of neurodegenerative disorders, as many studies report the presence of high levels of reactive oxidants and an impaired antioxidant defense response in the brain of patients affected by neurodegenerative diseases. Depending on the specific neurodegenerative disease, oxidative stress may be the consequence of a number of factors such as pathological protein aggregation, mitochondrial dysfunction, deregulated antioxidant pathways, specific neurotransmitter metabolism or inflammation . ExosomeExosomes also contain miRNAs and, more specifically, several miRNAs released from exosomes have been associated to neurodegenerative disorders, including AD. For instance, recent studies indicated that exosome-derived miRNAs can modulate the expression of both the amyloid precursor and tau proteins, suggesting that deregulation of exosome-associated miRNAs could be implicated in AD progression ,186. It Oxidative stress is considered one of the most prevalent mechanisms driving neuronal death in Parkinson’s disease (PD), an age-related multifactorial neurodegenerative disorder mainly characterized by dopaminergic neuronal degeneration in the substantia nigra pars compacta. A PD hallmark is the presence in the brain of Lewy bodies that are prevalently formed by the abnormal accumulation and aggregation of misfolded α-synuclein. In PD dopaminergic neurons, oxidative stress is derived from various sources such as mitochondrial dysfunction, dopamine metabolism, impairment of the antioxidant defense mechanisms and α-synuclein aggregation . Recent Oxidative stress plays a complex role in tumorigenesis and cancer development . The altIn addition to chemoresistance, EVs have also been implicated in radioresistance. Radiation therapy is one of the main therapeutic treatments for cancer. The cytotoxicity of ionizing radiation (IR) has been attributed mainly to the production of ROS, which then cause DNA damage and organelles dysfunction . RadioreAlthough the involvement of EVs in the pathophysiology of oxidative stress-related diseases has clearly emerged and many molecules responsible for this correlation, including proteins, transcription factors and noncoding RNAs, have been identified, the molecular mechanisms underlying the complex role of EVs in the development and progression of these diseases have yet to be fully elucidated.In this review, we report evidence showing that EVs play an important role in the cellular response to oxidative stress. The release and the cargo of EVs are markedly affected by redox status of the originating cell. However, no universal biochemical features of EVs released under oxidative stress has been identified, as EV molecular profiles depend on different oxidative stress conditions, on the cell type and on subclass of EVs analyzed. Nevertheless, most of the studies reported in this review reveal that EVs released under oxidative stress conditions are enriched in oxidatively damaged molecules and in transcription factors, RNAs and enzymes regulating the cellular response to oxidative stress. The oxidative stress-related molecular cargo of EVs can be transferred to neighboring or distant cells, thus modulating proliferation and survival, energy metabolism and the response to oxidative stress. The evidence from in-vitro studies is in line with biochemical analyses of circulating EVs from patients with oxidative stress-related diseases, showing that in these pathological situations circulating EVs are carriers of oxidized molecules and/or molecules involved in redox metabolism. The detailed knowledge of the biochemical and biological properties of EVs released under oxidative stress is an important target for therapeutic and diagnostic purposes. The pharmacological induction of EV release might alleviate cellular damage caused by an overloading of oxidized harmful molecules. Conversely, it has been taken into account that the impact that oxidative stress-related EVs could have on target cells that is strictly dependent on EV cargo. Consistently, the detailed information regarding the specific cargo of oxidative stress-related EVs can permit us to predict their effects on target cells and could be useful as diagnostic tools, as oxidative stress is a common molecular mechanism underlying several pathological conditions.


