Neurodegenerative diseases resulting from the progressive loss of structure and/or function of neurons contribute to different paralysis degrees and loss of cognition and sensation. The lack of successful curative therapies for neurodegenerative disorders leads to a considerable burden on society and a high economic impact. Over the past 20 years, regenerative cell therapy, also known as stem cell therapy, has provided an excellent opportunity to investigate potentially powerful innovative strategies for treating neurodegenerative diseases. This is due to stem cells’ capability to repair injured neuronal tissue by replacing the damaged or lost cells with differentiated cells, providing a conducive environment that is in favor of regeneration, or protecting the existing healthy neurons and glial cells from further damage. Thus, in this review, the various types of stem cells, the current knowledge of stem-cell-based therapies in neurodegenerative diseases, and the recent advances in this field are summarized. Indeed, a better understanding and further studies of stem cell technologies cause progress into realistic and efficacious treatments of neurodegenerative disorders.  Neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), are characterized by a progressive loss of structure, function, or number of neurons in the brain or spinal cord. Unfortunately, the currently available treatment options are insufficient in arresting the neurodegenerative processes . The comStem cell therapy, also known as regenerative therapy, improves the repair response of dysfunctional and damaged tissue using stem cells or their derivatives. The objectives of stem cell therapies typically focus either on cellular replacement or on providing environmental enrichment. Stem cell therapy has revolutionized medicine over the years since its therapeutic applications have provided invaluable and attractive options for treating numerous disorders, including neurodegenerative diseases . The potThis review provides encompassing information on the various types of stem cells and then discusses the existing data, progress, and status of using stem cells to treat neurodegenerative diseases. We also describe the remaining challenges associated with transitioning stem cell therapies from bench to bedside. Stem cells are characterized by the capacity to proliferate, self-renew, and differentiate into various mature cell lineages. There are different classifications of stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and neural stem cells (NSCs). The classification is based on the range of possible cell type production and derivation methods. Therefore, it is essential to understand the characteristics of the various available stem cell types and the potential effect of cellular therapies on disease mechanisms see . The ratESCs are a class of pluripotent stem cells derived from the inner cell mass of blastocysts , a key neurotransmitter that transmits signals between neurons, plays an essential role in motor control. PD is the second-most-common age-related progressive neurodegenerative disorder caused by DA deficits in the striatum due to the destruction of DA-producing neurons located in the substantia nigra ,53,54. TA specific diagnosis of PD includes the presence of Lewy bodies in the brains, which are abnormal protein aggregates developing inside nerve cells and found in both familial and sporadic PD patients. The main constituent of Lewy bodies is α-synuclein protein, a small protein with 140 amino acids that are abundantly found in presynaptic nerve terminals and play a role in synaptic transmission and DA level adjustments. α-Synuclein primarily affects tyrosine hydroxylase phosphorylation, activity, and DA transporter expression on the cell membrane .The current treatment options for PD include deep brain stimulation or therapies to increase DA levels by providing a DA precursor (Levodopa) to compensate for the deficit in DA caused by the destroyed dopaminergic neurons . AlthougOver the past two decades, researchers have been looking for alternative strategies to supplement DA by replacing the dopaminergic neurons lost in the disease with stem-cell-derived equivalents. ESCs, NSCs, and iPSCs are among the stem cell typologies that scientists are working with to induce their differentiation into the mature dopaminergic cell . Stem ceFurthermore, Takagi et al. demonstrated functional recovery after successfully transplanting dopaminergic neurons from monkey ESCs into PD patients’ brains . AnotherMoreover, ESCs have shown excellent outcomes in mice models due to their ability to form dopaminergic neurons for treating PD, which has not been accomplished while using adult neural stem cells. Moreover, in rat models with spinal cord injuries, ESCs showed migration into the parenchyma and spinal cord. They led to partial motor recovery, giving rise to the ultimate goal of reversing motor degeneration in PD. While these studies initially revealed promising results, they were banned due to ethical and religious concerns and the risk of tumorigenesis . BesidesMSCs have also been shown to reduce dopamine depletion and rebuild the damaged striatal dopaminergic nerve terminal network in a PD animal model . A more iPSCs have proven to be very useful for promoting dopamine replacement in