A), long-interval intracortical inhibition (GABAB), and the corticospinal silent period (GABAA+B). Net intracortical facilitation involving glutamate was assessed with intracortical facilitation, and corticospinal excitability was measured with the resting motor threshold. Results showed that FXS patients had significantly reduced short-interval intracortical inhibition, increased long-interval intracortical inhibition, and increased intracortical facilitation compared to healthy controls. In the FXS group, reduced short-interval intracortical inhibition was associated with heightened intracortical facilitation. Taken together, these results suggest that reduced GABAA inhibition is a plausible mechanism underlying cortical hyperexcitability in patients with FXS. These findings closely match those observed in animal models, supporting the translational validity of these markers for clinical research.Fragile-X syndrome (FXS) is characterized by neurological and psychiatric problems symptomatic of cortical hyperexcitability. Recent animal studies identified deficient γ-aminobutyricacid (GABA) inhibition as a key mechanism for hyperexcitability in FXS, but the GABA system remains largely unexplored in humans with the disorder. The primary objective of this study was to assess GABA-mediated inhibition and its relationship with hyperexcitability in patients with FXS. Transcranial magnetic stimulation (TMS) was used to assess cortical and corticospinal inhibitory and excitatory mechanisms in 18 patients with a molecular diagnosis of FXS and 18 healthy controls. GABA-mediated inhibition was measured with short-interval intracortical inhibition (GABA While the clinical phenotype may vary considerably between individuals affected by the disorder, it typically involves psychiatric and neurological manifestations indicative of neuronal hyperexcitability, including seizures, anxiety, hyperactivity, hypersensitivity, and hyperarousal3. Accumulating EEG evidence supports the existence of cortical hyperexcitability in patients with FXS7. However, the physiological mechanisms involved in hyperexcitability in FXS remain poorly understood. Research in this field is of prime clinical importance since correcting the alterations involved in neuronal and circuit excitability could alleviate several core symptoms of the disorder2.Fragile-X syndrome (FXS) is a rare disorder, but the leading monogenic cause of autism spectrum disorder and the first hereditary cause of intellectual disability. FXS results from the methylation of the Fmr1 gene, which leads to a marked reduction or absence of the fragile-X mental retardation protein (FMRP), an important regulator of protein synthesis involved in brain development and synaptic function9, and is believed to result from an imbalance between excitatory and inhibitory drives in intracortical circuits10. The dominant view over the past 15 years has been that the absence of FMRP in FXS induces an overactivation of metabotropic glutamate receptors (mGluRs), thereby increasing neuronal excitability11. Although experimental data from Fmr1-ko animals support the idea that mGluRs play a role in the process13, clinical trials using mGlurR antagonist for FXS have been unsuccessful15. Moreover, there is evidence suggesting that mGluR overactivity alone is insufficient to fully explain hyperexcitability in FXS18. In that regard, several recent studies show the presence of dysfunctions affecting diverse elements of the GABAergic system in various animal models of FXS21, suggesting that reduced inhibition is a key mechanism for circuit hyperexcitability. This theory is further supported by in vivo evidence confirming that defective inhibition from GABAergic interneurons is causally involved in the behavioral and sensory phenotype of the fly and mouse FXS models18. These findings suggest that the GABAergic system may be a promising therapeutic target to correct circuit hyperexcitability in FXS2.Hyperexcitability is a consistent observation across animal models of the disorder23 . To date, the most significant evidence of a GABA dysfunction in humans with FXS comes from Hulst and colleagues (2015), who showed diminished GABAA receptor binding throughout the brain in a small group of patients using positron-emission tomography (PET) (n = 10). Yet, it is unknown if this reduction in receptor availability translates into functional alterations at the cortical level. Transcranial magnetic stimulation (TMS) is valuable tool to answer this question, as this technique is capable of probing the excitatory and inhibitory mechanisms in the cortex of awake humans26. TMS is a painless and non-invasive neurostimulation technique that uses magnetic fields to stimulate a restricted part of the brain. TMS acts on the cortex through the recruitment of interneurons in upper cortical layers mediating GABAergic inhibition onto pyramidal neurons27, whose resulting activity closely match what is seen non-invasively and at a macroscopic level in humans following stimulation of the primary motor cortex28. Abundant evidence demonstrates the sensitivity of TMS to GABAergic mechanisms in healthy and clinical populations30. Because the technique is safe, well tolerated, and does not require extensive training or sedation to be used with people with intellectual disability, TMS is well suited for the study of the GABA system in FXS.The GABA system remains surprisingly unexplored in humans with FXS, despite several past and ongoing clinical trials aiming to modulate itn = 18). We assessed three measures of GABAergic inhibition, including short-interval intracortical inhibition , long-interval intracortical inhibition , and the corticospinal silent period . Resting motor threshold (rMT) was used to assess corticospinal excitability, and net intracortical excitation involving glutamate was measured with intracortical facilitation (ICF). We hypothesized that GABA-mediated inhibition would be reduced, while corticospinal excitability and ICF would be enhanced in individuals with FXS compared to healthy controls.In the present study, we used TMS to investigate intracortical inhibition and excitation in a cohort of individuals with a molecular diagnosis of FXS , resulting in 18 patients in each group . Power analysis showed that this sample size allowed the detection of an effect size of f  = 0.5 (large effect) in 83% of the cases with an alpha level of 0.05 (two-tails). Seven FXS patients were taking psychoactive medication, four of them taking two types of medications or more , and 20 age and sex-matched healthy controls were recruited. Data from one male FXS patient were rejected due to technical EMG recording issues, and multivariate outliers were identified and removed using the Mahalanobis distance  until a motor-evoked potential was obtained in the contralateral first dorsal interosseus (FDI) muscle. The location was then entered in the neuronavigation system , and stimulations of decreasing intensity (2% MSO steps) were administered in the vicinity (±1 cm) to identify the most sensitive location. Electromyographic signals (EMG) were recorded with surface electrodes, amplified using a Powerlab 4/30 system, digitized at 4 KHz, and recorded with Scope v4.0 for offline analysis.TMS was performed with a Magstim BiStim-2 using a 70 mm figure-of-eight coil . To identify the optimal stimulation site, the coil was initially positioned over the C3 location of the International 10–20 EEG system at an intensity of 35% of maximum stimulator output (MSO). Single pulse stimulations were administered at a 1 cm interval covering a 16 cm31 and administered following the guidelines from the International Federation of Clinical Neurophysiology32. Briefly, resting motor threshold (rMT) was established as the minimal intensity to induce MEPs of at least 50 µV amplitude in five out of ten consecutive trials using the relative frequency criterion33, and intracortical facilitation ) was acquired to measure net intracortical excitability. Tests for intracortical inhibitory functions included short-interval intracortical inhibition  and long-interval intracortical inhibition . Corticospinal silent period  was acquired while participants performed an isometric voluntary contraction of the FDI corresponding to 20% of maximal muscular contraction. In a subset of participants , short-interval intracortical facilitation (SICF) was acquired using the same interval as SICI (3 ms IPI) to assess the potential contribution of I-wave facilitatory components on SICI34. Conditioning stimuli (CS) for SICI, ICF, and the test stimulus for SICF, were set to an intensity corresponding to 80% of rMT; baseline MEPs and all other stimulations were administered using a test stimulus (TS) intensity set to induce MEPs of 1 mV peak-to-peak amplitude (≈125% of rMT). Fifteen trials were acquired for each measure, except for CSP (n = 5). A frameless neuronavigation system  was used to ensure stable coil positioning during testing. The procedure was well tolerated by all participants and there was no serious adverse event. Typical EMG responses for each TMS protocol are depicted in Fig. Standard TMS procedures were used35. CSP was measured independently by two raters as the duration from the MEP onset to the return to baseline EMG activity . For each measure, a minimum of 10 valid trials were kept, resulting in Cronbach’s alpha being superior to 0.9036. Between-group differences were assessed using independent samples two-tailed t-tests  and multiple comparisons were handled with false-discovery rate . Data are available from the authors upon request.Individual EMG epochs were visually inspected and rejected if visible muscular activity was present in the 100 ms time window preceding the TMS pulse. Peak-to-peak MEP amplitude was calculated and averaged for baseline, SICI, ICF, and SICF independently. Ratios were then computed for SICI, ICF, and SICF, using the mean baseline MEP as the denominator. For LICI, the amplitude of the MEP evoked by the TS was divided by the one induced by the CS within each trial, and the resulting ratios were averagedT32.4 = 1.97, q = 0.43), amplitude of baseline MEP , or CSP length  , increased LICI , and reduced SICI  compared with controls. Groups did not differ significantly on SICF  , indicating a positive relationship between intracortical hypo-inhibition and hyperexcitability. This relationship was significantly different from the one observed in the control group   comparing only non-medicated FXS with healthy controls. Results corroborated observations made at the whole-group level, with non-medicated patients differing significantly from controls on SICI  and ICF , and LICI showing a similar trend . The correlation between SICI and ICF remained significant in the non-medicated FXS group , and significantly different from the control group . Comparing medicated FXS patients with non-medicated FXS patients and controls did not show significant differences on any TMS measures (Mann–Whitney–Wilcoxon tests), possibly due to the small number of medicated FXS patients (n = 7). Excluding FXS females from the analysis did not change the overall pattern of results. LICI and ICF remained significant , the relationship between SICI and ICF remained significant in the FXS group , and significantly different from the control group . The most notable difference was that the p value for SICI was now slightly above the significance threshold .FXS patients and controls did not differ regarding rMT (43) Fig. . However43) Fig. . Since d11) Fig. . Because37. However, it remained unclear how these abnormalities translated to the human brain. The current study bridges this gap by providing evidence that GABAA-mediated intracortical inhibition is reduced in humans with FXS, and that this alteration co-occurs with increased intracortical circuit excitability.Recent evidence from animal studies showed the pivotal role of GABAergic inhibitory interneurons in the neurophysiological phenotype of FXSA receptor29. Importantly, since groups did not differ on SICF, it is unlikely that between-group differences in SICI level result from a contamination by indirect facilitatory waves34. The presence of altered GABAA mediated inhibition concurs with the only previous study directly assessing GABAergic function in FXS patients, where [11C]-flumazenil PET was used to show a reduction in GABAA receptor binding38. These results are in line with preclinical evidence showing a reduction of GABA synthesizing enzyme and GABAA receptor mRNA levels in animal models of the disorder2. Similarly, the number of parvalbumin expressing interneurons, the most abundant type of GABAergic interneurons in the cortex40, is reduced in Fmr1 KO animals41. Considering