Here, we review the current knowledge on genes and mechanisms critically involved in developmental syndromes and pediatric cancers, revealed by genomic sequencing and in vivo models such as insects, worms and fish. We focus on the monomeric GTPases of the RAS superfamily and their influence on crucial developmental signals and processes. We next discuss the effectiveness of exponentially growing functional assays employing tractable models to identify regulatory crossroads. Unprecedented sophistications are now possible in zebrafish, i.e., genome editing with single-nucleotide precision, nanoimaging, highly resolved recording of multiple small molecules activity, and simultaneous monitoring of brain circuits and complex behavioral response. These assets permit accurate real-time reporting of dynamic small GTPases-controlled processes in entire organisms, owning the potential to tackle rare disease mechanisms.While individually rare, disorders affecting development collectively represent a substantial clinical, psychological, and socioeconomic burden to patients, families, and society. Insights into the molecular mechanisms underlying these disorders are required to speed up diagnosis, improve counseling, and optimize management toward targeted therapies. Genome sequencing is now unveiling previously unexplored genetic variations in undiagnosed patients, which require functional validation and mechanistic understanding, particularly when dealing with novel nosologic entities. Functional perturbations of key regulators acting on signals’ intersections of evolutionarily conserved pathways in these pathological conditions hinder the fine balance between various developmental inputs governing morphogenesis and homeostasis. However, the distinct mechanisms by which these hubs orchestrate pathways to ensure the developmental coordinates are poorly understood. Integrative functional genomics implementing quantitative  Rare diseases are individually uncommon but collectively frequent, affecting approximately 25 million people in Europe and impacting between 263 million and 446 million people worldwide , with a A significant proportion of these disorders underlie one or more genetic alterations causing functional dysregulation of master regulators involved at various levels and stages of complex and dynamic developmental programs  of virtually any growing tissue or organ. Molecularly, a relatively small number of signaling pathways and networks (Wingless/Integrated (Wnt), hedgehog (Hh), Notch, Bone morphogenetic protein (BMP), fibroblast growth factor (FGF), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), etc.) are responsible for directing developmental programs. The crosstalk among these pathways, together with positive and negative control loop stations mediated by highly conserved molecular nodes, accounts for the pleiotropy of signaling, which ultimately shapes organismal development. These pathways’ interplays ensure differential responses to converging – and sometimes conflicting – messages and thereby multiorgan morphogenesis and homeostasis . Therefoin silico wide genome search for disease–gene/pathogenic variants in undiagnosed patients enrolled in international networks and (ii) functional genomics approaches using ad hoc in vitro systems , supported and enhanced by (iii) both vertebrate and invertebrate animal disease models. Along this line, in the past decade, others and we have contributed to decisive advancements in the understanding of the pathophysiological role of a number of small GTPases belonging to the large RAS superfamily. In an international research framework dedicated to undiagnosed patients started at the “Ospedale Pediatrico Bambino Gesù” children’s hospital, functional genomic studies employing WES analysis and complementary in vitro experimental approaches, as well as invertebrate and vertebrate models, have allowed us to identify new nosologic entities caused by mutations affecting several genes, including a subset of which encode small GTPases. For instance, we associated a uniquely behaving genetic alteration in CDC42 [OMIM: 116952] with a severe autoinflammatory condition  and somatically acquired (cancer-associated) mutations in this gene. The expansion of such paradigm in modern biomedical research clearly represents a valid tool for deepening our understanding of healthy and diseased mechanisms as well as core developmental hubs.Following the EU call for action, revolutionary sophistication and rapid implementation of next-generation sequencing (NGS) techniques, especially whole-exome sequencing (WES), have allowed a considerable boost in the identification of genomic modifications and signaling pathways’ alterations in the field of rare diseases. Recently, the National Institutes of Health (NIH)–supported Centers for Mendelian Genomics noted an unprecedented increase in the number of novel diseases discovered per year, estimated to be more than 200 . Clearlyondition . The speondition . Of noteondition , emphasispectrum . Again, in vivo to validate the putative pathogenic variants. Global conservation of the genes exists across taxa such that smartly chosen tractable model systems and ad hoc in vivo tools are and will be crucial for the majority of the newly discovered diseases for which we still fail to understand the impact on the signaling networks during development. In this context, we briefly discuss the advantages in using zebrafish for functional genomics of rare diseases and examine the latest tools, which enable highly resolved in vivo whole-embryo real-time reporting of dynamic small GTPase-regulated processes during development.Here, we review recent functional genomics findings proving mutations in some among the large group of small GTPases molecules to be critically involved in rare developmental syndromes and cancers. We next discuss the current knowledge on the interplay with signaling pathways networks, whose tightly regulated activity is essential in many developmental