patients with PD. Several studies have shown that dopaminergic neurons derived from iPSCs grafted into PD model systems can survive and integrate into the host network with remarkable functional improvements . Other sAlthough cellular replacement is considered a viable approach for treating PD, environmental enrichment through growth factor delivery may also support existing DA neurons’ ability to slow or prevent further degeneration. More recent work has focused on encapsulating the desired cell line into a semipermeable polymer, along with a pool of neurotransmitters and neurotrophic factors. This approach combines cellular replacement and environmental enrichment strategies. For example, the transplantation of NSCs derived from the SVZ and the nerve growth factor (NGF) in the striatum of animal PD models showed remarkable recovery . Taken tAD is the most common progressive neurodegenerative disorder characterized by the degeneration of synapses and loss of neurons in the hippocampus and the neocortex . AD progWhile AD’s exact pathology remains unclear, the pathologic hallmarks of AD, which lead to decreased synaptic signaling and eventually neuronal death, involve the formation of extracellular senile plaques  and intracellular neurofibrillary tangles (NFTs). NFTs deposited inside the cell are made of hyperphosphorylated tau proteins that form tangles after dissociating from destabilized microtubules . Aβ plaqAlzheimer’s is the sixth-leading cause of death, with a substantial economic burden on society ,81. ApprEvidence supports that stem cell therapies may play a beneficial role in slowing AD’s progression through enhancing neurogenesis, replacing lost neurons, and providing environmental enrichment. Human NSCs have been proven to be a promising source, improving synaptic plasticity, reducing the pathology’s burden, and ameliorating spatial learning and memory dysfunction by increasing the expression of multiple cognition-related proteins in vivo in AD mouse models ,84,85. OThe use of patient-specific iPSCs in the treatment of AD is still a work in progress. Scientists have employed a novel way to directly create functional neurons from AD patients’ skin cells converted into ESCs ,90. In aSeveral studies reported that MSCs stimulate neurogenesis, synaptogenesis, neuronal differentiation and influence amyloidogenesis and/or microglial activation, thus reducing Aβ accumulation and cognitive recovery ,95,96,97Recently, combining stem cells with NGF was recognized as a useful strategy for preventing cell death, stimulating the growth of cholinergic neurons, and facilitating the generation of specific neural populations in AD treatment. NGF gene therapy has been studied in many animal models and has led to successful clinical trials in AD patients . HD is a fatal progressive neurodegenerative disorder of autosomal dominant inheritance characterized by the loss of GABAergic inhibitory spiny neurons in the striatum of the forebrain, accompanied by degeneration in the cortex, brain stem, and hippocampus . The HD’Stem-cell-based therapeutic approaches have received considerable attention as potential treatments for HD. The objective of stem cell therapy for HD is to replace the damaged or lost neurons and modify the mutant genes containing the expanded CAG repeats. Based on recent studies, NSCs have been the type of stem cells most used for HD treatments. NSCs have been derived and induced from various sources, such as the brain itself and the somatic cells of HD patients. Despite the early stage of stem-cell-based preclinical and clinical trials in HD, there is convincing evidence regarding stem cells’ transplantation or their derivatives in HD animal models. Initial stem cell therapies focused on ESC-derived NSCs grafted into HD models have demonstrated the integration of motor neurons and circuitry formation in the host. However, the ethical and religious implications of fetal tissues are still crucial issues . The danA study on the brains of murine models of HD by Ebert et al. observed that mouse-derived NSCs act as GDNF delivery vehicles, playing a beneficial role in reducing neuronal death and the resultant motor impairment . To addrAt present, MSCs offer a promising source of cells for treating HD because of their ability to decrease immune cell dysfunction, enhance compensatory neurogenesis, reduce apoptosis, activate mitochondrial function, and promote cell survival .In 2010, Dey et al. reported that MSCs genetically engineered to overexpress BDNF or NGF decreased behavioral deficits and neuronal loss in the striatum in the YAC 128 mouse model of HD . Thus, tThere are only two reported studies investigating iPSC-derived NSCs for cell replacement therapy for HD. In the first study, Jeon et al. transplanted hiPSC-derived NSCs generated from an HD patient with 72 CAG repeats into an HD mice model and reported an improved functional outcome and no aggregates of human mutant huntingtin (mHTT) in transplanted cells . In subsTo date, stem cell therapy is still far away from the clinical setting for the treatment of HD since almost all the studies have been conducted in animal models. More in-depth, comprehensive preclinical studies will be needed to confirm its therapeutic potential.ALS  is a progressive, incurable neurodegenerative disease characterized