Based on the known responsiveness of TSPO expression to a wide range of noxious stimuli, we discuss TSPO as a potential biomarker of radiation-induced effects.Responses of the central nervous system (CNS) to stressors and injuries, such as ionising radiation, are modulated by the concomitant responses of the brains innate immune effector cells, microglia. Exposure to high doses of ionising radiation in brain tissue leads to the expression and release of biochemical mediators of ‘neuroinflammation’, such as pro-inflammatory cytokines and reactive oxygen species (ROS), leading to tissue destruction. Contrastingly, low dose ionising radiation may reduce vulnerability to subsequent exposure of ionising radiation, largely through the stimulation of adaptive responses, such as antioxidant defences. These disparate responses may be reflective of non-linear differential microglial activation at low and high doses, manifesting as an anti-inflammatory or pro-inflammatory functional state. Biomarkers of pathology in the brain, such as the mitochondrial Translocator Protein 18 kDa (TSPO), have facilitated   •Ionising radiation can modulate responses of microglial cells in the CNS.•High doses can induce ROS formation, oxidative stress and neuroinflammation.•Low doses can mitigate tissue damage via antioxidant defences.•TSPO as a potential biomarker and modulator of radiation induced effects in the CNS.•Non-linear differential microglial activation to high and low doses is proposed. This review aims to synthesise the important aspects of CNS functionality under conditions of stress, injury and pathology, which can elucidate the neurobiological responses to ionising radiation at different doses. In order to enhance understanding in this field and dissect the subcellular and molecular events that drive radiation-induced neurobiological responses, a key biomarker of CNS pathology, the mitochondrial Translocator Protein 18 kDa (TSPO), will be examined. We introduce TSPO as a novel perspective in clarifying the responses of the CNS to ionising radiation, and highlight its centrality to a comprehensive understanding of the complex network linking neuroinflammation and mitochondrial redox balance. Its utility as a sensitive 2The coordination of responses to insults and stressors in the CNS are complex and multifaceted. Neurobiological mechanisms of inflammation, protection, defence and repair comprise of networks of cells and molecular mediators that respond to alterations in homeostasis 2O2 from NADPH oxidases, which also generate large amounts of oxidants in activated phagocytic cells Importantly, classically activated M1 microglia and its associated pro-inflammatory functions are intrinsically linked to the production of free radicals 3One of the earliest physiological responses to ionising radiation is the production of ROS The presence of cognitive decline in patients after clinical exposure to high dose irradiation has led to a research focus on the hippocampal microenvironment and its population of mature cells and proliferating progenitor cells in the subgranular zone of the dentate gyrus. This region of neurogenesis is particularly sensitive to ionising radiation A search using the SCOPUS database for ‘ionising radiation’ and ‘microglia’ yields a steady increase in publication numbers since the earliest recorded data by Estable-Puig et al. in 1964 refer to . Such waThe induction of pro-inflammatory factors can occur through pronounced activation of transcription factors such as NF-κb, causing aberrant regulation of genes involved in pathological inflammatory states Oxidative stress and redox balance play critical roles in the responses of cells to higher doses of ionising radiation in vitro and in vivo methodologies used in studies, it is apparent that responses of the CNS to higher doses of ionising radiation are underscored by excess ROS production, contributing to oxidative stress related damage and neuroinflammation. It is interesting to note that many of the studies examined here, and indeed throughout the literature in general, apply a single dose irradiation to tissue and examine the effects thereafter  utilised a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s Disease to demonstrate acute increases in the levels of glutathione and catalase 3 h after exposure to a single dose of 0.5 Gy gamma radiation, with concomitant neutralisation of lipid peroxidation SOD2 and SOD activity facilitated through the NF-κB and SOD signalling network persisted 8 days after exposure. Notably, antioxidant upregulation was not present after exposure to 0.02 Gy, suggesting that the mitochondrial machinery behind this response requires sufficient exogenous stimulation by low dose ionising radiation Antioxidative mechanisms are evidently implicated in the neuroprotective and reparative responses that low dose ionising radiation can confer in vivo and in vitro methodology, all of which are varied across the majority of studies. Dose fractionation, protraction or acute single dose delivery can all manifest different neurobiological outcomes, from the molecular to the cellular level, which needs to be considered in future studies. Despite the heterogeneity in experimental conditions throughout the literature, it is clear that a salient trend across the radiobiological data points to a protective and adaptive effect of low dose ionising radiation. In order to further characterise CNS responses to low dose irradiation, and address the remaining uncertainties, a direct examination of microglial responses, as well as the machinery driving antioxidant stimulation, will need to be determined. The potential induction of an M2 microglial activation state following low dose irradiation, which can down-regulate inflammatory responses and induce reparative mechanisms in response to injury, should also be explored in future studies. This may also delineate the phenotypes of microglia in response to varying stimuli in the CNS.There is a growing body of evidence to suggest that low dose ionising radiation exposure to the CNS produces responses that are consistent with the radiation hormesis model, though inconsistencies in the literature emphasise the need for further research in this field. Recently, Katsura et al. 5in vivo neuroimaging markers, such as the mitochondrial Translocator Protein 18 kDa (TSPO), which has garnered considerable attention An understanding of the mitochondrial mechanisms that drive oxidant/antioxidant production and microglial activation after irradiation remains elusive. Given the remaining uncertainties surrounding the susceptibility of the CNS to ionising radiation at different doses, new research modalities and approaches to the study of mitochondrial biology are needed. Enhanced understanding of the interdependence between mitochondrial mechanisms and neuroinflammation has been facilitated through sensitive in vivo neuroimaging techniques such as Positron Emission Tomography (PET) have implicated TSPO in multifarious disease states of the nervous system, including neurodegenerative disorders Interestingly, in the CNS, TSPO expression is quiescent under normal physiological conditions, yet is predominantly, if not exclusively, upregulated in activated microglia under conditions of stress or pathology oF1-ATP(synth)ase, leading to the activation of caspase-3, collapse of mitochondrial membrane potential, and the induction of apoptosis Tspo homolog in Drosophila decreased caspase 3/7 activity and inhibited apoptosis triggered by 30 Gy gamma radiation and H2O2 exposure. Moreover, Aβ42-induced neurodegeneration was mitigated after Tspo depletion and male lifespan increased in diseased flies, providing novel evidence of the role of TSPO after radiation exposure and oxidative stress Functional characterisation of this protein within mitochondria remains elusive. Furthermore, the mechanisms by which TSPO contributes to microglial activation remains a topic of great interest, and enhanced understanding may be useful for uncovering novel insights into responses after ionising radiation exposure. Currently, TSPO deletion in mice has uncovered the role of TSPO in mitochondrial bioenergetics, as TSPO deficient mice display significant decreases in oxygen consumption Other putative links between TSPO and ROS formation involve protoporphyrin IX (PPIX), which promotes cell death through ROS formation in complex with TSPO in vivo neuroimaging of TSPO using high affinity ligands, will facilitate new approaches to research in this field. Not only will this enhance understanding of the responses of the CNS to ionising radiation, it will also clarify the enigmatic role of TSPO in cell functioning.Further research directed in this field may lead to novel insights into the postulated link between TPSO, ROS and neuroinflammation in the coordination of responses to ionising radiation refer to . As ther6The complex responses of the CNS under conditions of stress or pathology are largely underscored by neuroinflammation and mitochondrial redox balance. These mechanisms also coordinate responses to high and low dose ionising radiation. Exposure to high doses can impact the functionality of the CNS by inducing ROS formation, oxidative stress, alterations in DNA and gene expression, and activation of a neuroinflammatory response, all of which can propagate cellular damage. Whilst there is greater uncertainty surrounding responses to low dose irradiation, considerable evidence suggests that low dose exposure can manifest repair and protection to cells. Though there is sizeable literature challenging the current paradigm of the linear no-threshold model, the remaining contention and uncertainty surrounding low dose effects necessitates further studies which directly examine the mitochondrial mechanisms that stimulate antioxidant defences, as well as directly characterising the responses of microglia after low dose irradiation. This may also aid in investigating the dichotomous phenotypes of microglial activation, transitioning between M1/M2 activation states in response to low or high doses. Due to the ‘anti-inflammatory’ effects of low dose irradiation, it can be postulated that low dose ionising radiation may produce an M2 type microglial activation, which contributes to the reparative and protective effects that are present in many studies. This would create a ‘non-linear differential threshold’ of microglial activation in response to ionising radiation, transitioning between anti-inflammatory and pro-inflammatory functional states.in vitro/in vivo experimental conditions. As mentioned prior, many studies focus on single dose irradiation schemes and measure acute cellular responses. In many cases, it has been reported that the outcomes of single dose exposure cannot predict or extend to outcomes after fractionated delivery, which produce disparate responses. Concomitantly, the effects of protracted/chronic dose delivery over a period time have different effects on cells, thus, further studies are needed in order to extrapolate radiation effects as a function of how doses are delivered. This may also be beneficial for studying the different responses of microglia to varying stimuli at varying doses and intensities, and may elucidate the cellular mechanisms that produce radioadaptive responses subsequent to priming with low dose irradiation.Collectively, animal radiobiological studies on the effects of high and low dose ionising radiation on the CNS highlight the variability in responses. It is most likely that responses are contingent not just on total dose, but dose rates, schedule of delivery and In order to advance understanding of the mechanisms of radiation responses in the CNS, biomarkers of pathology may be of high utility moving forward in research. More comprehensive studies that exploit the exclusive upregulation of TSPO in activated microglia under conditions of stress in the brain will aid in explicating the role of neuroinflammatory responses after ionising radiation exposure, whilst also clarifying the exact function of TSPO and its role in cellular functioning. As more evidence emerges regarding the role of TSPO in modulating ROS, it may by extension lead to new insights into the role of TSPO in coordinating responses after ionising radiation that should be addressed in further studies. Furthermore, the controversial nature of low dose irradiation in conferring neuroprotection and the current lack of understanding of this phenomenon necessitate further investigation which will benefit from utilising TSPO, particularly its role in mitochondrial dependent processes. Future studies that elucidate the beneficial impact of low dose ionising radiation may ultimately uncover therapeutic potential in the prevention and treatment of sub-syndromal disorders associated with neuroinflammation, or may ameliorate the deleterious effects of neurodegenerative disorders. They may also pave the way for different approaches to setting radiation protection standards and regulatory bodies in making informed decisions, as well as a reassessment of the validity of the linear no-threshold model.