the fact that TMS is believed to interact predominantly with parvalbumin interneurons43, a reduction in this cell population could also contribute to the aberrant intracortical inhibition observed in FXS patients. Interestingly, recent evidence suggests a causal role between parvalbumin dysfunctions and impairments in sensory processing in FXS, a behavioral phenotype that is common to both animals and humans18. Sensory processing was not evaluated in the present study, thus the association between TMS measures of inhibition and the magnitude of sensory abnormalities in FXS patients remains to be investigated.It is recognized that SICI reflects the activity of cortical interneurons on post-synaptic GABA44. Because ICF reflects, at least in part, the activity of NMDA receptors45, one explanation for this observation is that the increase in ICF is directly linked to the exacerbation of glutamate signaling described in animal models of the disorder11. However, considering that it is also modulated by other neurotransmitter systems, ICF can more accurately be conceived as “glutamatergic facilitation tempered by GABAergic inhibition”46, namely because the time interval at which ICF can be observed (6–20 ms) encompasses the tail of the GABAA-mediated inhibition measured by SICI47. Hence, while it is established that ICF and SICI depend on distinct neuronal populations, it is their interaction that ultimately controls the corticospinal output48. Further supporting a relationship between GABAA-mediated inhibition and intracortical excitability, pharmacological manipulations that reduce or enhance SICI have the opposite effect on ICF (reviewed in29), and changes in SICI can mediate variations in ICF49. Here, the fact that reduced SICI is associated with increased circuit excitability in FXS patients, as expressed by ICF, concurs with recent evidence obtained from in vivo animals pointing to faulty GABAA inhibition as a primary cause for circuit hyperexcitablity in the absence of FMRP18. Although the present data cannot ascertain that the alteration in GABAA signaling is a causative factor of hyperexcitability in FXS, it nonetheless supports the significance of the GABAA system as a potential target to correct a core neurophysiological feature of FXS.The between-group difference observed on ICF confirms the presence of circuit hyperexcitability in FXS patients, as was previously inferred by EEG studiesB receptors52. The increase in LICI may explain why CSP values did not differ between both groups despite reduced SICI, as CSP amalgamates both GABAA and GABAB inhibition29. Although the mechanism responsible for the increase in LICI observed in FXS patients is elusive, it could reflect a compensatory mechanism. Indeed, there is cross talk between GABAA and GABAB receptors at the post-synaptic site in such a way that activation of GABAB receptors increases GABAA inhibition53. Whether such cross talk is at play in FXS remains to be established. However, the preservation of LICI can be related to evidence from mice models showing that post-synaptic GABAB receptors are spared in FXS, in contrast with what is observed at the presynaptic level20. Specifically, reduced presynaptic GABAB signaling has been reported at glutamatergic synapses19, while excessive presynaptic GABAB signaling appears to be present at inhibitory synapses20. Since presynaptic GABAB receptors are involved in auto-inhibition and regulate neurotransmitter release, both alterations are susceptible to contribute to circuit hyperexcitability. The presence of increased GABAB mediated inhibition described here, coupled with the apparent heterogeneity in terms of GABAB receptor function may explain, at least in part, the limited efficacy of selective GABAB agonists for treating FXS in recent clinical trials23.Surprisingly, FXS patients showed enhanced LICI, which is primarily mediated through the activity of post-synaptic GABA54, including voltage gated sodium channels which are believed to play a role in the cortical hyperexcitability of FXS56. It is however known that rMT is normal in several disorders associated with cortical hyperexcitability57, including in patients with Dravet syndrome, which is caused by a mutation of a gene directly involved in the proper function of voltage gated sodium channels24. It is possible that variables unrelated to neuronal function, such as the scalp-to-cortex distance58 and cerebral tissue structure60, may have obscured potential between-group differences. These evidence, coupled with abundant TMS data from epilepsy patients30, suggest that the rMT is less sensitive to hyperexcitability than paired-pulses protocols. Figure The lack of difference in rMT between controls and FXS patients is intriguing considering that FMRP influences the activity of ion channelsA receptor (GABRG2(R43Q)), which is associated with an hereditary form of generalized epilepsy57. These patients exhibit normal rMT and CSP, but display reduced SICI, and an increase in ICF of similar magnitude to the one observed in FXS patients57. Furthermore, patients with a missense mutation of the γ2 subunit show reduced [11C]-flumazenil binding61, like FXS patients38. Given that [11C]-flumazenil interacts with benzodiazepine binding sites, it is possible that the reduction in binding observed in FXS patients does not result from an under expression of the γ2 subunit per se, but from the diminished expression of other GABAA receptor subunits composing this binding site, including α1,3, β1,2 and γ2 subunits, which are all under expressed in animal models of FXS63. It thus remains to be seen if the same alterations are responsible for the reduction in SICI and [11C]-flumazenil binding in FXS. It is also noteworthy that the TMS alterations common to FXS and GABRG2(R43Q) patients differ markedly from those seen in disorders involving other aspects of the GABAergic system, including patients with succinic semialdehyde dehydrogenase (SSADH) deficiency25, who lack an enzyme involved in GABA metabolism, and Prader-Willi syndrome, caused by deletion or imprinting defect of genes encoding α5, β3, and γ3 subunits of GABAA receptors64. Considering that all of these disorders show a ubiquitous reduction of [11C]-flumazenil binding66, the sensitivity of TMS to specific elements of the GABAergic system emerges as being particularly enlightening for clinical research in neurogenetic disorders associated with inhibitory dysfunctions.Interestingly, the overall pattern of TMS abnormalities of FXS patients is strikingly similar to what is seen in patients with a missense mutation of the γ2 subunit of the GABA69. Biomarkers would benefit clinical trials by allowing a stratification of the clinical population and providing non-biased outcome measures. Eventually, they could be used to predict response to treatment and design personalized therapies. Because the consequences of FXS are primarily cognitive, emotional, and behavioral, there is tremendous interest in objectively assessing the impact of interventions on brain function. In that regards, interesting advances have been made with EEG70. Namely, auditory evoked EEG responses appeared partially normalized in FXS children treated with minocycline71, and recent data showed remarkably similar electrocortical responses in terms of neural synchronization between FXS patients and Fmr1 KO mice73, an important step towards the implementation of translational-relevant biomarkers in clinical trials. While additional research is required to assess the translational value of TMS markers for FXS, the upcoming results from two ongoing clinical trials including TMS as a secondary outcome could provide some answers . The unique features of TMS provide an interesting complement to the widely available EEG, notably by allowing for the discrimination of which inhibitory and excitatory processes aggregated in the EEG response are being modulated by an intervention. This is particularly relevant considering that all EEG markers put forth for FXS are hypothesized to involve, directly or indirectly, faulty GABAergic inhibition as an underlying mechanism73.The lack of objective outcome measures and biomarkers sensitive to the mechanisms targeted by experimental drugs is a major hurdle for clinical trials in FXS74, could not be performed. As it is usually the case in clinical studies with TMS, measures were obtained from stimulation of the primary motor cortex. It thus remains to be seen whether the alterations reported here extend to other cortical areas. This might well be the case, as the decrease in GABAA receptor binding reported by Hulst and colleagues38 seems fairly homogenous across the brain of FXS patients. The use of simultaneous TMS-EEG protocols could help assess the functional impact of this decrease in regions beyond the primary motor cortex. Indeed, using EEG to measure the brain response to TMS stimulations, recent studies have shown that paired-pulse TMS applied to the dorsolateral prefrontal cortex of healthy individuals induces EEG evoked potentials that are consistent with the electrophysiological responses typically derived from MEPs using SICI, ICF, and LICI protocols76. Although technically challenging, this approach could provide valuable insight on the inhibition-excitation imbalance across the cerebral cortex of patients with FXS.Although large for a study involving FXS patients, the sample size remains limited, which might have induced type II errors. The heterogeneity of the sample in terms of medication status can also be considered as a limitation. However, more than half of our sample was medication free and still displayed alterations identical to those observed at the whole-group level, showing that our results were not due to the presence of psychoactive drugs. Due to time constraint and to maximize collaboration from low functioning patients, relevant measures such as input-output recruitment curves and late cortical disinhibitionB inhibition illustrates the complex dynamics that are likely to be at play in terms of inhibitory mechanisms in FXS. These findings illustrate the usefulness of TMS in assessing intracortical excitability in FXS and, possibly, in monitoring it following treatment. These results also highlight the crucial need to better characterize the neurophysiology of humans with the disorder in order to accelerate the discovery of new treatment avenues.This study shows the presence of aberrant inhibitory mechanisms in patients with FXS. These alterations offer a plausible explanation for the cortical hyperexcitability typical of this disorder, and suggest new avenues for pharmacological interventions targeting the GABAergic system. Further research is required to elucidate the precise neurochemical mechanisms responsible for the physiological alterations reported here. While the GABAergic system holds promises as a therapeutic target, the increase in GABA