by motor neurons’ deaths in the spinal cord’s ventral horn and the motor cortex. With the disease progression, symptoms such as motor weakness, twitching, stiffness, and loss of voluntary movement control become apparent ,52. The Stem cell therapies have generated widespread interest as a potential therapeutic approach. However, no conclusive results have yet been reported, either from preclinical or clinical studies. The unknown pathogenesis and the lack of proper knowledge of the disease’s spreading mechanism in the human body are among the most critical barriers to overcome regarding choosing the ideal cell type and optimal anatomical site for implantation.Theoretically, the underlying strategies for treating ALS using stem cell therapies are similar to those previously described for other neurodegenerative diseases: (1) replace the damaged/dead motor neurons, (2) regulate inflammation, and (3) promote the expression of neurotrophic factors. The treatment’s end goal is equally similar to those previously described: provide both an integrated neural component and the necessary environmental enrichment to prevent existing motor neurons from degenerating . The firAltogether, although stem cell therapy in ALS treatment is still in its infancy, researchers worldwide hope treatments like this will stop the disease’s progression and improve the efficacy of current drug therapies .MAPT gene encoding the microtubule-associated protein tau [MAPT), progranulin (PGRN), and C9ORF72 are among the most common causes of FTD known up to now [MAPT to study hereditary FTD and tau pathologies in vitro [MAPT [GRN mutation [GRN-dependent pathogenic mechanisms and develop potential therapies. Ehrlich et al. also derived iPSCs from patients with FTD-associated MAPT mutations and differentiated them into mature neurons to provide an in vitro model for identifying distinct neurodegenerative changes in frontotemporal dementia with parkinsonism-17 (FTDP-17) [FTD, an insidious neurodegenerative clinical syndrome, is the second-most-common leading cause of neurodegenerative dementia and is usually found in people younger than 65 ,123,124.tein tau ,130,131.p to now . These gp to now . Interesp to now . iPSCs hp to now . For exap to now . In two in vitro ,137. Nimro [MAPT . Almeidamutation . They suFTDP-17) . In anotFTDP-17) . Based oFTDP-17) . AltogetDespite the remarkable advances in stem cell research for neurodegenerative disorders, several critical issues must be addressed. A major hindrance in stem cell therapies’ progression is learning how stem cells work in the body and how they integrate with the targeted tissue/organ. Furthermore, generating specialized cell typologies to overcome tissue- and environmental-specific hurdles are critical requirements. The safe and cost-effective generation of these cells in adequate quantities is also another factor to be considered. For example, ESCs and iPSCs can be grown indefinitely in the lab, but the procedures are very complex and demanding, limiting these cells’ overall availability.Furthermore, there is the problem of reducing the risk of post-implant rejection, which adds the burden of needing a close compatible donor for the cells’ recipient. Identifying the proper conditions to culture these cells, the most suitable route of administration, delivery, and the target site is also crucial to maximizing the benefit of the treatment and improving the outcome. Unfortunately, most of the data available to researchers were derived from animal studies; therefore, it is unclear whether these human stem cells will afford similar results when administered to a heterogeneous patient population . UnfortuOn the other hand, translating stem cell therapy to clinical trials is practically impossible without a suitable stem cell source for therapeutic applications. The cost, time, and labor-intensive nature of stem cell therapy limit its use, especially in developing countries. Additionally, safety considerations, such as the potential for malignant transformation and side effects, such as epilepsy, immune allergic reactions, and injection site injuries, remain significant concerns .The outcome of stem cell therapy can be improved by combining adjunct treatments . For exaDespite all the limitations and challenges, stem cell therapy is still a promising approach for treating neurodegenerative diseases in the future . The useNeurodegenerative diseases have devastating sequelae with conventional pharmacological therapies. To date, progress in the research area of stem cell therapies offers excellent promising methods for the treatment of these disorders. Although much work remains to be done, the growing focus on preclinical studies and the recent translation of some of these therapies to clinical trials has set the stage to continue progress. Using stem cells appears likely to become a key feature of future clinical strategies for treating neurodegenerative disease by replacing dysfunctional neurons and affording neuroprotective and neurorestorative functions. Furthermore, stem cells’ recent technological developments involving nanoparticles and hydrogels have made drug delivery and regeneration treatments more efficient. Thus, neural replacement and regenerative therapies are soon expected to be successfully translated into the clinical setting.