It has been proposed that synchronous and convergent afferent input arising from repetitive motor tasks may play an important role in driving the maladaptive cortical plasticity seen in focal hand dystonia (FHD). This hypothesis receives support from several sources. First, it has been reported that in subjects with FHD, paired associative stimulation produces an abnormal increase in corticospinal excitability, which was not confined to stimulated muscles. These findings provide support for the role of excessive plasticity in FHD. Second, the genetic contribution to the dystonias is increasingly recognized indicating that repetitive, stereotyped afferent inputs may lead to late-onset dystonia, such as FHD, more rapidly in genetically susceptible individuals. It can be postulated, according to the two factor hypothesis that dystonia is triggered and maintained by the concurrence of environmental factors such as repetitive training and subtle abnormal mechanisms of plasticity within somatosensory loop. In the present review, we examine the contribution of sensory-motor integration in the pathophysiology of primary dystonia. In addition, we will discuss the role of non-invasive brain stimulation as therapeutic approach in FHD. Task-specific focal dystonia is characterized by excessive and inappropriate muscle activation during highly skilled fine motor tasks, resulting in slow, clumsy movements, and impaired task performance. The condition typically affects highly trained, stereotypical movement patterns, such as writing (writer’s cramp) or playing a musical instrument (musician’s dystonia) Byl, . DespiteIn the present paper, we would like to review sensory abnormalities in dystonia and to explore how these informations can be exploited for new therapeutic strategies employing non-invasive brain stimulation (NIBS).The integration of inhibitory, mainly proprioceptive inputs from adjacent body parts is impaired in dystonia both at cortical and spinal level. Reciprocal inhibition is the central nervous system process in which a muscle is inhibited when its antagonist is activated. It has several components and the second longer phase of reciprocal inhibition, tested at the spinal level, is absent in patients with focal hand dystonia (FHD)  over primary motor cortex may normalize intracortical inhibition and induce a mild improvement in motor performance (Siebner et al., Despite NIBS represents a promising therapeutic tool in dystonia, the overall improvements so far have not been sustained in all studies. This is not surprising since as we discussed in the previous sections enhanced plasticity causes inappropriate association between sensorimotor inputs and outputs. In this case, a short session of NIBS might have little immediate effect on dystonic movements, because bad motor memories have already been “learned,” and takes time to be erased (Quartarone and Hallett, 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.


In ecology, biodiversity-ecosystem functioning (BEF) research has seen a shift in perspective from taxonomy to function in the last two decades, with successful application of trait-based approaches. This shift offers opportunities for a deeper mechanistic understanding of the role of biodiversity in maintaining multiple ecosystem processes and services. In this paper, we highlight studies that have focused on BEF of microbial communities with an emphasis on integrating trait-based approaches to microbial ecology. In doing so, we explore some of the inherent challenges and opportunities of understanding BEF using microbial systems. For example, microbial biologists characterize communities using gene phylogenies that are often unable to resolve functional traits. Additionally, experimental designs of existing microbial BEF studies are often inadequate to unravel BEF relationships. We argue that combining eco-physiological studies with contemporary molecular tools in a trait-based framework can reinforce our ability to link microbial diversity to ecosystem processes. We conclude that such trait-based approaches are a promising framework to increase the understanding of microbial BEF relationships and thus generating systematic principles in microbial ecology and more generally ecology. The relationship between biodiversity and ecosystem functioning (BEF) Table  is complBiodiversity effects on ecosystem functioning mainly arise from niche-related mechanisms that shape interactions of the biological units  that vary genetically and in the expressed functional traits  in published datasets in various environmental gradients  phylogenetic relatedness or functional gene/protein sequence relatedness . Community mean traits are not useful for this purpose, since the information about effects of the local trait diversity  will be lost by averaging.The analysis of metabolic processes offers great potential to evaluate aggregated trait values at the community scale. Functional traits can be assessed by high-throughput assays, such as Biolog or Ecoplates. These cultivation-based metabolic assays can be used to characterize the community capacity to oxidize a range of C sources Garland,  or to meWe see some advantage for microbial studies correlating community-mean traits to functional capabilities of the community as a whole, since these are more easily measurable than for higher organisms. Indeed, aggregated trait values would boil down to a metric sizing of “meta-species,” which can illuminate responses along environmental gradients and possible effects on ecosystem functioning. A drawback is that the combination of traits of all microbial individuals that compose the community can hardly be characterized.In situ enrichments  and other incubation methods can be used to determine cell-specific metabolic rates, even at extremely low rates  to reinforce experimentally-sound studies of the role of microbial (trait-based) diversity on ecosystem functioning, and (ii) to promote efforts for measuring and archiving microbial traits in a way suitable for the highly diverse and dynamic microbial communities that make up the biosphere.The first priority arises from the current paucity of microbial ecology in terms of BEF studies that directly manipulate diversity using a trait-based approach. While assembled communities clearly differ from complex communities from natural environments, this does not diminish the value and potential of such studies to disentangle the possible key mechanisms underlying BEF relationships.Concerning the second priority, we call for more innovative physiological studies in order to measure traits and their relevant unit . More specifically, by measuring traits in a standardized manner, e.g., incubation condition and media, and by applying analogous tests also to organisms we cannot get in pure culture, we may be able to reveal important trait distributions and generate a microbial trait database similar to, e.g., the TRY global traits initiative for plants (Kattge et al., Microbial ecologists can provide new insights and concepts to trait-based BEF studies, according to the particularities of microbial systems and the tools available in microbial ecology. For instance, BEF studies over many microbial generations allow researchers to reveal the effect of eco-evolutionary feedbacks on BEF relationships over reasonable time scales. Also, accounting for spatial and temporal niche variability as well as assessing the role of diversity in multiple related ecosystem functions, microbial trait-based approaches may deliver mechanistic insights in areas practically not feasible in higher organisms, thus providing benefits to ecology as a whole, which is still a major challenge for microbial ecologists (Prosser et al., 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.