Journal of Translational Medicine 2018, 16(Suppl 2):A1The Cystic Fibrosis Foundation’s venture philanthropy model has been highly successful in bringing transformational therapies to people with CF. The CF Foundation established its care center network, which concentrated the patient population in centers of excellence and improved CF care, coupled with existing therapies, the care center network contributed to significant gains in survival and a CF patient registry was started at the same time. Together, these programs enabled clinical trials in adults with CF, with study design guided by registry epidemiological data. In 1998 began the first-ever discovery efforts to repair the dysfunctional CFTR. Three components of the program proved critical to its ultimate success: The CF Foundation offset companies’ risk by providing funding for discovery programs. CFTR assays were developed using human bronchial epithelial cells obtained from individuals with CF who had undergone lung transplant, and other scientific tools were readily shared. Academic scientists with relevant expertise were embedded in each industry program. In 2017, the CF Foundation met with 140 companies interested in entering the CF field and 50 clinical trials were conducted in its clinical trials network. Through its ongoing collaborative approach, the CF Foundation’s venture philanthropy program has brought hope to individuals with CF and their families.Journal of Translational Medicine 2018, 16(Suppl 2):A2In 2005, the Jain Foundation was founded with the mission of finding a treatment for a member of the Jain family diagnosed with a rare genetic muscle wasting disease called Limb Girdle Muscular Dystrophy 2B/Miyoshi Myopathy (LGMD2B/MM). The rarity of LGMD2B/MM meant there were no known treatments, or even answers to most of the questions the Jain family had following their son’s initial diagnosis. Since then, the Jain Foundation has pursued its mission with a focus on repurposing approved therapeutics and decreasing the risk profile of LGMD2B/MM, to attract the attention of pharma companies. The Jain Foundation has focused its efforts on two primary areas: improving methods to assess the efficacy of candidate therapies through a better understanding of the disease process that occurs in LGMD2B/MM, and preparing the LGMD2B/MM patient community for participating in clinical trials. A retrospective analysis of the Jain Foundation’s programs that have led to a first therapeutic clinical trial for LGMD2B/MM will be covered.Journal of Translational Medicine 2018, 16(Suppl 2):A3Sometimes a successful clinical trial just isn’t enough. As a company brings its newly-approved treatment to market, it faces questions from payers and doctors. Does the drug work in older patients, patients with poor kidney function, certain comorbidities, etc? It would be too risky to include those patients in the trial, so often these questions are left to be answered in post-market studies, which are costly and time-consuming. Another pathway exists, providing treatment access to patients in trial-adjacent populations and learning from them. This talk will describe a combined approach of expanded access and real-world data capture that can make treatment development and commercialization more efficient.Journal of Translational Medicine 2018, 16(Suppl 2):A4As we continue to expand our knowledge of the specific genetic mutations which cause many diseases, we have sub-divided what were once considered large unmet medical needs into smaller subsets amenable to precision medicines. However, as the patient populations of each of these subsets decrease, the cost of discovering, developing and commercializing the targeted medicines has remained the same. M4K Pharma, was created to address this problem by using an open science business model, which commits to, and encourages its partners, to share and publish their work and data without patents. In doing so, M4K intends to align and aggregate the work of granting institutions, academics, foundations and corporations in a cost-effective manner to discover, develop and register new therapeutics for orphan diseases.Journal of Translational Medicine 2018, 16(Suppl 2):A5Gaucher disease type 1 and Fabry disease represent the two most prevalent sphingolipid storage disorders. The standard for treatment of these diseases has been enzyme replacement therapy, limited by its expense, requirement for intra-venous infusion, loss of activity due to antibody formation, and lack of CNS penetrance. An alternative treatment strategy is the inhibition of glucosylceramide synthesis which blocks the accumulation of lysosomal glycosphingolipids. Early efforts in the pursuit of this strategy led to the identification of potent inhibitors of glucosylceramide synthase with limited off target effects. Eliglustat tartrate, the clinical lead that resulted from these efforts and was subsequently the basis for multiple phase 1, 2, and 3 trials leading to FDA approval in 2014 as the first stand-alone oral therapy for Gaucher disease type 1. However, eliglustat is unsuited for use in CNS associated glycosphingolipid storage disorders due to its inability to cross the blood brain barrier. Recent efforts have been focused on the design eliglustat analogues with good brain penetrance with the goal of developing the next generation of drugs that can be used for Gaucher disease type 3, the GM2 gangliosidoses that include Tay-Sachs and Sandhoff disease, and GM1 gangliosidosis.Journal of Translational Medicine 2018, 16(Suppl 2):A6Patients and caregivers are driving more rare disease research than ever. Inspiring stories and insightful data from the world’s largest health community show the true impact people can have on each other by sharing their experiences. Learn the approaches researchers can use to better collaborate with the people whose lives they are trying to improve. Let’s help each other.Journal of Translational Medicine 2018, 16(Suppl 2):A7Prion disease is an incurable and fatal neurodegenerative disease caused by misfolding of the prion protein (PrP). In individuals with mutations in the PrP gene, predictive genetic testing provides an opportunity to intervene years or decades before the onset of disease and preserve full quality of life, but therapeutic trials in this healthy population cannot rely on a conventional clinical endpoint. Instead, we will present a path to prevention trials centered around a surrogate biomarker endpoint informed by our clear understanding of the biological mechanism of prion disease.Journal of Translational Medicine 2018, 16(Suppl 2):A8AFMTelethon has raised > $100 M/year for 29 years, which helped to reframe the whole field, bringing it from a rather remote scientific and commercially unattractive area to a very active and innovative ground. A striking demonstration is the comprehensive genetic map of the human genome published in 1992 and 1996 by its non-for-profit biotech Genethon; a quantum leap in the human genetics field. Not only patient organizations can serve as disease-specific experts, they more importantly (1) work on reducing bottlenecks/barriers for all levels of the process of product development, (2) initiate and drive research projects, (3) provide tools , and (5) represent a voice for all aspects  of innovative drug developments.Through AFMTelethon initiatives, examples will be given of successful ecosystems, which also serve as models for frequent disorders. This includes shared scientific and financial risks in translational research by (1) directly funding and supporting drug development programs in industry or in academia (2) putting in place its own Research and Development as well as GMP biotherapeutic facilities, generating innovative drugs that are now either in the clinic or approved (3) founding start-up companies out of its own portfolio (4) taking equity shares in biotech companies, (5) launching a venture capital fund, and (6) incorporating a new type of private company dedicated to cell and gene therapy. The different actions presented are also driven by the growing challenge of fair price, which aims at ensuring patient access to the therapeutics.Journal of Translational Medicine 2018, 16(Suppl 2):A9CLN6-Batten disease is a progressive rare disease characterized by motor and cognitive impairments, seizures, and ultimately premature death. Pathologically, there is massive accumulation of autofluorescent storage material in lysosomes, gliosis and neurodegeneration. Over the last five years, our team has taken a team approach to move the needle towards having effective treatments for this fatal disorder, including development of a first-in-man gene therapy program for CLN6-Batten disease. In this presentation, we will discuss some of our successes as well as hurdles yet to be overcome. With the push towards more clinical trials for Batten disease patients, one consistent hurdle that has yet to be overcome is how to non-invasively monitor patient on trial for disease progression. Recently, our team has partnered with Charles Rivers to come up with a comprehensive biomarker scoring system in a mouse model of CLN6-Batten disease that combines imaging and behavior data to provide a biomarker scoring system for longitudinal disease monitoring. Identifying biomarkers associated with the multiple forms of Batten disease is an integral step towards the goal of improving early diagnosis, tracking progression of the disease, and monitoring response to treatment in both clinical and research environments.Journal of Translational Medicine 2018, 16(Suppl 2):A10Phenotypic screening in living cells offers an approach to identification of new drug targets and molecules for treatment of neurodegenerative disease. Sophisticated chemical genetics analyses offer an approach to identifying the pathways and targets for molecules identified through phenotypic screening. Human patient derived neurons offer a substrate to determine that molecules identified in phenotypic screens modulate cellular processes that are relevant to neurodegenerative disease.Journal of Translational Medicine 2018, 16(Suppl 2):A11Homology Medicines, Inc. is a genetic medicines company dedicated to transforming the lives of patients suffering from rare genetic diseases with significant unmet medical needs by curing the underlying cause of the disease. Homology’s proprietary platform is designed to utilize its human hematopoietic stem cell-derived adeno-associated virus vectors (AAVHSCs) to precisely and efficiently deliver genetic medicines in vivo either through a gene therapy or nuclease-free gene editing modality across a broad range of genetic disorders. Homology has a management team with a successful track record of discovering, developing and commercializing therapeutics with a particular focus on rare diseases, and intellectual property covering its suite of 15 AAVHSCs. Homology believes that its compelling preclinical data, scientific expertise, product development strategy, manufacturing capabilities and intellectual property position it as a leader in the development of genetic medicines.Journal of Translational Medicine 2018, 16(Suppl 2):A12Myotonic Dystrophy type 1 (DM1), is an RNA-based disease caused by a transcribed CTG-repeat expansion within the 3’ UTR of the DMPK gene. Mutant repeat expansion transcripts remain in the nucleus of patient’s cells, forming foci that contribute substantially to the pathophysiology of the condition. Previously we developed a high throughput screening assay based around this disease feature and identified kinase inhibitors that were successful in removing these nuclear foci. We have sought to identify the kinase target responsible. We utilised a high content imaging screen to assess a large, well-characterised kinase inhibitor library for compounds that reduce nuclear foci to identify their common targets. A combination of chemoproteomics and mass spectrometry revealed a novel kinase target in DM pathophysiology. This kinase was analysed by western blot analysis and by detailed expression analysis in patient derived cells and biopsy samples. A compound inhibitor of this kinase was tested in the HSALR mouse model to assess phenotypic effects. The novel kinase is elevated in DM1 cell lines and patient muscle biopsies. Inhibition leads to the dissolution of nuclear foci and degradation of repeat expansion transcripts. Inhibitor treatment in the HSALR mouse led to beneficial phenotypic effects on myotonia and improvement of key splice isoforms. Our methods validate the use of phenotypic screening in drug discovery and highlight a collection of molecules suitable for further development in the therapeutic effort against DM1.Journal of Translational Medicine 2018, 16(Suppl 2):A13With now multiple modalities of gene therapy achieving regulatory approval, a growing body of clinical experience has supported commercial investment in gene therapy technologies. However, given the prohibitive costs of vector development, this investment has largely been limited to indications for which sufficiently large patient populations exist to support a financially viable model of commercialization. There exist many therapies for rare disorders that have robust pre-clinical proof-of-concept but due to ultra-rare prevalence lack a viable commercial model. Ultra-rare disease needs to establish new pre-clinical and clinical models that recognize the costs associated with bringing therapies to the clinic. Odylia Therapeutics, a non-profit gene therapy company, bridges the gap between researchers and drug developers by conducting preclinical and clinical development of gene therapies. Odylia hopes to usher in novel efficiencies and economies of scale in pre-clinical and clinical vector development. Odylia has designed a strategic plan that focuses on redefining a lean yet safe commercialization strategy, allowing therapies for rare disease to progress to the clinic more efficiently. Odylia precisely and iteratively determines how to increase efficiency and effectively move more therapies from the lab to the clinic. Odylia seeks to redefine lean clinical trials, including a new paradigm of a combined phase I/II/III clinical trial, which will increase patient access to emerging therapies. Odylia’s pre-clinical and clinical processes are made available for collaboration with for-profit development companies. Through these for-profit/non-profit collaborations, Odylia represents a new paradigm of drug development which will enable previously non-viable therapies to reach clinical viability.


Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction and is commonly seen in the elderly and diabetic and hypertensive patients. Despite its rising prevalence, the pathophysiology of HFpEF is poorly understood and its optimal treatment remains undefined. Recent clinical studies indicate that coronary microvascular rarefaction  with reduced coronary flow reserve (CFR) is a major contributor to diastolic dysfunction in HFpEF patients. On a molecular level, endothelial cells (EC) are dependent on glycolysis for supporting their functions and vascular homeostasis. Sirtuin 3 (SIRT3) has a critical role in the regulation of endothelial glycolytic metabolism and thus affects angiogenesis. Disruption of SIRT3-mediated EC metabolism and impairment of angiogenesis may promote cardiomyocyte hypoxia and myocardial fibrosis, leading to diastolic dysfunction and HFpEF. This review summarizes current knowledge of SIRT3 in EC metabolism, coronary microvascular rarefaction and HFpEF. Heart failure (HF) is a leading cause of death in the United States and worldwide. HF is a progressive disease that develops with advanced age, hypertension and diabetes. Each year over 600,000 patients are diagnosed with HF in the United States. More than half of these patients are diagnosed as heart failure with preserved ejection fraction (HFpEF) , 2. Dias+) dependent Class III histone deacetylases. They comprise of seven different proteins (Sirt1-7) and have been shown to regulate a broad extent of physiological and pathological processes, including energy production, stress resistance, reactive oxygen species (ROS), mitochondrial homeostasis, apoptosis, and aging  and impaired CFR is a powerful independent correlate of cardiac mortality and coronary microvascular rarefaction in patients with heart failure, especially HFpEF . PatientPrevious studies showed that overexpression of SIRT3 blocks cardiac hypertrophy whereas knockout of SIRT3 in aged mice promotes cardiac hypertrophy , 53. EmeECKO mice manifested reduced myocardial capillary density with impairment of CFR and diastolic function  mice were developed and their cardiac function was assessed. Similarly, SIRT3 ECKO mice developed diastolic dysfunction along with decreased CFR, but without reduced systolic function probably due to the younger age of the mice (6–8 months old other than 12 months old) , 37. TheSIRT3 has great potential in the modulation of a variety of cellular processes, through the link between protein acetylation and various physiological functions and diseases. Although SIRT3-mediated cardiovascular diseases have been intensively investigated and our understanding of the biology of SIRT3 has expanded remarkably over the past decade , the rolAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Titin, the largest protein known, has attracted a lot of interest in the cardiovascular field in recent years, since the discovery that truncating variants in titin are commonly found in patients with dilated cardiomyopathy. This review will discuss the contribution of variants in titin to inherited cardiac conditions (cardiomyopathies) and how model systems, such as animals and cellular systems, can help to provide insights into underlying disease mechanisms. It will also give an outlook onto exciting technological developments, such as in the field of CRISPR, which may facilitate future research on titin variants and their contributions to cardiomyopathies. Titin, historically also known as connectin Maruyama , is the TTN, 363 exons) makes it difficult, expensive and time-consuming to interrogate by conventional mutation screening approaches such as Sanger sequencing. Additional complicating factors include (i) alternative splicing, generating various differentially expressed isoforms in tissues and (ii) that a large portion of TTN consists of repetitive tandem Immunoglobulin-like encoding sequences and that variants in these regions may have very little effect on TTN length and function (Greaser The enormous size of the human titin gene ( Greaser .TTN locus (2q31) was associated with familial autosomal dominant dilated cardiomyopathy (DCM) for the first time   are a common cause of DCM, occurring in approximately 25% of familial and 18% of sporadic cases, but only present in 1% of HCM cases. Spatial distribution of variants found in HCM subjects and controls was distinct from that of DCM subjects. The DCM variants were predominantly located in the A-band region. This data has been integrated with RNA sequencing (RNAseq) data from diseased left ventricular samples to help understanding the role of TTNtv  and they estimated that 50% of the TTNtv identified in healthy controls occur in low PSI exons. Therefore, variants located in high PSI exons, including both N2BA and N2B isoforms, are strongly enriched in DCM patients versus controls. The penetrance of TTNtv in DCM appears to be strongly correlated with its position; distal I-band and all A-band variants show larger odd ratios compared to very proximal or distal ones  and that both the N2B-exon and N2A-exon containing isoforms of ttna are required for sarcomere assembly in the heart, while ttnb is dispensable for cardiac contractility  into pluripotency using reprogramming factors  /CRISPR-associated protein 9 (Cas9) or Transcription activator-like effector nucleases  exhibited a marked reduction in contraction amplitude, alterations in calcium transients and sarcomeric structural disruption, with less iPSC-CM displaying fully organised sarcomeres compared to iPSC-CM from a healthy control individual  and guided to the target locus without genetic perturbation , blocks transcriptional activity by a steric hindrance between dCas9 and the transcriptional machinery (Larson et al. In summary, the CRISPR/Cas9 system provides exciting tools beyond conventional genome-editing to study e.g. the consequences of titin variants in the cardiac system.While a causal role for TTNtv in DCM is now well established, the underlying disease pathways are still not fully understood. Animal models have highlighted a crucial role of titin for the integrity of the heart and have helped to gain insight into aspects of titin’s function, such as sarcomere assembly and signalling and their perturbations by titin variants. However, animal models are limited by species-related differences in morphology and physiology to humans. As an alternative, studies of human cardiac tissue can also provide insights into the patho-mechanisms of titin variants, but such tissue is rarely available in sufficient quality and quantity. Therefore iPSC-CM emerge as a novel model system to study titin-related cardiomyopathy. These cells allow mimicking human genetic disease in a human cardiac cellular model. While still in its infancy and currently plagued by the issue of immaturity, it provides exciting approaches to study the role of titin variants in cardiomyopathies and we are looking forward to future developments in the field.