In this Research Topic, High Performance Cognition (HPC) emerges as a generic term for the study of human performance and skill acquisition, from novices on an unfamiliar task  to experts displaying superior levels of domain-specific skill. We received 11 pieces of empirical work across several experimental tasks and three articles describing theoretical/conceptual work on flow, cognitive training, and Deliberate Practice framework for expert performance. The papers received in this Research Topic encompassed a range of methodologies including reports of phenomenology, measurement of behavior, and psychological traits, and measures based on psychophysiology and neurophysiology.Cowley et al., to the proactive control of executive function shown by “open”-skill athletes  in Yu et al.. This theme also reflects in theoretical contributions which call for revision of the longstanding constructs of deliberate practice and Flow, to either account for interdependency in a more holistic framework , or control for it (Abuhamdeh), respectively. Clearly, even to make such revisions is challenging; beyond which is the yet more-challenging task to build a unified framework on the revised constructs, coherent with existing evidence. Yet such a framework will refine the generic term HPC to a definition of cognitive performance, and thereby provide grounds for empirical predictions and a direction for future work for many years.What were the commonalities underlying the various contributions received for the topic? Most empirical papers report that the cognitive process under investigation showed interdepencies with other processes: from the longitudinal relationship between performance anticipation and Flow shown in Thus, while we cannot yet answer the question: “do cognitive processes that are specific to contexts of high performance also generalize across task domains?,” this Topic clarifies somewhat how we might start to address the question. A summary of the contributions follows to provide an overview of the specific articles received as part of this Research Topic.Chin and Kales investigate whether autonomic arousal  influences self-reported flow and performance in the Stroop task. To increase between-subjects HRV, participants first performed techniques in either nasal respiration, arm-muscle contraction, both combined, or read emotionally-neutral articles. The results showed an inverted-U pattern: performance and flow were maximal at moderate arousal levels. Optimal performance was also associated with predominantly sympathetic autonomic activity.Cowley et al. ask: How does subjective feeling of high performance relate to development of  optimal performance? They report a longitudinal  study, recording Flow and behavior as participants learned to perform a visuomotor steering task. Participants did not experience more Flow over sessions; instead, their trial-wise subjective anticipation of performance  was strongly related to Flow.Murch et al. report on three experiments which aim to identify objective markers of flow while gambling. The authors collected self-reported gambling flow and physiological data as measured by the cardiac pre-ejection period (PEP). The authors did not find evidence of changes in PEP when interacting with the electronic gambling machines but only interactions between subjective and objective measures during the first block of each experiment.Peifer et al. hypothesize that self-efficacy acts as a mediator, whereby positive feedback on one task imparts a positive effect upon performance and flow in subsequent tasks. They report evidence to support the hypothesis and propose multiple methods of intervention to further increase these positive effects.In a study examining the effect of positive feedback on perceived self-efficacy, flow and performance, Sinnett et al. ask: can increased flow experience can improve perception? The authors implemented a within-subjects design involving groups of athletes and musicians facing a temporal order judgement task paradigm. This task encompasses a measure of temporal processing measure and of spatial attention. Their results disclosed a positive relationship between the self-reported value of experienced flow and the efficiency of spatiotemporal information processing.Here we order the studies by participant expertise, starting with naïve-subject designs.Kee et al. investigate the effects of non-striving states, induced by a repetitive water-pouring task, on a word-length comparison task. The authors reported that the experimental group tended to exhibit lower performance and was significantly faster to perform the word comparison task than the control group. Though the authors did not use self-report questionnaires, they suggest their experimental design could help to improve mindfulness practice.Liu et al. investigate the motivating effect of monetary rewards (given during this low-vigilance state) on performance in a flanker task, and concomitant electrophysiological measures linked to selective visual attention, primarily P300. Monetary reward did improve performance during low vigilance, but neural measures recovered selectively, indicating that some constraints on higher cognitive performance are non-volitional.Prolonged task execution brings mental fatigue, characterized by “indolence, reduced motivation, and impaired performance.” Gong et al. investigate how EEG neurofeedback training (NFT) affects shooting performance and neuroplasticity among police students. They found that pre-post shooting performance increased for participants using sensorimotor-rhythm NFT, but slightly decreased for participants using alpha-rhythm NFT. Participants learned to alter their EEG patterns in both NFT groups. Pre-post measures also showed that