Cancer immunotherapy has a long history in the clinic. William Coley was an orthopedic surgeon in the late 1800’s who noted marked tumor regression in a patient with severe erysipelas. Based on this observation, he hypothesized that stimulating the immune system could effectively treat cancer. Thus, over the next decades he gave various bacterial products, including live bacteria, to patients with advanced cancers [In contrast to his relatively unsystematic approach, drug development has historically followed a carefully delineated pathway of sequential trials that systematically generate data characterizing the safety profile and clinical activity of a therapeutic agent in increasing numbers of patients . These tThe need for rapid and efficient cancer drug development has never been greater. The sequencing of cancer genomes has identified multiple rearranged or mutated targets that are druggable, ushering in the era of precision medicine. Concomitantly, advances in our knowledge of the molecular and cellular basis of the antitumor immune response has driven the development of multiple single-agent cancer immunotherapies. These agents target nonmalignant immune cells, and have often shown striking clinical activity across multiple tumor histologies. Importantly, the activities of targeted therapies and immunotherapies frequently intersect, with molecularly targeted therapies often promoting the antitumor immune response. Moreover, the activity of both precision medicines targeting genomic aberrations in tumor cells and immunotherapies targeting the antitumor immune response is maximized by the development of companion diagnostics that identify patients most likely to benefit from them. This may require the development of a companion diagnostic through a parallel regulatory pathway, or may be done simultaneously in registration clinical trials. Given the array of available targets, drugs, and biomarkers of response and resistance to therapy it is clear that traditional approaches to drug development are unable to support the speed and level of sophistication required for the development of effective cancer therapies today.New trial designs more effectively and efficiently tie the biologic activity of new agents to clinical activity and are more appropriate in today’s landscape than the traditional phased approach based on associations between toxicity and clinical activity . PlatforStrategies for meeting the challenges and mitigating the possible risks of this strategy must be identified and developed with the input from all stakeholders. This is particularly true for combination cancer immunotherapies that integrate immuno-oncology agents with standard chemotherapy or radiation, molecularly targeted therapies, or other immuno-oncology agents. These combination immunotherapy strategies are rapidly entering the clinic. Importantly, they have the potential for therapeutic synergy, but also a significant risk of enhanced and unexpected toxicities. The optimal integration of modern translational, clinical, and regulatory science is essential for delivering new single agent and combination cancer immunotherapies to patients as safely and efficiently as possible.The clinical success of immune checkpoint antagonists targeting the PD-1 pathway in multiple tumor types provides clear evidence that harnessing the immune system can lead to durable tumor regressions in cancer patients. As a result, enthusiasm for cancer immunotherapy has never been higher, and complex trials of single agent and combination cancer immunotherapies are entering the clinic at a dizzying pace. George Santayana once said, “Those who fail to learn from history are doomed to repeat it.” This may be true for the prior failures and risks of novel cancer immunotherapies. We also argue that those who fail to learn from history will not develop better ways of moving forward. We should apply lessons from our successes to create the most effective pathway for developing cancer immunotherapies. Here we present several case studies in cancer immunotherapy drug development to highlight important lessons from the past. These lessons illustrate  innovative, rapid clinical development pathways for novel immunotherapy agents with clinical benefit highlight the need for appropriate cautions to detect and manage potential adverse events early in the drug development process.The clinical development of pembrolizumab is an example of a modern, seamless drug development strategy that facilitates the rapid testing and approval of a novel, highly promising agent under the umbrella of a single clinical trial , 3. The Similar to pembrolizumab, the clinical development path of nivolumab, another PD-1 inhibitor, was also shortened. Ultimately, accelerated approval in unresectable or advanced melanoma was granted in December 2014 based on clinical efficacy data derived from a subset of 120 patients enrolled on a randomized Phase 3 clinical trial that screened 631 patients at 90 sites across the world [T-VEC, a genetically-modified oncolytic herpesvirus encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), is the first oncolytic virus to demonstrate clinical benefit in a prospective, randomized phase III clinical trial . T-VEC hThe clinical application of agents that target regulatory molecules within the immunologic synapse began well before the current era of immune checkpoint modulation targeting the CTLA-4 and PD-1 pathways (among others). TeGenero developed a first-in-class humanized superagonist monoclonal antibody specific for CD28, TGN1412 . CD28 isEM) were the source of cytokines  resulting in CRS. Because the accumulation of TEM is driven by repeated exposure to infectious agents, it does not occur in rodents housed in clean rooms. Thus, the relative TEM/Treg ratio in laboratory rodents and in humans differs, and favors immune hyperstimulation in patients. Moreover, low dose superagonist CD28 only expands Tregs, whereas doses at the level used in the trial expands both TEM and Tregs. Thus, the dose selected for study compounded the risks associated with the biological difference between rodents and humans. Second, TGN1412 had been given to cynomolgus macaques at 500-fold higher doses than to the human volunteers without adverse effect. Human and macaque CD28 has the same sequence and binds TGN1412 with the same affinity. However, macaque CD4+ T cells, but not human CD4+ T cells, lose CD28 expression as they differentiate into TEM cells. This difference was overlooked during primate testing. Finally, treating human peripheral blood mononuclear cells with TGN1412 did not induce cytokine release or proliferation. Subsequent investigations showed that when PBMC were “pre-conditioned” at high density to facilitate cell-cell contact mimicking tissue residence, the sub-threshold, tonic level of T cell activation required for the activity of TGN1412 that T cells lose during trafficking through the circulation was restored. Pre-culturing PBMC under high density conditions suggests that if TGN1412 had been given at the minimum anticipated biological effect level (MABEL), the first dose for the trial would have been 200-fold lower. This trial taught us that “the absence of evidence” is not the same as “evidence of absence” for drug-related toxicity [In preclinical models, CD28 superagonists result in polyclonal T cell activation that is rapidly counter-regulated by the expansion and activation of regulatory T cells (Treg) to prevent over-exuberant immune activation. Several peculiarities of the preclinical data lend insight into what happened. First, in the healthy volunteers, tissue-resident CD4+ effector memory T cells  cell therapy is a highly active cancer immunotherapy that yields dramatic tumor regressions. CAR-T cells are T cells engineered to express an artificial antigen receptor derived from the variable region of an antigen-specific antibody and linked to signaling components of the T cell receptor and co-stimulatory molecules for T cell activation. Like TGN1412, CAR-T cell therapy can result in massive T cell expansion in vivo, leading to CRS . CAR-T cListeria monocytogenes-based bacterial vaccine targeting human papilloma virus (HPV) has been tested in patients. In one clinical trial, a patient with cervical cancer was treated on study, with the last dose given in early 2013. In July 2015 the patient was hospitalized, and tests discovered the engineered Listeria bacteria in her bloodstream. The patient died in August 2015. The death was attributed to disease progression, but the FDA put four clinical trials testing axalimogene filolisbac on clinical hold in October 2015. The presence of axalimogene filolisbac in the bloodstream of this patient was ultimately attributed to colonization of the biofilm overlying an implanted medical device, seeding the device for re-emergence of the agent at a later date. Based on evaluation of the event, the protocol inclusion/exclusion criteria were modified to exclude patients with indwelling medical devices that are a high risk for biofilm colonization and cannot be easily removed. Study procedures were modified to require the strict use of a dedicated indwelling line for administration of the agent, a 6-month course of antibiotics after the last dose of axalimogene filolsibac was implemented, and additional patient surveillance and longer term monitoring measures were put in place. The clinical hold was lifted in December 2015.A The accelerated approvals described above illustrate how the timeline of drug development can be compressed using innovative adaptive clinical trial designs that enroll large numbers of patients in early trials coupled effectively with ongoing consultation with regulatory authorities. In large part due to these triumphs of drug development, there are now dozens of clinical trials testing novel immunotherapies alone and in combination with other therapies for the first time. This may include the first in human testing of two novel immunotherapy agents with the aim of testing them in combination under the umbrella of a single protocol. The trials often grow to include sites that span the globe with investigators who may have relatively limited experience in cancer immunotherapy. The vignettes above also highlight the importance of early detection of potential safety issues, the need to implement effective communication strategies amongst the study team, and the importance of efficiently implementing adverse event management guidelines into clinical trials as new data is accumulated. The progress we have made and the number of immunotherapy agents entering the pipeline thus poses challenges in how to prioritize them, study them most safely and effectively, and develop the most rational immunotherapy combinations for clinical testing. Here, we discuss these challenges and propose some strategies for meeting them.Advances in tumor immunotherapy have been dramatic over the last five years. We now understand that the kinetics of immune-mediated anti-tumor activity may be delayed compared to that of cytotoxic chemotherapy or targeted therapy. Immunotherapy mediates tumor regression indirectly through activation of immune responses and/or inhibition of suppressive immune elements. This may result in delayed tumor regression, with some patients even experiencing the progression of existing disease or the appearance of new lesions prior to eventual disease regression. Furthermore, many immunotherapy agents do not impact progression-free survival, but are associated with significant improvements in overall survival . StandarThe use of alternative clinical endpoints for immunotherapy has already been widely discussed. First, the atypical response patterns of cancer immunotherapy led to the development of the immune-related response criteria (irRECIST), which captures disease in bidimensional measurements and allows for continued treatment in the face of clinically insignificant disease progression . Immune-The selection of patients for cancer immunotherapy trials is key to their success. Four considerations are relevant to cancer immunotherapy trials today. First, in the past most trials enrolled patients who were immunotherapy naïve. As these trials became more widespread, newer trials started to exclude patients who had received prior cancer immunotherapies. Given the growth of the field and recent marketing approvals, this is likely no longer feasible as a matter of course, and patients previously treated with one agent may respond to re-challenge with the same agent, or to another drug. Thus, the exclusion of patients who have received prior immunotherapy should be based on a strong scientific rationale, or immunotherapy naïve and experienced patients could be studied in separate study cohorts. Second, after initial reports that PD-L1 expression within the tumor microenvironment had potential as a predictive biomarker, many sponsors defined eligibility for trials of PD-1 or PD-L1 antagonists based on expression of PD-L1 within the tumor using a pre-defined assay. Selection for intratumoral PD-L1 expression does appear to enrich for responses, and likely helped to ferret out clinical signals in early studies. However, PD-L1 expression is not a perfect predictive biomarker. Four Phase 3 clinical trials in melanoma, NSCLC, and RCC have evaluated PD-L1 expression as a predictive biomarker of response to therapy with PD-1 pathway antagonists –28. In tThe rapid growth and sheer size of these trials present challenges to communication, ensuring that investigators, sponsors, contract research organizations, investigational review boards and regulatory authorities remain up-to-date on the current data and modifications to the protocol, that patients are fully informed, and that patient safety and data quality are maximized. As trials are modified, the rationale, supporting data, and hypotheses to be tested in the amendment must be clearly defined. This could include evaluating additional tumor types for signals of clinical activity, modified drug doses or schedules, and/or predictive biomarkers of response or resistance (among other questions). The sample size justification and statistical analysis plan should be described, and early stopping rules for futility and/or toxicity clearly defined. Study participants should be kept informed of new developments in the study that warrant major changes to the study design through updated informed consent documents. Scientific review committees, institutional review boards, and the appropriate regulatory authorities should be notified of major changes to the study design and sample size/patient cohorts being evaluated. A plan for patient follow up should also be clearly delineated to collect late safety and efficacy signals.In cancer immunotherapy trials, unexpected (and expected) toxicities can quickly emerge, requiring an informed, early, and nimble response from the investigator for maximal patient safety. These events may also provide a limited window for evaluating patient samples that can elucidate the pathophysiology of an unexpected or rare event. Effective real-time communication between the study investigators and the sponsor will facilitate dissecting the science in the patient to inform the development of the most effective therapy for a specific adverse event (such as the use of tocilizumab for CRS). Additionally, the atypical clinical response patterns that may be associated with cancer immunotherapy can be tricky to manage for investigators, and difficult for patients to understand. For investigators with less experience in immunotherapy, having defined management algorithms and an infrastructure to fall back on to guide both toxicity management and the management of atypical clinical responses is critical. A defined communications strategy for cascading information about important adverse events (expected or unexpected) as well as atypical response patterns should be implemented early in the study. Regularly scheduled “state of the study” calls have proven very useful to make sure all stakeholders are optimally informed. The communications between the sponsor and investigators across multiple sites that occur on these calls are highly valuable. Monthly trial newsletters and toxicity management hotlines are other strategies that have been used to inform and support study investigators faced with serious immune-related toxicities new to them.An excellent, well-defined communications plan including all trial stakeholders is the first step to rigorous trial oversight. A steering committee that includes study investigators should also be considered to provide critical advice and insights from individuals with expertise in the field and “on-the-ground” experience with the study, particularly for new agents or novel immunotherapy combinations. Once a study incorporates advanced design features  and/or matures sufficiently that it could support a registration for marketing approval, it may be appropriate to consider an oversight committee that includes external experts with subject matter expertise who are not directly involved in the study. These individuals can provide an objective voice in monitoring the data and the evolution of the study design to ensure that the safety and clinical activity signals seen are considered appropriately across all disease cohorts, and the clinical science is maintained at the highest level of quality. These strategies are similar to historic data safety monitoring boards but demand more frequent updates and interaction among all stakeholders.Recent advances in cancer immunotherapy clearly illustrate virtually unprecedented long-term clinical benefit in some advanced cancer patients, and the potential for cure seems tangible. In lock step with transforming patient care, we have also already begun to revolutionize the drug development enterprise. Given the sheer number of investigational agents and potential combination cancer immunotherapies before us, the opportunity to make a major impact on the world’s cancer burden and on patient’s lives has never been greater. This is an exciting time in the field, and it is critical to expedite the development of new cancer immunotherapies. It is also imperative to remain vigilant in order to detect safety signals early, and incorporate adverse event management procedures rapidly to prevent avoidable toxicity. We should continue to re-structure and adapt the system to fit the opportunities before us, working as a team across the drug development enterprise to safely deliver transformative cancer immunotherapies to the patients and families that need them. We should learn from past experiences and recent successes, and communicate emerging clinical trial data often and clearly among investigators, local review boards and federal regulators. These strategies will help to ensure that we maintain the highest level of ethical and scientific rigor as the clinical development of cancer immunotherapy evolves and we continue to innovate to eradicate cancer.