Recent research indicates crucial roles of autophagy during sepsis. In animal models of sepsis induced by cecal ligation and puncture (CLP) or the systemic administration of lipopolysaccharides (LPS), autophagy is implicated in the activation and/or damage of various cells/organs, such as immune cells, heart, lung, kidney, and liver. Since sepsis is associated with an increased production of pro- as well as anti-inflammatory cytokines, it has long been considered that hypercytokinemia is a fetal immune response leading to multiple organ failure (MOF) and mortality of humans during sepsis. However, a recent paradigm illuminates the crucial roles of mitochondrial dysfunction as well as the perturbation of autophagy in the pathogenesis of sepsis. In the livers of animal models of sepsis, autophagy is involved in the elimination of damaged mitochondria to prevent the generation of mitochondrial ROS and the initiation of the mitochondrial apoptotic pathway. In addition, many reports now indicate that the role of autophagy is not restricted to the elimination of hazardous malfunctioning mitochondria within the cells; autophagy has been shown to be involved in the regulation of inflammasome activation and the release of cytokines as well as other inflammatory substances. In this review, we summarize recent literature describing the versatile role of autophagy and its possible implications in the pathogenesis of sepsis in the liver. Autophagy, derived from the Greek ("auto" meaning "self" and "phagy" meaning "eat"), is a cellular system to degrade intracellular unnecessary materials, including proteins and organelles, for recycling 2autophagy related) genes 55Autophagy is executed through a set of genes called atg  animals, it has been demonstrated that basal autophagy is required to maintain liver homeostasis. Komatsu, Sepsis caused by infection, trauma, or endotoxins leads to an excessive and uncontrolled immune cell response 912Concurrent with the rising suspicion that uncontrolled immune responses might not play a central role in the pathogenesis of sepsis, mitochondrial dysfunction has come into the spotlight. Examinations of skeletal muscle biopsy samples from critically ill patients suffering from sepsis have shown that mitochondrial damage is more severe in patients who subsequently die than in those who eventually survive The molecular mechanisms underlying mitochondrial dysfunction caused by LPS (lipopolysaccharide)stimulation in the liver are poorly understood. On the other hand, a possible mechanism for mitochondrial reprogramming in immune cells has recently been proposed. Macrophages undergo metabolic reprogramming upon activation by immunostimulation such as LPS stimulation 16et al.Direct evidence of autophagy in liver injury during sepsis has been reported by Watanabe, Although its role in protecting hepatocytes against septic insult has been established, the cellular degradation activity of autophagy might not be fully activated in the liver during sepsis: several reports have pointed out perturbations in autophagy. There is a report indicating that in the hearts of sepsis model mice, autophagic flux is increased during the initial phase of sepsis, but declines at a later phase Fig. 1).Several studies have proposed TNFα as a crucial mediator of liver toxicity during sepsis 2324Fig. 2). During the first step, NF-kB is activated to induce the expression of NLRP3 as well as cytokines. Then, in the second step, NLPR3 forms a complex, the so-called "inflammasome", with other molecules including caspase-1  and ASC. Indeed, it has been shown that LPS stimulation rapidly induces the expression of NLRP3 and other inflammasome components in hepatocytes 333335LPS binds to its cognate receptor, TLR4, and promotes the synthesis of cytokines, chemokines, and type-I interferons In addition to the canonical pathway for intracellular LPS signaling, which is initiated by the ligation of LPS to TLR4, LPS also binds directly to and activates cytosolic caspase-11 in mice (caspase-4 and -5 are the human homologues of murine caspase-11) 38404343et al., using a comparative proteomics approach, found CPS-1 et al. also confirmed the release of CPS-1 into circulation in CLP-sepsis model mice Carbamoylphosphate synthase1 (CPS-1) is a urea cycle enzyme that catalyzes the generation of carbamoyl phosphate from ammonia and bicarbonate. CPS-1 expression is restricted to the liver and to some extent the intestine. In an experiment to find liver proteins whose expressions are dysregulated during sepsis, Struck CPS-1 release into the circulation prior to ALT during sepsis has also been confirmed in the LPS model Table 1). Carbamazepine (CBZ), which is an anti-epileptic drug that was recently identified as an autophagy enhancer, can alleviate liver injury in mice with CLP-sepsis 2+-signaling in LPS-induced autophagy in the liver. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+-mobilizing molecule that is different from cyclic ADP-ribose (cADPR) and myo-inositol 1,4,5-triphosphate (IP3) 2+ from different intracellular storage sites, including ER and lysosomes In accordance with the supposed perturbation of autophagy at least in the later stages of sepsis 25Table 2). Wortmannin is a PI3K inhibitor that is frequently used as an inhibitor of autophagy +-channel On the other hand, there are several reports demonstrating autophagy as an exacerbating factor in sepsis . Unconventional functions of autophagy include a broad range of cellular activities, among which is the secretion of intracellular contents. Recent research suggests that autophagy plays important roles in septic liver failure, not only through intracellular degradation, but also via the secretion of cell contents.Although autophagy is considered to be an intracellular degradation mechanism, recent research progress has shown it plays roles in other processes than the intracellular degradation of excess and/or unnecessary materials , Ushio Nakajima and colleagues showed that MEFs from c-Flip KO mice stimulated by TNFα extrude mitochondria from the cells et al. showed that the secretion of ATP is executed through the exocytosis of lysosomes in which ATP is stored ATP is often released from damaged cells and acts in DAMPs (damage-associated molecular patterns) to activate immune cells. Tumor cells often show immunological responses, such as the release of ATP, during chemotherapy-induced cell death et al. have provided evidence for the release of autolysosomal contents from cells Takenouchi Necroptosis is a form of programmed cell death that is executed by defined molecules such as receptor-interacting serine/threonine-protein (RIP) 1 and 3, and MLKL (mixed lineage kinase domain like pseudokinase), but morphologically resembles necrosis rather than apoptosis Many literatures have shown that autophagy is essential to maintain liver homeostasis under both healthy and disease conditions. Autophagy is consisted from multiple steps and the completion of the whole processes of autophagy is the prerequisite for its cytoprotective role. Although mitochondrial dysfunction and subsequent induction of autophagy to eliminate damaged mitochondria should be important to protect liver and other organs/tissues from septic injuries, the fate of damaged mitochondria might be dependent on lysosomes. There seems to be at least two destinations of mitochondria during sepsis: digestion within the cells and secretion into extracellular space. Factors determining the fate of mitochondria should be examined in the future studies. In addition, chaperone-mediated autophagy has also been shown to be necessary for liver homeostasis The extrusion of intracellular contents/substances from immunologically activated cells raises the question as to why cells actively secrete these contents. One explanation is that the intracellular capacity to degrade dysfunctional materials reaches its limit. Indeed, several studies have pointed out that the flux of autophagy is prone to stagnate in the liver during sepsis 4761


This istabolism , as implmBio study, in another mouse model of CDI indomethacin use was associated with reduced colon PGE2 levels .Noori and colleagues conclude their letter with the idea that indomethacin might have induced gut microbiota dysbiosis in association with CDI-induced proinflammatory changes that triggered the induction of inducible cyclooxygenase 2 (COX-2) enzyme and thereby enhanced the production of PGE2 are clearly important in gastrointestinal homeostasis, and understanding the complex mechanisms underlying the therapeutic and detrimental effects of NSAIDs in this context is important, given the significant popularity of this class of drugs in clinical medicine.Eicosanoids such as PGE


Schistosoma mansoni (S. mansoni)-derived lipids are agonists of innate pattern recognition receptors on eosinophils, mediating a pro-fibrotic phenotype. Indeed, the release of lipids from Schistosoma could be a key factor driving disease pathogenesis in hepatosplenic forms of the infection, where excessive hepatic fibrosis is linked to significant morbidity. A fundamental question that remains is how are lipids derived from the tegumental outer surface of S. mansoni adult worms, cercariae and eggs, transported and protected from the inflammatory milieu to target and activate surface receptors on eosinophils. The recent identification of lipid-enriched extracellular vesicles (EVs) as an evolutionarily conserved form of host-pathogen communication, has led us to speculate that S. mansoni-derived extracellular vesicles are responsible for the targeting of bioactive lipids to eosinophils, and we argue that these cargo delivery systems may be an influential factor in both tissue repair and fibrosis during helminth infection.The co-evolution of helminths with their hosts has required these parasites to develop a range of sophisticated molecular mechanisms and adaptations to evade, suppress and activate host cells to maximize survival and maintain infection within their chosen niche. Recent studies have revealed that Schistosoma. The schistosmiasis lifecycle involves free-living larval cerceriae that penetrate human skin, mature into schistosomula in the tissues then migrate through the lungs and systemic circulation before residing in either vesical (S. haematobium) or mesenteric  veins as sexually mature adults where they can evade the immune system for years  and prostaglandin (PG)D2, can activate eosinophils via toll-like receptor 2 (TLR2) and prostaglandin D2 receptor 1 (DP1) promoting the release of TGF-β to support both fibrosis and tissue repair  , so asseS. mansoni lipidome study found LPC to be enriched in cercariae and eggs, although only present as a minor phospholipid in the adult worm suggesting the amount of LPC within the worm tegument may not be sufficient to engage and ligate TLRs to drive cellular activation  highlights their potential clinical applications 2-fold. Enrichment of LPC and PDG2 lipids within the serum above levels of a healthy subject could be used as diagnostic biomarkers and it would be of interest to see if serum levels of these lipids correlated with disease severity or more excitingly, increased fibrosis. Moreover, LPC and PDG2 have been identified as promising new vaccine targets in masonic and japonica schistosomiasis. Now it is of importance to determine whether targeting these schistosomal lipids through vaccination will alter the recruitment and activation of eosinophils to the site of inflammation and influence or limit disease pathology during schistosome infection, which could have broader implications for infections in which eosinophils play a key role.JB and GC drafted the manuscript. JB, GC, and MW edited the manuscript. JB, GC, and MW approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genetic studies have revealed that many epilepsy susceptibility genes encode ion channels, including voltage-gated sodium, potassium and calcium channels. The high prevalence of ion channels in epilepsy pathogenesis led to the causative concept of “ion channelopathies,” which can be elicited by specific mutations in the coding or promoter regions of genes in genetic epilepsies. Intriguingly, expression changes of the same ion channel genes by augmentation of specific transcription factors (TFs) early after an insult can underlie acquired epilepsies. In this study, we review how the transcriptional regulation of ion channels in both genetic and acquired epilepsies can be controlled, and compare these epilepsy “ion channelopathies” with other neurodevelopmental disorders.Epilepsy is a common neurological disorder characterized by recurrent uncontrolled seizures and has an idiopathic “ Epilepsy is a severe chronic brain disorder characterized by recurrent seizure activity due to aberrant neuronal network activity  and hippocampal sclerosis Engel,  can playShorvon, .In the last decades, enormous progress has been made in the discovery of epilepsy genes, resulting in a current list of approximately 1,000 epilepsy-associated genes  correlated with autosomal dominant nocturnal frontal lobe epilepsy  rare variants with high penetrance  or of (ii) common variants with low penetrance  epilepsy with febrile Seizures Plus , as well as nonsense mutations (mutations causing a premature stop codon), can underlie epilepsy pathogenesis by inducing a loss-of-function (LOF) or a gain-of-function (GOF) channelopathy. In addition, also deletions and duplications of (part of) the gene can strongly affect normal channel function  and tonic (perisynaptic) inhibition  compared to the GABRB3-haplotype-1 promoter (overrepresented in controls). The difference in expression could be explained by reduced binding of the TF N-Oct3 to the GABRB3-haplotype-2 promoter, resulting in decreased expression of the GABRB3 gene  or a repressor, it may result in altered regulation of the transcriptional machinery. For example, four different haplotypes, consisting of 13 SNPs located in the 5’ region of the Acquired epilepsies are epilepsies, which are on the consequence of an environmental factor. These epilepsies can be: (i) completely dependent on environmental factors; or (ii) can be caused by an interaction of environmental factor(s) with a predisposition genome. In the latter case, the presence of common susceptibility variants  can lower the threshold of the environmental factor for inducing an epileptic outcome. Most of our current knowledge of acquired epilepsy pathogenesis comes from the use of animal models, in which insults causing TLE can be mimicked in rodents using approaches like traumatic brain injury, kindling, or by applying one of the chemo-convulsants pilocarpine or kainic acid to induce SE , which can as a consequence dysregulate the transcriptional machinery of many genes, including ion channel genes  upon a rise in intracellular zinc ([Zn2+]i); denoted as the Zn2+-MTF1-CaV3.2 cascade  can strongly affect the transcriptional machinery . miRNAs are 22 nucleotides noncoding RNAs that can regulate gene expression by associating with the RNA-induced silencing complex (RISC). The RISC complex then uses the miRNA as a template for recognizing the complementary mRNA of the ion channel gene , schizophrenia, bipolar disorder, major depressive disorder and migraine  general underlying transcriptional phenomenon, possibly explaining the comorbidity between epilepsy and other neurodevelopmental disorders.genetic and acquired epilepsies. Although a large amount of data exists, our understanding of transcriptional mechanisms governing ion channel expression is far from complete and requires further detailed investigation, not only for epilepsy pathogenesis, but also for other neurodevelopmental disorders. A better understanding of the underlying mechanisms might result in the development of novel drugs and may provide opportunities for better-individualized treatment strategies.In this study, we reviewed the mechanisms involved in the transcriptional regulation of channelopathies in Both authors contributed to the writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors AB.