neuroplasticity was affected by NFT. Thus, NFT may facilitate training for activities requiring sensorimotor accuracy.Muñoz et al. present work toward a psychophysiological model of firearm training for real police officers using a combination of head-mounted virtual reality and non-intrusive physiological sensors. The authors demonstrate how changes in frontal theta activation and heart rate variability were affected by changes in task difficulty levels, which they articulate as metrics of “concentration” and “calmness.”Savostyanov et al. utilizes ERPs to examine the effect of long-term meditation on comprehension of statements about oneself and others. Three groups  completed a reading task containing self or non-self-related evocative statements and grammatical errors. They demonstrate that meditation increases negative affect processing and an ERP analysis that potentially shows an increase of voluntary control over emotional states for meditators.The study by Yu et al. investigates how motor-skills experience modulates proactive and reactive control of executive function, using a cued task-switching protocol with “open”- and “closed”-skilled athletes and non-athlete controls. Their findings highlight that open-skilled participants showed significantly less positive-going parietal cue-locked P3 and better predictive task-switching performance. They conclude that proactive control may be enhanced in open-skilled participants when compared to the other groups.Work presented by Aidman proposes a framework for Cognitive Fitness: trainable cognitive abilities  that can be improved and maintained by specific cognitive fitness training, analogous to physical-fitness training for strength, endurance, and flexibility. Aidman proposes his framework would be valuable for training performance in sport, arts, emergency services—indeed, any domain where individuals must deliver high performance on demand under stressful conditions.Hambrick et al. and Abuhamdeh present critical opinion pieces on two highly-influential theoretical constructs in study of HPC: Deliberate Practice (DP, Ericsson et al., Hambrick et al. paper continues their ongoing interrogation of the internal consistency of existing DP research, building on the authors' earlier meta-analysis (Macnamara et al., Abuhamdeh paper looks critically at how the original definition of flow has since been operationally defined.Both contributions share an underlying message: after decades of research, both lines of investigation remain pre-paradigmatic, in the sense of Kuhn . Each isThe studies presented in this Research Topic illustrate both current diversity, and a bright future, for research in performance cognition. Researchers investigating flow states posit new experiments to model the conditions and relationships involved and call for a consistent operationalization of the flow state. Neurofeedback researchers propose to move into real-time adaptation of training protocols; those investigating meditation propose novel interventions to further increase the benefits of the practice. Extension and improvement of human cognition through augmented and directed training comprises a common vision for the future. To achieve it, we must unify this diverse field around a common and parsimonious framework, such that we can move toward measuring and understanding high performance cognition as it develops through learning and training across various domains.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.


Most ovarian cancer patients are diagnosed only at advanced stages when survival outcomes are worse, andwhen therapeutic decisions might prove challenging. The fundamental treatment for women with ovarian cancerincludes debulking surgery whenever possible and appropriate systemic therapy . In the last few years, knowledge about histological and molecular characteristics of ovariancancer subtypes and stages has increased considerably. This has enabled the development and improvement ofseveral options for the diagnosis and treatment of ovarian cancer in a patient-tailored approach. Accordingly,therapeutic decisions are guided by the characteristics of the patient and the tumour, especially the molecularfeatures of the cancer subtype and disease stage. Particularly relevant are the advances in early genetic testing ofgermline and somatic mutations involved in DNA repair, and the clinical development of targeted agents. In orderto implement the best individual medical strategies, in this article, we present an algorithm of treatment options,including recently developed targeted agents, for primary and recurrent ovarian cancer patients in Belgium. Ovarian cancer is the fifth leading cause of cancer-related deaths among women in Europe and theUnited States of America, and is the most frequentcause of death among all gynaecological cancersin Europe . In 2018Ovarian cancer is often asymptomatic, resulting in diagnosis at advanced stages in most patients. This trend is reflected in the Belgian cancer statistics from 2016; 18.9%, 5.6%, 28.7%, and 25.1% of ovarian cancer patients were diagnosed with stage I,II, III, and IV disease, respectively (21.6% of patients had an unreported stage) . In BelgHere, we aim to provide an overview of the current treatment landscape for ovarian cancer in Belgium to help physicians translate evidence and guidelines into individualised medical strategies for their patients.Ovarian cancer is a heterogeneous disease: it is comprised of tumours that can show very different morphologies, histologies, grades, and molecular characteristics . EpithelThe term “EOC” does not refer to a single disease but encompasses a group of tumours. According to their morphology, several histological subtypes of EOC have been defined, with the most common