RNA Binding Protein (RBP) Expression and Disease Dynamics database (READ DB) is a non-redundant, curated database of human RBPs. RBPs curated from different experimental studies are reported with their annotation, tissue-wide RNA and protein expression levels, evolutionary conservation, disease associations, protein–protein interactions, microRNA predictions, their known RNA recognition sequence motifs as well as predicted binding targets and associated functional themes, providing a one stop portal for understanding the expression, evolutionary trajectories and disease dynamics of RBPs in the context of post-transcriptional regulatory networks.Database URL: READ DB is freely available on the web at http://darwin.soic.iupui.edu/ with all major browsers supported. RNA Binding Proteins (RBPs) have a primary role in the post-transcriptional regulation of genes by adding an extra level of plasticity in controlling gene expression . They fomRNA interactome of HeLa cell ,mRNA bound proteome identified from photoreactive nucleoside-enhanced UV crosslinking and oligo(dT) affinity purification approach ,Human orthologs of proteins identified in mouse embryonic stem cells to be bound to RNA ,RBPs screened in a RNA compete study ,RBPs reported in RBPDB .Each RBP in the database can be queried for or is associated with the following levels of information as summarized in A non-redundant and curated list of 1344 RBPs in the human genome was constructed from various experimental sources  as descrThis section provides for each RBP, its synonyms from HGNC annotations, Ensembl accession ID and a description from the NCBI Refseq. We have also included subcellular localization from Uniprot database .We extracted domain information for every RBP from Pfam database  and presThe orthologs of human RBPs in 62 different species are extracted from Ensembl (v73) compara . These sThe expression levels of all the protein isoforms corresponding to each RBP gene are extracted from the human proteome map for 24 different tissues .Transcripts annotated as ‘protein_coding’ in Ensembl are extracted and their expression levels across 16 different tissues are obtained from the Human Body Map . Our analysis revealed that the chosen thresholds compared few other tested thresholds, are appropriate to identify relatively high precision and recall values for miRNA predictions and hence they were considered for reporting the predictions. These predictions are represented as downloadable tracks generated using UCSC genome browser in our database.MicroRNAs (miRNAs) targeting the 3′UTR of each protein-coding transcript of a given RBP are predicted using Miranda  and Targ.et al  in which an orthologous gene was identified to be present.Protein Expression: This section shows the list of protein isoforms (denoted by RefSeq ids) encoded by the gene and their expression levels in 24 different tissues. Additionally, the expression levels of each isoform can be visualized as barplots.Transcript Expression: For each protein-coding transcript encoded by the gene, we present the expression levels in 16 tissues. These expression levels measured as Reads Per Kilobase per Million mapped reads are also visualized as bar plots.Disease Association: For each RBP, we provide the list of disease terms associated with it and the corresponding relevance score.Protein–Protein Interactions: The physical interactions of a given RBP with other proteins is provided under this section. In addition to the gene symbol of the interactor, its corresponding synonyms, evidence code for the physical interaction which describes the experimental method used to identify the interaction and the reference to the identified interaction is also provided.miRNA predictions: The miRNAs predicted to be targeting the 3′UTRs of the protein coding transcripts of RBPs are represented as genomic tracks. Each track image shows the transcript (ENSEMBL ID) and the binding locations of different miRNAs on its 3′UTR. MicroRNA predictions associated with each transcript of an RBP where available, are represented as downloadable tracks generated using UCSC genome browser in our database.Binding Motifs: The sequence motif to which the RBP binds on its target genes is depicted as sequence logo and a list of target genes in the human genome which contain the top 4000 most significant occurrences of the motif are made available as a viewable table. Enriched processes and pathways corresponding to the gene list for each motif are also made available as a table for the end user.We anticipate including tissue-specific protein interactions, increasing the number of RBPs with experimentally available sequence motifs as well as providing expression of RBPs in disease contexts such as in cancer samples  in futurSupplementary data are available at Database Online.School of Informatics and Computing at Indiana University Purdue University Indianapolis (IUPUI) in the form of start-up funds for SCJ. Funding for open access charge: SCJ from IUPUI.This work was supported by Conflict of interest. None declared.


Despite the potential for inflammatory immune responses, this complex interface contains host immune cells and epithelial cells interacting with the microbiota in a manner that promotes symbiosis. Due to the complexity of the cell types and microorganisms involved, this process requires elaborate regulatory mechanisms to ensure mutualism and prevent disease. While many studies have described critical roles for protein regulators of intestinal homeostasis, recent reports indicate that non-coding RNAs are also major contributors to optimal host-commensal interactions. In particular, there is emerging evidence that microRNAs (miRNAs) have evolved to fine tune host gene expression networks and signaling pathways that modulate cellular physiology in the intestinal tract. Here, we review our present knowledge of the influence miRNAs have on both immune and epithelial cell biology in the mammalian intestines and the impact this has on the microbiota. We also discuss a need for further studies to decipher the functions of specific miRNAs within the gut to better understand cellular mechanisms that promote intestinal homeostasis and to identify potential molecular targets underlying diseases such as inflammatory bowel disease and colorectal cancer.The mammalian intestinal tract is a unique site in which a large portion of our immune system and the 10 A major goal of the biomedical research community is to understand mechanisms that regulate the gut immune system in a manner that maintains homeostasis, despite the presence of trillions of bacteria and other microorganisms that reside on and within the host. Gut homeostasis is defined by a proper balance of pro-inflammatory responses against harmful and/or invading microbes while tolerating non-invasive and beneficial microbes . During Figure 1). These regions include: the outer and inner mucus layers, in which invading microbes become trapped to prevent spread of infection  have recently emerged as important mediators of immune development and responses. miRNAs are short (21–25 nucleotide), non-coding RNA molecules that are most commonly transcribed by RNA polymerase II and processed by proteins such as Drosha and Dicer . In theiFigure 1). These non-coding RNAs function as rheostats to the immune response as opposed to binary switches as they act to adjust the magnitude of gene expression. miRNAs also work in feedback loops, ensuring that the immune system does not produce inappropriately strong responses while also promoting protective immunity when needed. In this way, miRNAs themselves can be considered mediators of homeostasis within the immune system as they act to buffer inflammation. Immune processes within the GI tract are coordinated differently when compared to other sites in the body as a result of regulatory mechanisms required to handle the constant exposure to microbes. Intestinal homeostasis is sensitive to even small disruptions, and therefore miRNAs make excellent candidate molecules for fine-tuning responses at this sensitive site. Multiple studies are beginning to demonstrate the importance of miRNAs within the intestine and identifying how the microbiota influences miRNA expression and function within the GI tract; some of this work will be reviewed here (Table 1).microRNAs modulate expression of genes involved in microbial recognition and downstream immune activity, and thus may play an important role within the intestinal immune system during its interactions with the gut microbiota. Their expression in various intestinal cell types, including hematopoietic-derived leukocytes, IECs, and other specialized gut cell types points to a role in mediating homeostasis with commensal microbes and overall intestinal health  are now recognized as non-hematopoietic cells that exhibit immunological functions, including the ability to directly recognize microbial products via PRRs  and to sDicer1 within IECs in mice  and interleukin1 receptor-associated kinase 1 (IRAK1) to downregulate inflammation , 2013. ImicroRNAs within non-hematopoietic cells may also a play a role in the development of and protection from intestinal cancers. The epithelium requires proper signaling mechanisms for normal development, differentiation, renewal, and repair of the intestinal tissue . Many ofAnother example, in which known miRNA functions are relevant in IECs, is the interplay of the RNA-binding protein LIN28 and the Let7 family of miRNAs. Let7 miRNA processing has been shown to be inhibited by LIN28 in models of cellular reprogramming, growth, and oncogenesis . One gro in vitro led to increased expression of miR-122  mice are markedly different from WT specific pathogen free (SPF) mice . New stuListeria monocytogenes infection  derived from commensal bacteria . These mAnother study demonstrated that miR-10a is highly expressed in the intestines and can be modulated by the gut microbiota through TLR signaling on dendritic cells, which suppresses expression of miR-10a . In expeOverall, signals from the microbiota have the ability to alter expression of miRNAs. In turn, miRNAs can target immune-related mRNAs that have the ability to impact responses to microbes and thus shape commensal communities. Thus, crosstalk between microbiota and miRNAs is required for shaping intestinal immune responses and maintaining homeostasis.Many human diseases are related to dysregulation of the intestinal immune system and of the microbiota, including allergies, various autoimmune diseases, and cancers . One of ∗  in miRNA genes may also play a role in susceptibility to intestinal diseases . SNPs winfection . Furthernfection . MiR-146nfection , 2013, aAs IBD patients carry an increased risk of developing CRC, miRNAs also have potential as biomarkers, prognostic tools, and therapeutic targets during cancers of the GI tract. While multiple factors may influence CRC, miRNAs have been shown to play important roles in the molecular pathways that can give rise to intestinal cancers. These include pathways involved in inflammation, chromatin formation, stem cell signaling, apoptosis, and others . ExpressIn general, these investigations suggest that miRNAs are likely indispensable regulators of host-commensal interactions that are required for proper intestinal homeostasis. Because miRNAs fine-tune targets by downregulating their expression 1.2–4-fold , they prTying together emerging concepts in the miRNA field with those in the GI tract and microbiota fields will bring about a comprehensive understanding of cellular processes that control intestinal homeostasis. One of these novel concepts is the transfer of miRNAs between cells via exosomes . In the miRNAs and host-commensal interactions have been independently linked to human development, health, and disease in the past. Now, as we begin to explore how these systems are integrated, a greater understanding of how host-commensal interactions are regulated will undoubtedly emerge. This will provide novel therapeutic insights that will help combat the wide range of diseases that are related to the gut.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.