A major impediment to the long-term in vivo vascular imaging is a lack of suitable probes and contrast agents. Our developed mercaptosuccinic acid (MSA) capped cadmium telluride/cadmium sulfide (CdTe/CdS) ultrasmall quantum dots (QDs) have high fluorescent quantum yield, long fluorescence lifetime and long half-life in blood, allowing high resolution long-term intravital vascular imaging. In this study, we showed that these QDs can be used to visualize the in vivo the vasculature in normal and cancerous livers in mice using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging (FLIM), with cellular resolution (~1 µm) up to 36 h after intravenous injection. Compared to highly regulated and controlled sinusoids in normal liver tissue, disordered, tortuous, and immature neovessels were observed in tumors. The utilized imaging methods have great potential as emerging tools in diagnosis and monitoring of treatment response in cancer. Optical-based intravital imaging of vasculature is an emerging modality for studying vascular structure, function, and angiogenesis. Changes in the number and spacing of vessels, permeability of the vasculature and vascular function have been implicated in many diseases, including malformation and cancer . AlthougWe have successfully synthesized long-circulating QDs in our laboratory and their in vivo fate has been fully investigated ,5,8. OurIlium xylazile and ketamine hydrochloride were purchased from Bayer Australia . Hepa 1-6 cells were obtained from ATCC  and maintained in vitro under cell culture conditions recommended by ATCC.QDs were mercaptosuccinic acid  capped cadmium telluride/cadmium sulfide  particles, recently developed in our laboratory . These QAll animal procedures were approved by the health sciences ethics committee of the University of Queensland (ethics number is 521/12 and approved on the year of 2013) and were carried out in accordance with the legislation of Australian authorities for the care and use of experimental animals.7 Hepa1-6 cells into BALB/c nude mice via open surgical technique and imaging procedures were performed after 14 days .Liver images were obtained at the excitation wavelength of 900 nm to selectively capture QD fluorescent signal since liver autofluorescence of NADH is absent at this excitation wavelength . As showIn addition to fluorescent signals, FLIM could map the spatial distribution of fluorophores based on fluorescence lifetime, the average time of an electron stays in the excited state before returning to the ground state . LifetimFinally, we did the histological analysis of key organs of mice treated by QDs, which allows for detailed microscopic evaluation and histological assessment of tissue interactions. Since QDs are mainly accumulated in the liver, spleen, and kidney for a long period, it is necessary to understand their toxicity in these organs. As shown in In this study, we found that the anionic ultrasmall QDs present new opportunities for intravital vascular imaging as they retain in vascular for a long time after systematic injection without uptake by parenchymal cells. Compared to conventional in vivo imaging, MPM has the advantages of less photobleaching and photodamage, and has considerably enhanced imaging penetration depth due to less scattered multiphoton excitation in samples . Fresh, Vascular imaging is of particular importance in tumor biology where imaging can be used to study the growth and progression of cancer, and assess the efficacy of anti-tumor and anti-angiogenesis drugs . Once thIn summary, we show that anionic ultrasmall QDs can be used to visualize the in vivo vasculature in normal and cancerous livers in mice, with cellular resolution (~1 µm), up to 36 h after intravenous injection. MPM coupled with FLIM has great potential as emerging tools in diagnosis and monitoring of treatment response in liver cancer.


For decades, Alzheimer's disease (AD) research has focused on the two pathological hallmarks of the disease: amyloid plaques and neurofibrillary tangles. Reactive astrocytes and activated microglia decorating amyloid plaques are other long known pathological features of the AD brain –4, yet oPerez-Nievas and Serrano-Pozo). Next, Garcia-Esparcia et al. compare the astrocyte reaction present in AD and dementia with Lewy bodies (DLB) brains by quantifying both protein and mRNA levels of several astrocyte markers such as glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 2 (EAAT2/GLT-1), and aldehyde dehydrogenase 1 L1 (ALDH1L1). They observed a non-significant reduction of EAAT2/GLT-1 protein levels and a normal EAAT2/GLT-1 immunoreactivity around plaques, suggesting limited consequences of astrocyte reaction for glutamate transport in AD.We start with a comprehensive review on the role of reactive astrocytes in the disease, highlighting the heterogeneity and complexity of astrocytes in the healthy brain, the molecular signaling pathways involved in astrocyte reaction in AD, the phenotypic changes exhibited by reactive astrocytes in the AD brain, and the consequences of this astrocyte reaction with respect to plaques, tangles, neurons, and synapses  and its implication in amyloid plaque and tangle formation from studies on Trem2 deficient AD mouse models. They also review their recent finding that TREM2 enhances microglial metabolism through the mammalian target of rapamycin (mTOR) pathway  to amyloid and tau pathologies. Thei et al. contribute with a review of the ion channels expressed by homeostatic microglia, their potential disruption in activated microglia in AD, and how human inducible pluripotent stem cell (hiPSC)-derived microglia could be a better tool than primary microglial cultures to elucidate the role of these ion channels. And, finally, Chun et al. summarize their experience with novel in vitro approaches to study glia, including microfluidic devices with human microglia exposed to Aβ to investigate microglial chemotaxis  have been widely used for almost two decades to image activated microglia in vivo (11C]deuterium-L-deprenyl ([11C]DED)—an irreversible inhibitor of monoamine oxidase B—has recently been introduced to image reactive astrocytes  to study astrocyte morphology and distribution (Lastly, the Frontiers Research Topic/eBook deals with the imaging methods available to study reactive glia. trocytes , which atrocytes . Hierro-ribution , and theribution . Practicin vitro and in vivo in AD patients and mouse models.In summary, although acknowledging that the topic of glial cells in AD is a rapidly evolving field, we believe that the present Frontiers Research Topic/eBook will provide the interested readers with the most recent developments on the role of reactive astrocytes and activated microglia to AD pathophysiology, and the latest technical advances to study and image these glial cells All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Purpose: To simulate numerically the collagen fibril reorientation observed experimentally in the cornea. Methods: Fibril distribution in corneal strip specimens was monitored using X-ray scattering while under gradually increasing axial loading. The data were analysed at each strain level in order to quantify the changes in the angular distribution of fibrils with strain growth. The resulting relationship between stain and fibril reorientation was adopted in a constitutive model to control the mechanical anisotropy of the tissue material. The outcome of the model was validated against the experimental measurements before using the model in simplified representations of two surgical procedures. Results: The numerical model was able to reproduce the experimental measurements of specimen deformation and fibril reorientation under uniaxial loading with errors below 8.0%. With tissue removal simulated in a full eye numerical model, fibril reorientation could be predicted around the affected area, and this change both increased with larger tissue removal and reduced gradually away from that area. Conclusion: The presented method can successfully simulate fibril reorientation with changes in the strain regime affecting cornea tissue. Analyses based on this method showed that fibrils tend to align parallel to the tissue cut following keratoplasty operations. With the ability to simulate fibril reorientation, numerical modelling can have a greater potential in modelling the behaviour following surgery and injury to the cornea. Soft tissue has an important role to bind and structurally support the components of the human body using an extracellular matrix (ECM) composed of proteins, glycosaminoglycan and water . The ECMA more recent challenge has been the growing evidence that the orientation of fibrils is not static but changes with variations in the strain, to which the tissue is subjected, which typically arise due to surgery, injury or disease ,12,13,14These changes are believed to be due to the biosynthesis and degradation in tissue with cells modulating their behaviour according to alterations in their biomechanical and biochemical environment ,21, and Several biomechanical models have been developed to account for changes in the microstructure characteristics of soft tissues and represent the resulting changes in tissue’s mechanical response to load. Most models considered fibril remodelling (or volume-fraction-based reorientation) as a response to the mechanical stimuli caused by changes in either stresses or strains ,24,25. HThis study presents a step forward in seeking to simulate the simultaneous changes that can take place in fibril quantity at any point in 16 equally-spaced orientations. It also builds on an earlier step where experimental measurements were taken of fibril density and orientation across strips of corneal tissue subjected to uniaxial loading . That stThe material model considered the nonlinear and anisotropic material behaviour using a strain energy function. Isochoric split into volume changing behaviour and volume distortional behaviour was performed on the energy function. The volume changing behaviour concerned the material deformation that led to density change. Decupled from the material volume changes, the volume distortional behaviour was subjected to deformation of collagen fibrils and the ground substance matrix of ocular tissue. Therefore, the material model adopted the strain energy function with 3 components for volume dilation W¯LamIn our earlier study, 13 strip specimens with 3.5 mm width and 20 mm length were obtained along the superior-inferior principal meridian from donor corneas aged between 21 and 90 years old. The specimens were clamped at the ends, leaving a distance of 16 mm in between, and tested under an axial tensile strain up to 8% . The ethComparisons between repeated measurements of total fibril content at each point revealed small variations (within 12.7% of the average), possibly due to measurement noise or small changes in location of scanned points from one strain level to another. As the ex-vivo tissue could not synthesise further collagen fibrils, a normalisation process was adopted whereby the total fibril content at each measurement point was equated to 1.0 in order to remove these small variations.While 256 fibril density measurements provided only 128 unique values, this amount of data at each scanned point presented a challenge in the analysis of reorientation. In earlier work, we conducted an optimisation study to determine the