being the serous (68%–71%) subtype, followed by clear cell (12%–13%), endometrioid (9%–11%), mucinous (3%), malignant Brenner (1%) and mixed histology (6%) subtypes . In a moOvarian cancers are staged surgically and pathologically according to two systems: the International Union Against Cancer (UICC) TNM  system of classification of malignant tumours, and the International Federation of Gynecology and Obstetrics (FIGO) staging system . FIGO stGermline or somatic mutations in arrays of genes coding for proteins involved in the homologous recombination (HR) repair of double-strand DNA breaks occur in up to 40–50% of patients with HGSC and more rarely in the other epithelial subtypes . The mosThe homologous recombination deficiency (HRD) diagnosis also has prognostic and therapeutic implications. ABRCA-mutated ovarian cancer has a better prognosis and an increased platinum sensitivity compared to a BRCA-wildtype cancer . MoreoveTherefore, the identification of somatic and germline mutations in the BRCA1 and BRCA2 genes (and other HR genes conferring sensitivity to PARP inhibitors [PARPis]) has become an essential guide for treatment decisions .Among other techniques, next-generation sequencing (NGS) of germline DNA can be used to identify mutations in genes associated with hereditary cancer risk, not only HR genes . A typicIn Belgium, the Personalised Medicine Commission (ComPerMed) recommends genetic testing for all patients with invasive EOC (excluding borderline or mucinous ovarian cancers), with fallopian tube cancer and with peritoneal cancer, ideally at diagnosis . NGS tesThe implementation of early systematic germline testing in ovarian cancer requires workflow changes in health care practices. While current standard clinical practice in many countries implies a pre- test consultation with a qualified geneticist and subsequent BRCA1/2 mutation status testing , it has Fundamental principles of treatment for women with ovarian cancer include surgery to reduce the tumour bulk to no residual disease whenever possible and appropriate systemic treatment based on the ovarian cancer subtype, disease stage, biology, and patient characteristics .Primary debulking surgery, which aims at complete resection , is a cornerstone of ovarian cancer therapy . OptimalIn women with poor general health, a high perioperative risk profile, or a low likelihood of achieving effective cytoreduction because of extensive disease (advanced stages), neo-adjuvant chemotherapy to reduce the tumour burden should precede surgery . The neoIn women with recurrent ovarian cancer, the benefit of secondary cytoreductive surgery depends on the patient population; its role remains controversial and may depend on the systemic treatment given . In the The current standard first-line systemic treatment, administered after primary debulking surgery or as neoadjuvant therapy is a platinum-based doublet intravenous chemotherapy  with bevacizumab .In case of intolerance to paclitaxel, alternative platinum-based doublet regimens are carboplatin with docetaxel or with pegylated liposomal doxorubicin (PLD),which should be used according to the patient’s/physician’s treatment preferences and the patient-specific drug tolerability since each of the regimens is associated with a specific toxicity profile .The carbAlthough surgical resection followed by first-line platinum‐based chemotherapy leads to complete remission in many patients, recurrences occur in up to 70% of cases .AlthoughThe management of recurrent ovarian cancer is less validated than that of newly diagnosed ovarian cancer .After reTreatment of first-line and recurrent ovarian cancer has now expanded to include targeted and antiangiogenic agents .The targIn Europe, olaparib, niraparib and rucaparib are indicated as monotherapy for the maintenance treatment of adult patients with platinum-sensitive, relapsed, high-grade, serous epithelial ovarian, fallopian tube, or primary peritoneal cancer (BRCA- mutated for olaparib) who are in response to platinum-based chemotherapy . RucaparMoreover, olaparib is reimbursed in Belgium as a single agent for maintenance treatment in patients with recurrent platinum-sensitive, PARP-naïve, BRCA-mutated, high-grade EOC, fallopian tube cancer or primary peritoneal cancer after at least two prior lines of platinum-based chemotherapy . NiraparBevacizumab is indicated and reimbursed in Belgium in combination with carboplatin and paclitaxel for the first-line treatment of adult patients with advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer . BevacizClinical trials are evaluating other indications for the above-mentioned targeted therapies and antiangiogenic agents as well as new agents. A phase III, randomised study  has shown that the addition of olaparib to bevacizumab maintenance therapy following first-line platinum- based chemotherapy plus bevacizumab led to a statistically significant and clinically meaningful PFS benefit in patients with advanced ovarian cancer; especially in those with a BRCA mutation and in HRD-positive patients . A seconAccurate pathological and molecular testing is essential to construct multifactorial, patient-tailored treatment approaches in ovarian cancer. Based on recent results, infrastructural and pragmatic workflow changes are necessary to implement early systematic genetic testing and counselling, and to timely plan the integration of targeted agents and antiangiogenic agents, which have now proven their efficacy in clinical practice, into treatment strategies. Since new treatments for ovarian cancer are currently under development and should be implemented in the coming years, ongoing research should further help refine the precise treatment algorithm and answer open questions in this rapidly evolving field.