We will examine the role of the hypothalamus through the complimentary lenses of (1) loss of function studies, including pharmacology and pharmacogenetics; (2) gain of function studies, including specific comparisons between results from classic electrical stimulation studies and more recent work using optogenetics; and (3) neural activity, including both immediate early gene and awake-behaving recordings. Lastly, we will outline current approaches to identifying the precise role of the hypothalamus in promoting aggressive motivation and aggressive action.The hypothalamus was first implicated in the classic “fight or flight” response nearly a century ago, and since then, many important strides have been made in understanding both the circuitry and the neural dynamics underlying the generation of these behaviors. In this review, we will focus on the role of the hypothalamus in aggression, paying particular attention to recent advances in the field that have allowed for functional identification of relevant hypothalamic subnuclei. Recent progress in this field has been aided by the development of new techniques for functional manipulation including optogenetics and pharmacogenetics, as well as advances in technology used for chronic  Specific actions associated with aggression depend on the species, but can include biting, kicking, hitting or pushing. In addition, threatening displays such as hisses, enlarged body size, and changes in facial expression are also parts of the aggressive repertoire of many species. Although humans express aggression in diverse physical and verbal forms , the underlying goal of human aggression—defense and competition for resources—remains the same. Increases in both human and animal aggression are accompanied by similar autonomic responses including raised heart rate and respiration, and both are influenced by changes in circulating hormones, such as testosterone , such as hissing and paw striking. In contrast, if the cut was made posterior to the caudal hypothalamus, these behaviors were absent, indicating that an intact hypothalamus is indispensable for the generation of these rage behaviors.Many studies have attempted to assess the role of the hypothalamus through inactivation experiments, using diverse surgical, pharmacological, and genetic methods. Some of the earliest experiments on aggression were knife cut experiments performed on cats during the 1920’s which demonstrated the importance of hypothalamus in the expression of rage Bard, . When thFurther experiments demonstrated that specific subnuclei within the hypothalamus may be preferentially involved in promoting aggression, though the precise effects remained unclear. Contradictory results were reported regarding the effects of electrolytic lesions of the medial hypothalamus on aggression: while some reports showed decreased aggression after lesioning, others showed the opposite trend -gated chloride channel (GluCL), which prevents the initiation of action potentials by hyperpolarizing the cells upon ligand binding. We found that attack, but not intermale social investigation, is strongly suppressed by VMHvl inhibition, and this decreased aggression returns to normal levels when VMHvl activity is restored , and these results have provided the most clear picture to date of the role of the hypothalamus in aggression though its role in mating behavior is less well understood Figure . SeveralOther studies have capitalized on the fact that the VMHvl is enriched in estrogen receptor alpha (ERα). Since ERα knockout mice exhibit a severe reduction in aggression, these studies examined the role of ERα cells within the VMHvl on male mouse aggression , often fused with fluorescent protein, is virally or genetically expressed in a specific brain region or a group of molecularly defined cells. Following expression, light pulses delivered through an implanted optic fiber Figure  can contIn most species, males are significantly more aggressive than females, and the vast majority of studies have focused on the role of the VMHvl in intermale aggression. However, although the VMHvl is anatomically sexually dimorphic, with males possessing a larger neural volume, several studies have probed whether there is an analogous hypothalamic substrate for female aggression ) or from the expression of immediate early genes (IEGs) such as Fos , AHN, VMHvl and premammillary nucleus ventral part , revealed importantly that mating and fighting induced Fos in male mice is mostly localized in intermingled but non-overlapping cells throughout the hypothalamus  translocate to the cytoplasm, the IEG pattern associated with two properly spaced behaviors can be distinguished based on mRNA localization Figure . This me Figures . Recentl Figures ; these min vivo recordings performed during ethologically relevant behaviors  have been difficult because these behaviors often involve brief violent bursts of action that are not ideal for conducting stable recordings, and also because previous recording technologies were too cumbersome for animals to perform complex, quick movement with these devices attached to their heads. Fortunately, recent advances in both the stability and size of chronic extracellular recording technology have allowed us to begin interrogating these nuclei during behaviors of interest.Perhaps because of its heterogeneity, small size and deep position in the brain, the hypothalamus has long resisted physiological scrutiny. In particular, in vivo recordings of populations of VMHvl neurons during fighting and mating , physiological activity in the VMHvl shows clear differences in activity during separable phases of male and female interactions Figure . During Aggressive motivation can be loosely defined as the internal state that drives animals to seek out opportunities to perform aggressive actions. Signals in the brain that convey information about aggressive motivation should precede and perhaps predict future aggressive actions. Recordings of VMHvl neurons during intermale aggression revealed that VMHvl activity appears to carry information regarding past, current and future attack events  is paired with an initially neutral motor response such as a nosepoke or lever press. By tuning the schedule of the reward delivery, the amount of work an animal is willing to expend to acquire the reward can be assayed, and this “work” reflects the motivation of the animal for the given reward. These paradigms have been commonly adapted to assay the motivation to acquire food, the motivational state that we call “hunger”, and several studies have found a clear link between hypothalamic activity and hunger. Most compellingly, optogenetic activation of agouti-related peptide (AGRP) neurons in the arcuate nucleus of the hypothalamus not only induces eating when food is readily available, but also promotes an increase in bar pressing to obtain food that is not immediately present  information, and neurons at the output level signal the upcoming movement. In this sensorimotor circuit, sensory information is mapped from visual coordinates into saccade coordinates by neurons in the parietal cortex, superior colliculus, and other key structures that contain neural representations of either the visual stimulus, the saccadic location, or both, and are subject to constant updating by changing environmental factors  and also can preferentially signal the future actions that are coupled with these cues . In addition, neurons that signal both sensory and action related information ” and by examining HAA stimulation of induced IEG expression or deoxyglucose uptake pattern (Roeling et al., The recent identification of ERα/PR cells in the VMHvl as a key population for aggression offers an opportunity to track other components in the circuit genetically (Yang et al., Aggression, which has evolved to resolve competition and secure resources, is an essential part of many species’ ethological repertoire. However, disregulated or pathological aggression poses huge risks to society. Although aggression studies thrived in the early and middle 20th century, this line of research has substantially declined since then, especially in comparison to other innate and “emotional” behaviors such as fear (Blanchard et al., in vivo electrophysiological recording devices, wired or wireless, and deep brain optical measuring devices make it possible to follow cell activity reliably over a long period (Fan et al., In addition to the political over-interpretation of aggression studies beyond the line of basic research, technical limitations also obstructed advances in the field. As mentioned, classic lesion and stimulation methods lack adequate spatial resolution to pinpoint aggression-relevant cells. However, the recent emergence of genetically based functional manipulation and tracing methods allows one to target neurons with specific functional relevance, projection patterns or molecular features, greatly improving precision in cell manipulation (Lima et al., 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.