minimum number of orientations that could be adopted without significantly affecting the outcomes of numerical models based on this microstructure data . In thatFollowing normalisation and reduction of the fibril density data to 16 orientations, the mean density in each orientation at each measurement point for all 9 specimens was calculated. By following this process at each strain , the change in fibril density with strain growth was determined as:The changes in angular fibril density between the 5 strain levels considered were assumed to vary linearly. This meant that the total value of Additionally, since specimens were aligned with the corneas’ vertical centre line, around which there was earlier evidence of symmetry in the central cornea, symmetrical fibril distribution was assumed around the specimens’ longitudinal axis. Therefore,As explained in our earlier publication, the regional variation and anisotropy of fibril distribution across the ocular globe were introduced in the material constitutive model through fitting the microstructure data to Zernike polynomials. The polynomials were then used to estimate the fibril density and angular distribution at the integration points of the model’s finite elements ,31, FiguConsidering the difference between the uniaxial loading condition, which was applied in the experiments, and the physiologic conditions where the intraocular pressure (IOP) affected all radial and tangential directions, a loading condition compensation process was needed. First, the trends of fibril reorientation were quantified by the uniaxial test analysis before being overlapped when implemented in each of the 16 fibril orientations at every integration point in the model, After deriving the fibril reorientation trends based on the X-ray scattering data, these trends were embedded in a finite element simulation of the uniaxial tissue test and the fibril reorientations predicted numerically at different strain levels were compared with those observed experimentally in order to validate the reorientation trends.A numerical model was created to simulate the uniaxial test, A model made of a single element was used to provide a further assessment of the fibril reorientation algorithm and to check the ability to maintain no change in total fibril content at individual integration points as reorientation takes place with strain growth. The model was composed of an 8-noded linear, hybrid brick element (C3D8H) with dimensions 10 × 10 × 10 mm, n points b followec = 7.8 mm [L = 5.85 mm [s = 11.5 mm [Full-eye numerical models were used to represent (1) an intact case, (2) a case with the anterior corneal stroma removed within the central 6 mm diameter zone, providing an approximate representation of tissue loss in laser-assisted in situ keratomileusis (LASIK), and (3) a case with the full corneal thickness removed within the central 6 mm diameter zone, representing penetrating keratoplasty. The first model included 12,696 15-noded continuum elements (C3D15H) arranged in 2 layers and 8 rings in the cornea and 38 rings in the sclera, = 7.8 mm ,33, cent= 7.8 mm , periphe 5.85 mm ,36, scle 11.5 mm , scleralThe mean values and standard deviations (SD) of fibril density recorded across the specimen width in the 16 discretised orientations and with strain growth from 0% to 8% are shown in Based on the evident symmetry of fibril density around the axial direction , the mean of the density data in each two opposite orientations  was plotted against strain in The fibril reorientation trends, developed in The single-element model simulations showed, in a simple case, that asymmetry of loading triggered fibril reorientations that became clearer with higher strain asymmetry and higher strain levels, This application included three cases with an intact globe, a globe with the anterior stroma  removed and one with full-thickness tissue removal from the middle 6 mm-diameter zone. The behaviour of the three cases in terms of fibril reorientation under the action of an IOP of 30 mmHg is presented in Ocular diseases and refractive surgery may result in significant changes in the tissue’s microstructure, making it necessary to quantify the associated changes in mechanical behaviour under the eye’s common loads, primarily IOP ,13,19,20In this study, it is further assumed, based on experimental evidence, that while fibril content at any particular location does not change with strain growth, fibril anisotropy does. This principle of fibril reorientation has been incorporated into earlier microstructure-based constitutive models in different ways. Earlier models considered only two preferred fibril orientations whose vectors changed direction with strain growth ,27, whilThe study adopted an approach whereby fibril density (content divided by tissue thickness) varied with strain in pre-determined 16 directions, instead of reorienting the directions themselves. The approach, while being simpler to implement in numerical modelling, should not lead to any significant variation in results due to the large number of orientations (16) adopted. The predictions of models, which used this approach to simulate fibril reorientation, matched closely the results of the ex-vivo tissue testing in terms of fibril distribution under different strain levels up to 8%.Following this limited validation, the implementation of the reorientation algorithm in whole globe models simulating intact cases and cases with partial or full loss of central corneal tissue showed how the models responded to the different strain distributions in the zone immediately next to the tissue loss zone. In that region, a growth in circumferential strain caused by tissue loss led to growth in fibril density in that direction that increased with internal load, proximity to the edge of the tissue loss zone and magnitude of tissue loss. However, the growth in density of circumferential fibrils (max 6%) remained below 15% of the total collagen fibril density, which was observed in a single cornea that underwent penetrating keratoplasty (PK) and whose microstructure was characterised using the X-ray scattering technique . This laTo conclude, a new method has been proposed to simulate the reorientation of collagen fibrils in corneal tissue following significant changes in strain regime. The method relied on experimental tests on ex-vivo corneal tissue subjected to uniaxial tension when being scanned using X-ray scattering. An algorithm representing the fibril reorientation trends, observed experimentally, was developed and implemented in numerical models of the cornea and whole eye globe, and shown to demonstrate reorientation of collagen towards directions of higher strain.


Spider monkeys are important dispersers of many hardwood trees that contribute greatly to the carbon sequestration of tropical forests. One way in which Spider monkeys influence tropical ecosystem structure and function is through the creation of visible terrestrial latrines beneath their “sleeping sites”—trees in which they frequently return to sleep. Spider monkey latrines are thought to create high quality resource patches for rainforest plants and other wildlife to exploit. We investigate this using camera traps placed in both the canopy and on the rainforest floor to determine which rainforest wildlife are attracted to the latrines beneath the sleeping sites of spider monkeys. We also assess the tree species and dung beetles found within the latrines compared with other areas of the forest. Our evidence suggests that spider monkey roosting sites are a hub of activity for other rainforest wildlife, and act as germinating beds for many rainforest trees. If rainforests were to lose spider monkeys, from intensive hunting for example, many other rainforest wildlife species would be affected, and forests would therefore be made up of different tree communities than landscapes where spider monkeys exist.The sleeping site behavior of Ateline primates has been of interest since the 1980s, yet limited focus has been given to their influence upon other rainforest species. Here, we use a combination of arboreal and terrestrial camera traps, and dung beetle pitfall traps, to characterize spider monkey sleeping site use and quantify the impact of their associated latrines on terrestrial vertebrate and dung beetle activity. We also characterize the physical characteristics of the sleeping sites and the floristic and soil composition of latrines beneath them. Spider monkey activity at sleeping sites peaked at dawn and dusk and group composition varied by sex of the adults detected. The habitat-use of terrestrial fauna (vertebrates and dung beetles) differed between latrine sites and non-latrine controls, underpinned by species-specific changes in the relative abundance of several seed-dispersing species (such as paca and great curassow). Seedling density was higher in latrines than in non-latrine controls. Although most soil properties were similar between latrines and controls, potassium and manganese concentrations were different. These results suggest that spider monkey sleeping site fidelity leads to a hotspot of ecological activity in latrines and downstream impacts on rainforest floristic composition and diversity. Ateles geofroyii) is an endangered Ateline primate and a major disperser of large-seeded hardwood trees, which contribute substantially to the biomass and carbon storage value of tropical forests [The Central American or Geoffroy’s spider monkey . Howeve forests .The sleeping site behavior of primates has been of interest to tropical ecologists since the 1950s , and thaAlthough movement behavior of spider monkeys has received substantial research attention , relativWhile we are aware of the importance of latrines in shaping floristic composition of the forest floor ,20,21, iHere, we carry out the first camera trap study of spider monkey sleeping sites within the lowland wet tropical forests of the Osa Peninsula, on the Pacific coast of Costa Rica. Specifically, we: (1) Use arboreal camera traps to quantify the intensity of use of sleeping sites by spider monkeys; (2) use terrestrial camera traps to determine the effect of sleeping site latrines on the presence on terrestrial vertebrates; (3) use pitfall traps to determine the effect of sleeping sites on dung beetles; and (4) use floristic and soil nutrient assessments to assess the physical characteristics of the forest surrounding sleeping sites. Our findings highlight cascading ecological impacts of latrine sites on terrestrial vertebrate and invertebrate communities and suggest ways in which the presence of latrines might generate heterogeneity in forest composition and soil quality.Ateles geoffroyi); with a population density estimate calculated from transect surveys in 2002–2003 of 68.45 (±26.25) individuals/km2 [The Osa Peninsula in southwest Costa Rica is home to the largest remaining tract of Pacific lowland wet forest in Mesoamerica  and hostuals/km2 . The paruals/km2 ,33,34. SOur study site was situated around the Osa Biological Station  of 2017. Individuals of these subgroups typically began traveling to sleeping sites after 17:00 and settled around 18:00. Before sunrise the following morning, we returned to the sleeping site in order to minimize disturbance and prevent monkeys fleeing the tree before defecation. Spider monkeys typically started their activity at 04:50 and, on most occasions, had