processes, from lateral inhibition  to cell polarity mechanisms (instructing gastrulation cell movements) and is being implicated in pediatric diseases. The cost reductions in sequencing and the extraordinary progress in functional imaging, signal biosensors optimization, and genome engineering in living organisms are opening long-awaited possibilities to combine in a single workflow (a) analyses directed to identify new disease genes with (b) sophisticated functional approaches in vivo models.The RAS superfamily of small GTPases comprises five main protein families grouped by structure and function. They include proteins belonging to the (i) RAS family, involved in cell proliferation, specification, and differentiation ; (ii) RHSince the identification of the RAS proteins in the 1980s, the biochemistry of their signaling and their role as regulators of multiple cellular processes  in development and homeostasis has been intensively examined . VariousConsidering the plethora of functions of Ras/MAPK signaling during development, it is clear that mutations affecting signaling backbone’s core components have various deleterious effects in terms of development. Indeed, germline mutations affecting different components of signaling cascade are responsible for RASopathies, a group of developmental disorders comprising Noonan syndrome (NS), LEOPARD syndrome (NS with multiple lentigines), Costello syndrome (CS), cardiofaciocutaneous syndrome (CFCS), neurofibromatosis type 1 (NF-1), and other clinically related disorders, displaying high genetic and clinical heterogeneity , the first gene involved in NS and LS   .Drosophila and murine models exist for this condition, which showed the involvement in morphogenesis  directly and possibly their ability to interact with regulators and effectors  to downmodulate Notch-controlled transcriptional output and likely that of other developmental pathways  during corticogenesis  in migrating skeletal precursor cells .shp2 mutant variants in zebrafish embryos, recapitulating human NS and LS traits, indicates a Wnt-dependent effect of Shp2 on embryonic convergence and extension (CE) movements, resembling phenotypes found by downregulating the non-canonical Wnt  mediates the crosstalk between Ras-, Notch-, and Wnt-induced pathways via interacting with disheveled (Dsh/Dvl). This Wnt-dependent mechanism seems to facilitate Ras induction and Notch signaling to finely modulate their relative signal intensities throughout mesoderm specification . SimilarDrosophila, multiple interplays were shown that instruct mesoderm migration and muscle specification  within the RAS superfamily. RAC1, RAC2, RHOA, and CDC42 are classical members regulated by cycling between an active and inactive state via hydrolysis of GTP . AtypicaThe importance of RHO proteins in early developmental schemes is illustrated by the embryo lethality often observed in mutant mice models . As discAt least 30% of neuroblastoma cases are indeed due to mutations that alter RHO and RAC activity , involvein vivo (Rac1 forebrain knockout (KO) mice showing microcephaly further confirmed a function in vertebrate neuronal migration, proliferation, and dendritic arborization  . Developin vivo . Accordiin vivo , and simin vivo , as wellin vivo . Rac1 forization .in vitro and in vivo functional approaches has recently contributed to map and validate a number of de novo mutations in RAC1 in patients affected by a range of developmental defects, including brain malformations , impairing hematological development  was shown in r models . In addir models . On the r models . In thisr models , similarelopment . It is aelopment .Drosophila wing and eye systems, as well as Xenopus embryo models, were useful to demonstrate the necessity of several RHO GTPases in mediating actin cytoskeleton modifications via non-canonical Wnt signaling during development  pathway is emerging also for neuroblastoma  and B-ceogenesis .Drosophila models showed that the modulatory activity on FGF signaling contributes substantially to these processes and ultimately to mesodermal cell motility  axis and neurogenesis , also ARL2 and ARL3 influence MT dynamics and GA stability, as well as protein trafficking to the cilium, with a major impact on photoreceptor development in mice . Accordi KO mice , and mut elegans , with anLastly, evidence for a crosstalk between ARF1 and actin–cytoskeleton regulators of RHO family was also shown. ARF1-mediated assembly of COPI complex is crucial for recruitment of CDC42 to the GA and for the local activation of N-WASP, Arp2/3, and actin polymerization, necessary to promote vesicle formation and scission . An ARF1Acting on shaping developmental signals strength and distribution and regulating cell behavior, ARF and RAB-controlled biosynthetic trafficking globally modulates morphogens’ distribution and function in developmental programs. The regulated process of polarized vesicular transport of morphogenes guarantees coordinated cell–cell signals and movements during development, ultimately leading to cell specification and organogenesis . Indeed,Here, we examine established and mounting evidence on the direct action of ARF and RAB-mediated intracellular trafficking on crucial developmental pathways, relevant for morphogenesis and disease. In general, these proteins have an active role in intracellular trafficking and cytoskeleton reorganization in cilia formation, directly regulating cilia development , their gDespite poor mechanistic understanding, a clear contribution of ARF function in the modulation of both canonical and non-canonical Wnt signaling is emerging to be directly relevant for pathology.Many developmental processes including proliferation and differentiation require controlled ARF-dependent biosynthetic pathways for establishing cell polarity. This was demonstrated in several developmental models and timings, i.e., gastrulation events , dendritARF1 result in a complex neurodevelopmental condition called “periventricular nodular