Combination therapy for the treatment of cancer is becoming increasingly important as the complexity of carcinogenesis and the availability of targeted therapies continues to expand. Successful combination therapy would target the primary tumor and distant metastases from multiple different angles to ensure a higher probability of cancer eradication. Immunotherapy is quickly gaining acceptance as an active therapeutic modality in a range of cancers, however cancer is generally thought of as an immunosuppressive disease and often it may be difficult to induce a successful antitumor immune response. Thus, combining cancer immunotherapy with other agents that have immunomodulatory capabilities could significantly improve its efficacy. We have evaluated the receptor tyrosine kinase inhibitor Cabozantinib for its ability to modulate the immune system as well as alter the phenotype of tumor cells to determine if a rationale exists for combining this targeted agent with cancer immunotherapy. Cabozantinib inhibits multiple receptor tyrosine kinases including MET, which has been shown to reduce dendritic cell function, and VEGFR2, which plays a major role in tumor vascularization thus affecting immune infiltration. Our studies indicated that Cabozantinib was able to alter the phenotype of MC38-CEA murine colon carcinoma cells, increasing their sensitivity to T cell-mediated killing. Cabozantinib treatment also modified the composition of the peripheral immune compartment, increasing the frequency of effector T cells while simultaneously reducing the frequency of suppressor cells. This alteration translated to the tumor microenvironment resulting in the generation of a more permissive immune environment. When combined with a poxviral-based cancer vaccine targeting a self-antigen, rMVA/F-CEA/TRICOM, Cabozantinib treatment also resulted in significantly reduced function of regulatory T cells and increased cytokine production from effector T cells in response to the antigen. In the MC38-CEA tumor model, Cabozantinib treatment alone significantly reduced tumor growth rate but was unable to induce complete and durable tumor regression at a dose of 10 mg/kg/day. However, the alterations to the immune landscape as well as the direct modification of the tumor cells led to markedly improved antitumor activity when Cabozantinib was combined with rMVA/F-CEA/TRICOM, resulting in complete and durable tumor regression in 50% of tumor-bearing mice. These data provide a scientific rationale for the clinical combination of Cabozantinib with immunotherapy for the treatment of cancer patients.


Peripheral nerve injury can be accompanied by long-term pain-related manifestations, such as affective and cognitive disturbances, suggesting the involvement of supraspinal mechanisms. One particular region of interest is the prefrontal cortex (PFC), an area implicated in depression, anxiety and cognitive impairment, all of which are frequently associated with chronic pain -4. CliniEpigenetics is a term used to describe modifications to genomic DNA that alter gene expression. DNA methylation is an epigenetic mechanism that is involved in gene regulation mainly by silencing promoter activity. We propose that long-term alterations in DNA methylation could provide a molecular substrate for chronic pain-related changes in the CNS, forming a ‘‘memory trace’’ for pain in the brain.in vitro promoter assay. Finally, mRNA levels of the target genes were measured by RT-PCR.Spared nerve injury or sham surgery was performed in male CD1 mice at three months of age. Six months after injury, mechanical hypersensitivity was confirmed, brains were collected and DNA and RNA were extracted. Global DNA methylation was measured by the luminometric methylation assay in various brain regions, including the PFC. Promoter methylation of individual genes was assessed by sodium bisulfite sequencing and functionally validated using an syt2, a known regulator of synaptic function. Furthermore, transcription of syt2 was regulated by differential methylation of its promoter in vitro and syt2 mRNA was upregulated in the PFC of injured animals. Thus chronic pain-induced changes in the PFC are detected long after the original injury, at a long distance from the site of injury.Six months following peripheral nerve injury, abnormal sensory thresholds and increased anxiety were accompanied by significant genomic DNA hypomethylation  and tranWe show that peripheral injury produces long-term changes in the PFC methylome and propose that DNA methylation mediates the changes in brain structure and cortical function that are associated with chronic pain.The authors declare no competing interests.


Hypertension, defined as a systolic/diastolic blood pressure of 140/90 mmHg or greater, is estimated to carry a 3-fold increased risk of developing cardiovascular disease (CVD), while treating hypertension significantly reduces CVD events, and stroke in particular. Among the many risk factors involved, there is much recent interest in the role of genetic factors that might predispose to hypertension.in vivo in homozygous individuals. In recent years we showed that CVD patients with the relevant MTHFR 677TT genotype (compared to CC or CT genotypes) had significantly higher blood pressure, and that blood pressure was highly responsive to riboflavin intervention, specifically in the TT genotype group [Evidence from genome-wide association studies has identified an association between blood pressure and the gene encoding the folate-metabolising enzyme, methylenetetrahydrofolate reductase (MTHFR), while recent meta-analyses of observational studies show an increased risk of hypertension in people homozygous for the 677C→T polymorphism in MTHFR. Riboflavin vitamin B2) in the form of FAD acts as a cofactor for MTHFR and we have been studying its modulating role in relation to this polymorphism. The variant enzyme is known from molecular studies to become inactive as a result of having an increased propensity to dissociate from FAD, but our earlier work suggested that supplementation with low-dose riboflavin could stabilise MTHFR activity pe group . Further in the fAlthough the precise mechanism linking this polymorphism to hypertension remains to be established, it would appear that the biological perturbation that leads to higher blood pressure in individuals with the MTHFR 677TT genotype is modifiable by correcting the variant MTHFR enzyme through enhancing riboflavin status. Thus riboflavin, targeted specifically at this genetically at-risk group, may offer a personalized non-drug approach to managing hypertension.


A disruptive approach to therapeutic discovery and development is required in order to significantly improve the success rate of drug discovery for central nervous system (CNS) disorders. In this review, we first assess the key factors contributing to the frequent clinical failures for novel drugs. Second, we discuss cancer translational research paradigms that addressed key issues in drug discovery and development and have resulted in delivering drugs with significantly improved outcomes for patients. Finally, we discuss two emerging technologies that could improve the success rate of CNS therapies: human induced pluripotent stem cell (hiPSC)-based studies and multiscale biology models. Coincident with advances in cellular technologies that enable the generation of hiPSCs directly from patient blood or skin cells, together with methods to differentiate these hiPSC lines into specific neural cell types relevant to neurological disease, it is also now possible to combine data from large-scale forward genetics and post-mortem global epigenetic and expression studies in order to generate novel predictive models. The application of systems biology approaches to account for the multiscale nature of different data types, from genetic to molecular and cellular to clinical, can lead to new insights into human diseases that are emergent properties of biological networks, not the result of changes to single genes. Such studies have demonstrated the heterogeneity in etiological pathways and the need for studies on model systems that are patient-derived and thereby recapitulate neurological disease pathways with higher fidelity. In the context of two common and presumably representative neurological diseases, the neurodegenerative disease Alzheimer’s Disease, and the psychiatric disorder schizophrenia, we propose the need for, and exemplify the impact of, a multiscale biology approach that can integrate panomic, clinical, imaging, and literature data in order to construct predictive disease network models that can (i) elucidate subtypes of syndromic diseases, (ii) provide insights into disease networks and targets and (iii) facilitate a novel drug screening strategy using patient-derived hiPSCs to discover novel therapeutics for CNS disorders. The disease burden on society is increasing at a dramatic rate. Focusing specifically on central nervous system (CNS disorders), the prevalence is growing at an alarming rate, with one in sixty-eight in the U.S. having some form of autism , 1.8 milConsiderable effort and resources are being expended on drug discovery research aimed at developing novel therapeutics that would address the unmet medical need across a broad spectrum of diseases. However, less than one out of every eleven drug discovery programs makes it to market . The sucFor SZ, existing treatments target a very limited number of putative mechanisms, which treat some of the symptoms of SZ in some of the patients some of the time. Although several new pharmacologic interventions have been tested clinically in the last decade, none have shown medically relevant efficacy required for approval by regulatory authorities. Similarly, the field of therapeutic development to slow the progression of AD is littered with clinical failures of multiple pharmacological mechanisms and treatment modalities. These failures have had somewhat sobering effect on the field, particularly since many of the interventions were founded on human genetics- and pathology-informed amyloid hypothesis. A bright spot and beacon of hope may be the field of cancer therapeutics, where personalized treatments with far superior efficacy than traditional chemotherapies have been developed successfully. It behooves us to understand the primary reasons for the emerging successes in cancer treatments in order to adapt the paradigm to drug discovery for CNS disorders.We will begin this review by discussing key factors that have likely contributed to the clinical failures for novel drugs, discuss the cancer research paradigms that have led to drugs with superior efficacy and diagnostic tests, and offer a perspective on the application of emerging technologies and tools that could improve the success rate of novel therapies. Specifically, we will focus on two potentially game-changing paradigms: (1) advances in human induced pluripotent stem cell (hiPSC)-based disease models and (2) multiscale predictive modeling. Together, these approaches can enable a more integrative biology approach to deriving insights into disease mechanisms, upon which drug screening and development may be founded in the near future. Although these approaches may be adapted broadly to many CNS indications, this review will focus primarily on SZ and AD.First, the selection of a target for novel drug therapies requires an in-depth understanding of disease biology, from the etiological factors to pathophysiological mechanisms, and their relationship to disease progression and duration. It is insufficient to move forward with only the knowledge that a particular target is expressed in the brain or that a specific DNA variant is associated to a neurological disease. For common, complex trait diseases, a more systems oriented view is emerging in which human diseases are demonstrated to be emergent properties of biological networks, not the result of changes to single genes . Hence, Secondly, other than rare or orphan diseases that are caused by Mendelian mutations, common CNS disorders are syndromic diseases diagnosed primarily by non-specific and blunt clinical diagnostic tools, often based on patient reported symptoms. Thus, both the so-called clinically diagnosed SZ or AD patient population represents a diverse patient population with respect to etiologies and associated