left the tree by 05:15, by which time they had defecated. Once the individuals had left, the area was searched to locate latrines. Latrines were identified in the field by three key features: (1) The presence of spider monkey feces; (2) a higher than normal density of young saplings; and (3) clear evidence of excreted seeds in the leaf litter immediately beneath the sleeping site. Latrines were difficult to identify after a night of rain, as feces were typically washed away. At each sleeping, site we measured the following variables: Canopy height (m), canopy cover (%), diameter at breast height (cm), tree species , and we mapped out the latrines using a GPS. Of the 39 sleeping sites identified, 10 of the most active (based on perceived continued sleeping site activity throughout the early dry season) were selected for a multi-strata assessment using three camera traps at each site. The camera traps were set up at each site between February and June 2018.Sleeping sites were located by following subgroups of In order to monitor spider monkey sleeping site use and behavior, one camera trap was placed in the canopy, either facing along a horizontal branch of the sleeping site, or when trees were unable to be climbed for safety reasons, facing at the whole sleeping site from a nearby suitable tree. Ideally, we would have more cameras in a tree to increase detection likelihood, just as every ground-based survey would want more observers on the ground to spot, count, and sex monkeys; however, it is not always logistically possible . Having a single camera at a survey site is a common limitation in the camera trap literature generally, but having at least one camera provided us with a long-term constant monitoring tool within the sleeping site, and we tried to ensure it was directed to where we had observed spider monkeys using the tree, based both on direct observations and upon the location of the latrine on the ground below. All cameras were programmed to take 13 s videos with a 30 s resting period to maximize battery life ,36,37. TTo gather information on terrestrial seed dispersers and predators, mammal and bird species were identified from the terrestrial camera footage. Camera traps were deployed in a paired design, with the first camera placed at ~30 cm height from the ground directed at the latrine, and the second camera placed 50 m away at the same height, at a random forest point (non-latrine control site). The detection zone of camera traps easily covered the average core area of latrines. Although all methods suffer from imperfect detection, particularly within tropical forests, one of the strengths of this survey is that we used a standardized method of surveying to examine habitat use within latrines in comparison with paired non-latrine controls. Independent records were defined by 30-min intervals, with any of the same species within 30 min being regarded as non-independent . We theAteles dung might have been the best-case scenario, finding a sufficient quantity of fresh primate dung was logistically impossible without access to a large captive population. However, as with the terrestrial cameras, we used a standardized method of surveying to examine habitat use within latrines in comparison to paired non-latrine controls. The only thing that differed between the latrine site and the non-latrine sites was the presence of the latrine. Everything else was standardized , so we were therefore confident that any differences observed were being driven by the presence of the Ateline sleeping site latrines and not the attractant. Traps were set for three rounds of 24-h trapping. At each location, the latrine and control traps were surveyed simultaneously for a direct comparison over the same time period. Dung beetles were trapped into alcohol for processing and identification. The abundance, species richness, and biomass of trap sites were determined. Biomass was calculated by multiplying the abundance of each respective species by the known average dry biomass calculated for each species and then aggregating to display the overall trap biomass.To determine the effects of latrine presence on dung beetle assemblages, pitfall sampling was carried out from August to December 2018. This was done after the camera trapping survey was completed to minimize anthropogenic disturbance at the site while cameras were active. At each latrine and non-latrine control site, pitfall traps were set baited with 25 g of human dung ,40. We c2 quadrats were surveyed at each sleeping site. The quadrats were placed within the latrine, beneath the focal tree outside of the latrine, and 4 m from the latrine outside the crown of the tree. The number of tree seedlings was counted in each quadrat.To quantify the effect of spider monkeys on the surrounding tree seedling density, four 2 mSoil samples were taken from latrine and the paired non-latrine control sites using a soil extracting device from five points at a depth of 10–15 cm. Before extracting the soil, the top layer of leaf litter was removed. The extracting device was cleaned between sampling points to ensure no cross-contamination. The soil from the five points was extracted into a bucket, mixed, and 500 g was placed in a zip-lock bag and transported to the University of Costa Rica (UCR) laboratory for analysis within 72 h. At the UCR laboratory, the samples were analyzed for micronutrient levels, including: Calcium (Ca), magnesium (Mg), potassium (K) in cmol/L; copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) in Mg/L. In addition, the percentage of macronutrients carbon (C) and nitrogen (N), and the pH of each sample were determined. We were especially interested in the levels of potassium, phosphorus, and nitrogen, as they are known plant-limiting nutrients from tropical forests .All data analysis was conducted in the R statistical environment . To charTo examine the diel variation in group size, we fitted a loess smoother  to the group size per detection data. To compare the community compositions between latrine sites and non-latrine controls, we used non-metric multidimensional based on Bray–Curtis similarities within the “vegan” package . To deteBrosimum utile (n = 3), Otoba novogranatensis (n = 3), and Tapirira guianensis (n = 3). The average diameter at breast height of sleeping sites was 59.5 cm . Twenty-eight of the sleeping sites had clear evidence of latrines beneath them. Latrines had a mean area of 5.7 ± 1.8 m2. One latrine shared by several sleeping sites had an area of 56.1 m2. The 10 sub-selected sleeping sites for further camera and trapping surveys consisted of eight different species.Overall, we located 39 sleeping sites and 28 latrines. The 39 sleeping sites belonged to 21 different tree species, 18 genera, and 12 families. In old-growth forest, spider monkeys slept in 25 trees belonging to 13 species and 10 families. In secondary forest, the 14 trees identified were comprised of 11 species and 6 families (see n = 108) or female (n = 432). Adult female spider monkeys were identified by a conspicuous clitoris, while adult male spider monkeys could be identified by male genitalia and associated aggressive behavior. Adult spider monkeys that could not be assigned a sex were classified as unidentified (n = 754). Where juveniles were detected (n = 256), they were more likely to be observed with adult females (81% of detections events) than adult males (1% of detection events). We only assigned age classes between adult and juvenile spider monkeys, as deeper classification from dependent to infant and juvenile to sub-adult was deemed unreliable from the footage. Juvenile spider monkeys were classified as infants still clinging to adult females and semi-independent smaller-sized spider monkeys climbing in the presence of adult females but with no assistance. We did not assign a sex to juvenile spider monkeys , which resulted in 4348 raw records of medium-to-large vertebrate species within latrines versus 2121 records in non-latrine controls. Nineteen species of mammal, nine species of bird, and a single species of reptile were detected at latrine cameras, and eighteen species of mammal and six bird species were detected at the non-latrine controls see , higher p = 0.03). The difference in composition was principally driven by three species: The paca (Cuniculus paca), which was found at a rate 427.2% higher in latrines than non-latrine controls , found at a rate 212.2% higher than non-latrine controls , which was found at a rate 68.6% higher than non-latrine controls, although this difference was not statistically significant , principally driven by three species. Canthon aequinoctalis was found at a rate 265.6% higher in latrines than non-latrine controls . Consistent with this, of the two soil parameters suggested to drive the differences between latrines and non-latrine controls, only one showed a statistically significant change in concentration: Manganese was present at 22.7% lower concentration than in controls  Arboreal cameras allowed us to characterize spider monkey sleeping site use and composition; (2) terrestrial cameras allowed us to confirm that latrines attract greater vertebrate visitation rates, some of which are important secondary seed dispersing species; (3) baited pitfall traps indicate that latrines associated with sleeping sites attract a greater abundance and biomass of secondary seed-dispersing dung beetles that shape brown food webs; (4) floristic analysis of latrines and direct observations determined a total of 111 plant species consumed by spider monkeys , Manu National Park (Peru), and Calakmul, in the Lacandonian forest of Mexico [Dialium and Brosimum), we also detected many different species and genera being utilized; including fast-growing figs, Tachigali and Symphonia, and ancient old-growth species of Caryocar, Hieronyma, and Vantanea. Many of the species we identified spider monkeys sleeping in represent their own key food resources . By usiIn addition to understanding the activity within sleeping sites, arboreal camera traps also allowed us to investigate the social dynamics of the groups using the trees, and to determine that males generally slept in different sleeping sites to females and young offspring. This segregation agrees, to some extent, with Chapman , who sugThis is the first time that spider monkey latrines have been directly proven to present a large and diverse food resource that is attractive to seed predators and secondary dispersers . AlthougThe potential for additional nutrient resources available as a result of feces deposition in latrines was also identified by Pouvelle et al.  with HowFuture work across more sleeping sites and a greater number of latrines would be able to further corroborate generally the value of Ateline (and other primate) sleeping–latrine sites for other rainforest wildlife and plants. Identifying sleeping sites and latrines was particularly difficult, as it took a significant amount of field time (three months) to detect the sleeping sites. Following large primates off-trail and in low light conditions is challenging and poses some safety risks. An effective solution to this could be to utilize drones and thermal cameras to detect spider monkeys in their sleeping sites . This coThis work adds support to the influential role which spider monkeys play in the formation of tropical forest diversity. Tropical forests without spider monkeys  will li