heterotopia.” This neuronal migration disorder is characterized by microcephaly with brain malformations and progressive cerebral atrophy and spasticity  within a single precursor cell, which guarantees correct morphogenesis. Furthermore, although the exact mechanism remains unproven in vertebrates, suggestive of the decisive impact on complex vertebrate embryogenesis events, constitutively active Arf1 (obtained by overexpressing the human variants) results in typical PCP-dependent phenotypes in zebrafish, i.e., body shortening and morphological alterations of the AP axis, likely caused by perturbed gastrulation cell movements  and the complex and pleiotropic ciliopathy Bardet–Biedl syndrome showing polarity defects, associated with ARL6  P2 , which anstrated . An invonstrated  caused bith ARL6 . Mutatioith ARL6 .in vitro data, a lack of Rab8b was found to block Wnt signaling during fish development  is involved in this morphogenetic process , as wellormation . On the ignaling . The exaARL13b are lined to altered Shh signaling and underlie Joubert syndrome, a condition causing midbrain–hindbrain developmental abnormalities and various other defects typical of impaired Shh signaling , while discovering novel mechanisms on multiple developmental contexts and tissues simultaneously. The utility of animal models also relies on the possibility of setting up preclinical systems for assessing potential targeted treatments, identifying development and physiology principles that can uncover evolutionary rules. Moreover, sophisticated xenografting in vivo models enable innovative studies of cancer cells’ heterogeneity  emitting molecules that improve the light penetration in deep tissues with little scattering  or strategies for stable modifications  barcoding strategies  method using Kaede under the control of nal cord . Moreovenal cord .in vivo  sensor has been established and successfully employed in zebrafish . Dynamicin vivo, with considerable applications for understanding of RASopathies.in vivo in the future.In parallel, other smart methods to visualize more dynamics are being developed , which win vivo demonstrated the sensitivity of early Drosophila blastula and gastrula stages to ectopic Erk expression for axial morphogenesis  domain is masked and cannot bind its receptor SsrB fused to a fluorescently tag SOS activator in the dark. The binding can be, however, triggered by blue light allowing strict spatiotemporal control of SOS membrane recruitment and thereby Ras/MAPK signaling activation. The use of this tool ogenesis . Furtherogenesis . Furtherogenesis .Drosophila and zebrafish  to manipulate the Ras/MAPK signaling using microscopy and Dronpa-based photo-dimerizable protein domains, already applicable to ebrafish . In the rocesses .in vivo  have been already used in combination to specific Cdc42 FRET sensor to assess the function of this small GTPases during angiogenesis . The system is expandable to a range of mutants and allows systematic functional investigation of RHO protein–specific cells and developmental windows of interest and possible crosstalks in fish cancer models  (UAS:EGFP-tuba2) ((XlEef1a1:dclk2DeltaK-GFP), the Eb3 + growing tip of the MT (bactin2:HsENSCONSIN17–282-3xEGFP) fish expressing GFP-tagged MT-binding region of ensconsin  are successfully used for dynamic analysis of recycling endosomes in vivo , as wellact-GFP)  and a nuP-tuba2) , the MT-f the MT , and Tg nsconsin .in vivo whole-embryo reporter and demonstrated the dynamics of formation, transport, and function and the trophic role of the EV secreted by cells of yolk syncytial layer during development. In addition, the FRET type of RAB biosensors also exists and has been developed and used to monitor, for instance, Rab5 activity in phagosome maturation of immune cells  awaiting in vivo applications, allowing direct observation of GTPase activation state, were recently used to detect RAB1 and K-RAS activity in vitro and quantification of RAB1 GTP-/GDP ratio at high spatial and temporal resolution . The tool was already implemented in rodent hippocampal neurons to study the contribution of RAB5 and RAB11 in influencing dendritic growth’s rate.The development of optogenetic devices to manipulate intracellular trafficking has been notoriously more challenging. Nevertheless, plant photoreceptors responsive to UV light have been genetically engineered for controlling protein secretion and used to investigate dendritic cargo secretion . More rein vivo studies are now unfolding, well exemplified by the zebrafish model. We anticipate that the expansion and optimization of these tools for multiplexing in vivo signal visualization and manipulations will have an unprecedented impact for the spatiotemporal investigation of developmental signaling networks modulated by small GTPases in health and disease. In conclusion, an integrated pipeline from patients back to precise organismal biology in the global context of embryo development represents the blueprint for a modern global health care response to the burden of the ever-increasing pediatric genetic diseases, critical for developing tailored measures in the rapidly emerging field of precision medicine.Next-generation sequencing of previously unrecognized pediatric conditions disclosed an unforeseen impact of small GTPases of the RAS superfamily on the pathogenicity of a growing number of developmental disorders. The precision of this approach is bringing up amazing possibilities for investigating unexplored mechanistic principles of developmental biology, re-employing classical animal models. Continuous advances in the field of high-resolution microscopy, genetic engineering, and synthetic biology for optimized biosensors and actuators for AL, GF, MT, and BD wrote the manuscript. AL conceived and generated all the illustrations. AL and GF conceived the table. GF and MV generated the table. All authors contributed to the article and approved the submitted version.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.