pathophysiological mechanisms, giving rise to similar sets of clinical symptoms but with distinct rates of disease progression. The clinical diagnostic tools, such as the Diagnostic and Statistical Manual (DSM) criteria for mental disorders or Mini-Mental State Examination (MMSE) for dementia, have served us well and led to approval of several drugs. However, the poor specificity and sensitivity of these tools require that they be supplemented with objective, diagnostic biomarkers with which to classify or enrich patient populations that are more homogenous in either their etiological or pathophysiological factors, or disease stage, being targeted by drugs.Third, chronic diseases have an additional inherent issue related to adaptive biological mechanisms that emerge with chronicity of the disease or drug treatment. For neurodegenerative disorders, there is also the complicated factor of loss of resiliency in surviving neurons affected by the disease process. Thus, interventional strategies have to be designed to be specific to the stage of the disease, and when possible, to target primary prevention. Therefore, an understanding of adaptive molecular mechanisms as well as objective biomarkers that can monitor the biologic processes associated with the disease stage or progression and adaptive processes induced by drugs or disease will be required to improve drug discovery and development.Figure 1). Systems biology approaches seek to presume less knowledge, capture more information, and interpret the information in a more data driven way.Finally, drug screening paradigms need to evolve so that disease biology mechanisms are monitored in cellular and animal models that more faithfully recapitulate human disease biology. Similarly, the systems biology/disease network approach will require that end-points of drug screens may have to be multi-parameter or phenotypic in nature, rather than those based on ease or throughput considerations alone. Thus, we must facilitate the development of cell-based systems derived from patients with disease as well as normal controls, in which the cell types are directly relevant to those implicated in human disease, so that we may garner insights into disease biology and design effective screening paradigms. Given that the large-scale generation and integration of panomic data has enabled the construction of complex gene networks that provide a new framework for understanding the molecular basis of disease , it is nThe four points discussed above require translational approaches that begin with patient-centered research to derive insights into disease biology. Of all the major therapeutic areas, cancer drug discovery research has led the way in translating the explosion of human genomic data into new therapeutics despite each clinically diagnosed malignancy being a heterogeneous mixture. Hence, we begin by reviewing the cancer drug discovery paradigm.in vitro and in vivo model systems, which are being shown to more faithfully recapitulate important aspects (notably uncontrolled proliferation) of the disease, and in many cases can be formatted for high throughput screening  although, as described below, the development of resistance has plagued each of these drugs.Multi-center genome sequencing endeavors  have revBRAF-mutant melanoma, where the defective oncogene is directly upstream of, and activates, MEK in the MAPK pathway. Conversely, however, MEK inhibitors have not thus far proven very effective in treating cancers with mutations that activate the oncoprotein, RAS, just one extra step further upstream of RAF in the canonical pathway. What has become evident now is that a comprehensive understanding of the entire defective network, rather than a single canonical pathway, is necessary to identify new drug targets for cancers harboring these undruggable oncogenes and tumor suppressors . Genetically characterized cancer cell lines and tumor grafts have been established from human disease specimens and generally preserve the genomic features observed in the disease. In many cases, mutated oncogenes and tumor suppressors that drive disease in animal models are also known to be critical for the growth and survival, in culture, of human cancer cell lines with the same mutations. This offered the opportunity to use proliferation assays as a facile but disease biology-relevant phenotypic assay on human cancer cell lines to identify vulnerabilities particular to the network reconfigured by specific mutations. A large number of n cancer . Such apnd BRCA2  and clinnd BRCA2 .Using high throughput screening methodologies coupled with statistical methods, large panels of genetically characterized tumor cell lines can be assembled to identify gene–drug interactions in an unbiased manner and thereby identify either drugs or drug–drug combinations effective for the treatment of cancers with the relevant mutation . In an ain vitro, to identify drug cocktails that are predicted to more effectively treat and prevent the emergence of drug resistance  de-risking target selection through identification of driver mutations or disease networks through multi-center studies on patient-derived tumor specimens, (ii) reducing patient heterogeneity by implementing molecular definition of disease taxonomy rather than clinical diagnosis alone, (iii) addressing drug resistance by targeting disease networks associated adaptive processes induced by drug and/or disease, and (iv) incorporating cellular and animal models with greater predictive validity in drug screens.Tracking with the great advances in cancer drug development that have benefitted from the genomics revolution, forward genetics strategies to elucidate the complexity of human disease have been accelerated as the cost of assaying nucleic acid sequences continues to drop exponentially and ever bigger cohorts of diseased individuals are assembled. Whether performing a genome-wide association study or whole exome/genome sequencing studies in case/control cohorts or families segregating diseases of interest, the quest has been to identify specific genes, pathways and networks that are critical for disease onset, progression and severity and thereby rationalizing the selection or prioritization of molecular targets or pathways for drug discovery.Schizophrenia is a complex and heterogeneous disorder with an estimated heritability of about 80% . Much lide novo CNVs are elevated 2–50 fold, (3) CNVs generally contain many genes and confer large relative risks (2–50), (4) specific sites of CNVs are often also found in multiple additional neurological diseases, and (5) CNVs are enriched in neuronal functions, particularly those that are involved in synaptic activity and neurodevelopmental processes. CNVs represent a polygenic burden of rare disruptive mutations, one that is particularly enriched in gene sets including the voltage-gated calcium ion channel and the post-synaptic density , for the first time, critical glutamatergic genes such as GRM3 and GRIN2A, and calcium channel subunits (CACNA1C and CACNA1l) have been associated with SZ.While individually penetrant CNVs are only found in a minority of patients (perhaps 5–10%), common DNA variants  are significant contributors to the heritability of SZ, accounting for ∼30% of the variance in liability . It is nde novo point mutations is also a powerful tool; it was recently shown that small de novo mutations affecting one or a few nucleotides are overrepresented among glutamatergic post-synaptic proteins  is sufficient to directly reprogram adult somatic cells into an induced pluripotent stem cell (iPSC) state , Shinya In previous publications, we directly reprogrammed fibroblasts from four SZ patients into hiPSCs and differentiated these disorder-specific hiPSCs into forebrain NPCs  and neurUntil recently, functional differences in SZ hiPSC neurons had not been identified, likely owing to the heterogeneity in hiPSC neuronal culture. Now, the first phenotypic characterization of a single neuronal subtype  shows reduced neuronal activity and spontaneous neurotransmitter release in SZ hiPSC-derived neurons . This deA growing body of evidence links SZ with abnormal functioning of dopaminergic, GABAergic and glutamatergic neurons. Although pharmacological modulation of dopamine transmission helps manage the positive symptoms of SZ for some patients , emerginBRN2, ASCL1, and MYTL1 – with the addition of NEUROD1 in human cells. Though rapid, the process is inefficient, occurring in just 2–4% of the original fibroblasts, and generates relatively immature neurons unable to form synapses on their own. The addition of key microRNAs improves the process, resulting in mature neurons capable of forming fully functional synapses in pure cultures .Given the enormous amount of panomic data that have been generated to characterize common human diseases, this data can be integrated in order to build predictive network models of normal and disease states, which can elucidate the key biological drivers of the disease state. To fully understand complex neurological diseases, we must link molecular biology to physiology . MultimoFigure 1).Integrative network models utilize panomic data to derive causal relationships among 1000s of intermediate molecular traits and between molecular and higher order physiological traits associated with disease . In thisHigh dimensional panomic data will increasingly be generated in hiPSC derived neurons, with 100s or even 1000s of samples generated from disease cohorts now possible for (relatively) low costs. This type of panomic data permits the construction of interaction and differential connectivity networks, which characterize the connectivity patterns of the molecular networks in disease relevant cell types between those with and without disease. Interaction networks and differential connectivity measures, such as the module-centric differential co-regulation (MDC) measure, provide for deeper insights into the molecular processes involved in disease. For example, by applying MDC to the molecular interaction networks generated from late-onset AD brain regions compared to these same brain regions in non-demented controls , we deteFigure 3). The identified components represent sub-networks that can in turn elucidate potential disease mechanisms defined by them. The master regulators of these sub-networks can in turn be identified using key driver analysis methods , AD, and other such common human diseases . The effect of any given perturbagen, whether a small molecule compound, natural product, RNAi-based construct and so on, on a specific network of interest can be assayed directly in cell-based systems (such as hiPSC-derived neurons derived from SZ or AD patients), which more accurately reflect the states of networks underlying disease. Complementing the network-based screens that use molecular network state as a readout, are cellular phenotyping assays that also aid in the linking of molecular states of disease to pathophysiological states. Screening carried out in this way can lead to the rapid identification of compounds that affect disease networks in favorable ways, while simultaneously identifying compounds that hit networks associated with toxicity or other adverse events . In this way, compounds can be identified that target specific subtypes of disease without targeting networks that can lead to toxicity or adverse events.Multiscale models defining networks for a given neurological disease can be used to construct gene expression assays for high throughput screening  that capture far more of the relevant biology for common human diseases may be performed. The prediction is that such screens will significantly improve discovery of targets and drugs, as well as diagnostic tests, to make personalized therapies a reality for CNS disorders.After considering the frequent clinical failures for novel drugs together with the novel cancer research paradigms that have led to improved drug discover, here we have discussed two transformational technologies that have and will continue to provide unprecedented insights into molecular mechanisms associated with complex diseases of the brain, and thereby de-risk drug discovery. First is the ability to generate on a large-scale hiPSCs derived neurons; this cellular model recapitulates disease mechanisms 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.