Chronological aging as well as biological aging accelerated by various pathologies such as diabetes and obesity contribute to cardiovascular aging, and structural and functional tissue damage of the heart and vasculature. Cardiovascular aging in humans is characterized by structural pathologic remodeling including cardiac and vascular fibrosis, hypertrophy, stiffness, micro- and macro-circulatory impairment, left ventricular diastolic dysfunction precipitating heart failure with either reduced or preserved ejection fraction, and cardiovascular cell death. Cellular senescence, an important hallmark of aging, is a critical factor that impairs repair and regeneration of damaged cells in cardiovascular tissues whereas autophagy, an intracellular catabolic process is an essential inherent mechanism that removes senescent cells throughout life time in all tissues. Several recent reviews have highlighted the fact that all longevity treatment paradigms to mitigate progression of aging-related pathologies converge in induction of autophagy, activation of AMP kinase (AMPK) and Sirtuin pathway, and inhibition of mechanistic target of rapamycin (mTOR). These longevity treatments include health style changes such as caloric restriction, and drug treatments using rapamycin, the first FDA-approved longevity drug, as well as other experimental longevity drugs such as metformin, rapamycin, aspirin, and resveratrol. However, in the heart tissue, autophagy induction has to be tightly regulated since evidence show excessive autophagy results in cardiomyopathy and heart failure. Here we discuss emerging evidence for microRNA-mediated tight regulation of autophagy in the heart in response to treatment with rapamycin, and novel approaches to monitor autophagy progression in a temporal manner to diagnose and regulate autophagy induction by longevity treatments. Global deaths from cardiovascular disease increased by 41% between 1990 and 2013 and population ageing alone contributed to an estimated 52.5% increase in these deaths . The 201Conversely, CVDs resulting from a variety of metabolic and environmental stressors or infections promote cellular senescence and accelerate cellular, organ-level, and organismal aging. Two well-established and preventable factors that accelerate biological aging independent of chronological age are obesity and type 2 diabetes. Currently 1.9 billion adults are overweight and 650 million people are obese . Age-adjvia SASP, and in conditions of diabetes, activation of DNA damage response (DDR) in cells induce senescence and thus activate a ‘malignant positive feedback loop' in which diabetes-DDR-cellular senescence further increases tissue damage and spreading of senescence  is an effective method to ameliorate cardiovascular pathologic remodeling and to improve cardiovascular function . For exaOne of the underlying mechanisms for the anti-aging effect of CR is induction of autophagy, a process that removes senescent cells from tissues and thus prevent spreading of cellular senescence. It is now well established that autophagy is a converging point for the beneficial effects of longevity drugs such as rapamycin, other rapalogs, metformin, and resveratrol . OptimalAutophagy encompasses highly regulated cellular processes to maintain cellular homeostasis and proteostasis, and eliminates potentially harmful cellular stressors that induce cell death . The higWhile basal level of autophagy is required to maintain normal cardiac function, autophagy becomes downregulated in the heart during the course of aging . Age-indvia administration of rapamycin inhibits the adverse effects of aging, increases lifespan, and promotes autophagy in the heart as well as in many other cell types and organs, even when autophagy was suppressed by aging  on the heart tissues of male healthy Zucker lean (ZL) and obese, diabetic Zucker obese (ZO) rats that exhibit accelerated aging, and reported that while rapamycin treatment was beneficial in reducing body weight and suppressing fasting triglycerides, insulin, and uric acid in ZO rats, it also increased blood glucose levels. In ZO rats, rapamycin initially improved multiple diastolic parameters , but by the end of treatment these beneficial effects were reversed. In ZL rats, rapamycin increased fibrosis and induced differential regulation of intracardiac cytokines . A cardiin vivo imaging was performed by planar imaging at 4 h after the co-injection of rapamycin (autophagy induction) and chloroquine (blocked flux). The authors reported a 23% increase in cardiac fluorescence signal compared to the baseline levels prior to rapamycin/chloroquine injection  may activate miRNA networks that can regulate progression of autophagy in the heart either as an adaptive mechanism to regulate excessive autophagy or a method for spreading of senescence. Temporal visualization of cardiac autophagy in the context of heart function is needed to ensure safety of longevity treatments.LP and HC wrote and edited manuscript and approved final version.This work was supported by NIH NHLBI 1R01HL138988-01A1 (LP) and NIH K99/R00HL121152 (HC).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.