Analysis of extracellular vesicles (EV) and their contents may be useful as disease biomarkers if they reflect the contents of cells of origin, differ between normal and diseased tissue and can be reliably detected. An increasing number of long noncoding RNA (lncRNA) are being reported to be aberrantly expressed in human cancers. These tumor-associated lncRNA may have potential as new biomarkers of disease. In this review, we highlight lncRNAs that are commonly associated with hepatocellular cancer, and summarize their potential biological roles and underlying molecular mechanisms. While lncRNA can be detected in the circulation, their low expression within circulating vesicles will require the use of highly sensitive detection technologies such as digital polymerase chain reaction or next-generation sequencing. While the integrity and functional role of tumor-specific lncRNAs within EV have yet to be established, their presence or enrichment within tumor cell-derived EV offers promise for their potential as disease biomarkers. Extracellular vesicles (EVs) are small membrane-bound vesicles that are released into the interstitial fluid from a wide variety of normal or diseased cells. The predominant types of EVs are exosomes, microvesicles and apoptotic bodies and these are distinguished on the basis of their size and biogenesis . ExosomeEVs can be detected and isolated from body fluids. Analysis of EVs and their contents may have additional utility as disease biomarkers if they reflect the contents of cells of origin, differ between normal and diseased tissue and can be reliably detected. LncRNAs are a group of noncoding RNAs with diverse functions that are becoming increasingly recognized. In this overview we will focus on the lncRNA content of EVs, and their potential application and utility as disease biomarkers, using hepatocellular cancer (HCC) as an example. HCC-derived EVs have been shown to have selective enrichment of lncRNA, and some of these such as TUC339, linc-RNA ROR and lincRNA-VLDLR have been implicated in tumor cell behavior . EnrichmHCC is the most common type of primary liver cancer in adults and is a leading cause of cancer-related deaths worldwide, resulting in close to 750 000 deaths worldwide every year , 7. WithDiagnostic tools for the detection of early HCC include serum biomarkers, radiological imaging such as abdominal ultrasound (US), computed tomography (CT), and magnetic resonance imaging (MRI) and liver biopsy. Ultrasonography is frequently used for screening, but has poor sensitivity for detection of smaller tumors . WidesprAnalysis of serum tumor markers is an attractive choice for screening for HCC. The most common marker used to detect HCC is alpha fetoprotein (AFP). Serum AFP levels are elevated in some, but not all persons with HCC, and AFP levels >400 ng/ml are highly correlated with HCC . HoweverThe analysis of tumor-associated RNA within EVs could provide an opportunity for identification of novel biomarker candidates. While much attention has been given to aberrantly expressed tumor-associated protein-coding mRNAs, genome sequencing studies have identified deregulated tumor-associated expression of several noncoding RNAs such as miRNAs, and lncRNAs. In contrast to the extensive studies on mRNAs and miRNAs, our understanding of the role of lncRNAs in disease remains limited . LncRNAsThis RNA gene was first identified as a lncRNA that was highly specifically upregulated in HCC . Highly This lncRNA is significantly overexpressed in HCC tissues and liver cancer cell lines  and has Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a large lncRNA and is >8000 nucleotides in length . This lnH19 is an imprinted gene that is normally expressed in fetal liver and placenta during embryonic development, but is repressed after birth in most tissues . OverexpHigh levels of LncRNA with high expression in HCC (lncRNA-HEIH) in HBV-related HCC are markedly associated with recurrence, and can function as an independent prognostic marker for survival . LncRNA-Maternally expressed gene 3 (MEG3) has been found to be highly expressed in normal tissue but is downregulated in a number of human tumors . The expUpregulation of microvascular invasion in HCC (MVIH) has been shown to be associated with increased microvascular invasion and metastasis in HCC tissues . FurtherThis lncRNA encompasses a sequence that is highly conserved across species and markedly upregulated in HCC cells compared with nonmalignant cells . TUC338 in vitro and in vivo [Downregulated expression by HBx (Dreh) is significantly downregulated in HBV-related HCC, and is associated with poor disease prognosis . This ln in vivo .Low expression in tumor (LET) levels were found to be decreased in tumor samples of HCC patients when compared with normal tissue . DownregThe expression of HBx-LINE, short for fusion of the HBV encoded X protein and human cellular long interspersed nuclear elements, is upregulated in HCC tissues and associated with poor patient prognosis, and thus could potentially serve as a predictor of patient survival . The HBxThis lncRNA is encoded within the X-inactivation center and has been implicated in HBV-associated HCC . Ftx maylncRNA activated by TGF-β (lncRNA-ATB) is a mediator of the transforming growth factor (TGF-β) signaling pathway, and is highly expressed in HCC tissues . LncRNA-Plasmacytoma variant translocation 1 (PVT1) was found to be highly upregulated in HCC tissues and was associated with poor clinical prognosis in patients . PVT1 inThe proliferating cell nuclear antigen antisense lncRNA (PCNA-AS1) transcript is located opposite to the PCNA gene, and may regulate PCNA activity via RNA hybridization . This anThis lincRNA was first identified as an epigenetic regulator involved in pluripotency and lineage commitment, and is significantly upregulated in malignant hepatocytes as well as enriched within EVs derived from tumor cells . Recent This is an ultraconserved RNA that is highly enriched within EVs released from HCC-derived tumor cells and has been implicated in modulating tumor cell growth and adhesion .The emerging data on lncRNA involved in HCC indicates the presence of several tumor-associated lncRNA, some of which have been functionally linked to processes involved in tumor growth. Given the large repertoire of transcribed lncRNA within the genome, many more lncRNA associated with HCC are likely to emerge. Once associations with the presence, behavior or outcomes of HCC are ascertained, these will represent attractive candidates for further evaluation as biomarkers of disease.Accurate and sensitive detection of candidate lncRNA biomarkers in the circulation is a prerequisite for their use as biomarkers of disease or as markers to monitor disease progression, treatment effect or prognosis . Startinm) is different, and this may negatively affect the specificity and sensitivity [Microarrays are commonly used for RNA detection and are based on nucleic acid hybridization between target molecules and their complementary probes . The sigsitivity . With thsitivity . An advasitivity .T), which is defined as the PCR cycle at which the fluorescent signal of the reported dye crosses an arbitrary threshold [T value is inversely related to the amount of amplicon in the reaction; hence, a lower CT value indicates greater gene expression [T values for specific genes are referenced to well-known housekeeping genes such as GAPDH. However, this could be problematic because the housekeeping genes vary in expression between cell populations. The use of qPCR is limited because it requires an adequate standard curve to be generated for each sequence of interest [Quantitative PCR (qPCR) is a well-established tool for quantifying gene expression that relies on an increase in fluorescence signal that is proportional to the polymerase reaction product or amplicon . Quantithreshold . The CT pression . Typicalinterest . Furtherinterest . A limitThe NanoString nCounter Gene Expression Assay is a novel technology to measure RNA expression . The assDigital PCR (dPCR) offers an alternative and more direct approach for detecting gene expression; dPCR involves the partitioning of a sample into multiple separate reactions, such that some reactions contain no nucleic acid template and others contain one or more template copies . The parNGS offers a powerful tool for detecting RNA molecules in biological samples. NGS initially requires the generation of a small RNA library in which the 5′ and 3′ RNA adaptors are ligated to either end of the noncoding RNA. The 3′ adaptors then bind to other small RNAs that carry the corresponding 3′ hydroxyl group. This is followed successively by reverse transcription and PCR amplification . NGS is An increasing number of lncRNA are being reported to be aberrantly expressed in human cancers. Characterization of these tumor-associated lncRNA offers the possibility of providing new insights into disease pathogenesis. lncRNA can be detected in EV released by tumor cells and can be detected in the circulation. However, the low expression of these in circulation and assays for clinical use will require highly sensitive detection technologies such as dPCR or next-generation sequencing. In addition to establishing tumor specificity, successful application of emerging knowledge of lncRNA will require the development of sensitive and reliable assays and a systematic demonstration of their clinical utility as biomarkers for disease diagnosis, prognosis, prediction of recurrence and therapeutic response. Such data are becoming available for several lncRNAs, for example HOTAIR and MALAT1 in predicting tumor recurrence following liver transplantation for HCC . While tKey PointsAlterations in the expression of several lncRNAs such as HOTAIR and MALAT-1 have been reported in many cancers.Aberrant expression of lncRNA that is specific to certain tumors, such as HULC in hepatocellular cancer, support a role for these in disease pathogenesis.The differences in lncRNA present in tumor cells and in extracellular vesicles derived from these cells supports the existence of mechanisms that selectively enrich lncRNA within extracellular vesicles.New quantitative methods for RNA gene analysis such as digital PCR are enabling sensitive measurements of RNA within extracellular vesicles that will facilitate their adoption as cancer biomarkers.DK 069370 and UH3-TR000884 from the National Institutes of Health.Supported by Grants


In humans and other mammals with XX females and XY males, the Y bears a gene (SRY) that induces testis differentiation in the embryo and switches on hormones that masculinize it. The human X has more than 1500 genes, but the tiny Y is a genetic wasteland and bears only 45 protein-coding genes, most active only in testis. To discover how human sex chromosomes got to be so weird, we compared the chromosomes, genes and DNA in distantly related mammals and even birds and reptiles (with completely different sex determining systems). Kangaroo sex chromosomes reveal the original mammal sex chromosomes, while the bizarre platypus sex chromosomes (more related to those of birds) tell us that human sex chromosomes and the SRY gene are relatively young. The human X and Y evolved from an ordinary chromosome pair as the Y degraded progressively. If Y degradation continues at this rate, it will disappear in just 5 million years. If humans don’t become extinct, new sex determining genes and chromosomes must evolve, maybe leading to the evolution of new hominid species.Where will our new sex genes and chromosomes come from? Whereas mammals (and birds) have rather rigid systems, other vertebrates (particularly reptiles) show great variation in sex determining systems, and we can find many examples of switches in sex determining systems. Using a model of dosage-dependent and temperature dependent sex determination, we can readily understand switches between temperature-dependent and chromosome-dependent sex determination, and even between XY and WZ systems. We also see many ways in which genes or gene copies (often of the same genes which seem to be particularly good at this role) have taken on a sex determining function, and defined new sex chromosome systems.