Shortcomings related to the treatment of bone diseases and consequent tissue regeneration such as transplants have been addressed to some extent by tissue engineering and regenerative medicine. Tissue engineering has promoted structures that can simulate the extracellular matrix and are capable of guiding natural bone repair using signaling molecules to promote osteoinduction and angiogenesis essential in the formation of new bone tissues. Although recent studies on developing novel growth factor delivery systems for bone repair have attracted great attention, taking into account the complexity of the extracellular matrix, scaffolding and growth factors should not be explored independently. Consequently, systems that combine both concepts have great potential to promote the effectiveness of bone regeneration methods. In this review, recent developments in bone regeneration that simultaneously consider scaffolding and growth factors are covered in detail. The main emphasis in this overview is on delivery strategies that employ polymer-based scaffolds for spatiotemporal-controlled delivery of both single and multiple growth factors in bone-regeneration approaches. From clinical applications to creating alternative structural materials, bone tissue engineering has been advancing constantly, and it is relevant to regularly update related topics. Nonhealing chronic bone tissue defects represent a major problem in healthcare. Despite numerous reports ,2, thereDifferent factors that promote tissue growth have been found at the skeletal damage site and have a physiologic role in healing bone fractures. Osteoinductive GFs such as platelet-derived growth factors (PDGFs), bone morphogenic proteins (BMPs), insulin-like growth factors (IGFs), transforming growth factors (TGFs-ß), and vascular endothelial growth factors (VEGFs) have presented great application potentials in bone healing and osteogenesis for regulating cell behavior, including recruitment, migration, adhesion, proliferation, and differentiation  7,8,9].,8,9.7,8,Biomechanical stability and biological activity that furnishes an appropriate background for new bone formation are the basis for triumphant GF therapy in bone tissue engineering . Thus, uSeveral in vivo and clinical studies showed that incorporating GFs into polymer carriers/scaffolds such as gelatin, chitosan, alginate, chitosan, collagen, and hyaluronic acid improved bone healing ,11,12,13A very short duration of action and systemic toxicity by over-release have prevented GFs from being developed into effective regenerative treatments . To circEmerging and trailblazing materials that modulate the biological presentation of GFs are promising analeptic agents to aid in treating diseases ,22. ThisStudies have shown the projected perspectives of tissue engineering. However, triumphant translations into the clinical application are still restricted owing to the shortfall of delivery systems with optimal signaling. Thus, engineers and scientists are promptly developing biomimetic drug delivery systems that can take advantage of reproducing signaling molecules released by the native ECM during healing or regeneration processes. Designed drug delivery systems aim to provide control over the localization, time, and kinetics of the release pattern of signaling molecules such as GFs according to the drug chemical properties and specific biological mechanisms .Biological signal molecules have a crucial function in modulating cellular activities and tissue regeneration. Bioactive compounds such as GFs are proteins that regulate many aspects of cellular function, including survival, proliferation, migration, and differentiation , and havIn the bone-repair process, angiogenesis precedes the onset of osteogenesis. A combination of angiogenic (VEGF), cell recruiting (platelet-derived growth factor (PDGF)), and osteogenic (BMPs) GFs has been designed and demonstrated a synergistic effect that is more beneficial to bone repair than any GF delivered alone . This syAt the surgical site, a specific delivery system should use GFs to exert and maintain biological activity in a controlled fashion and to avoid any systemic diffusion. Therefore, a delivery system is imperative to stabilize GFs and to provide long-term sustained release for in vivo efficacy. Understanding the biomolecular processes during the healing of injured organs is essential for developing GF-based therapeutics for tissue regeneration. An aspect of the natural healing process is the continuous delivery of GFs throughout recovery, avoiding a high variability of GF concentration at the target tissue and rapid clearance .A successful delivery system can deliver GFs to areas besides the target spot through surgery. This system can maintain enough bioactive factors during the time needed to promote osteogenesis and low fundamental doses to prevent side effects due to supraphysiological GF doses . DeliverEvidenced by the wide range of inflammatory, osteogenic, and angiogenic factors involved in all bone tissue regeneration processes, these processes can be directly related to biomolecular and cellular processes . GFs’ thSuch structures provide initial biomechanical support to the implanted tissue until cells can develop a proper ECM to support the regeneration process. It is expected that the scaffold is gradually degraded and metabolized during the formation, deposition, and organization of the ECM, allowing for the tissue to be reestablished with the same or improved function. Thus, such scaffolds are engineered to be biocompatible, biodegradable, and porous to assure vascularization, to show mechanical reinforcement, and to allow functional and bioactive responses . Bone grCollagen is the most studied natural polymer for bone tissue engineering scaffolds, as this biopolymer integrates about 90 wt.% of natural bone ECM proteins . CollageLinh et al.  conjugatIn addition to collagen, other natural biopolymers such as silk fibroin can also be effective in bone tissue engineering applications ,80. Silk2+) due to the anionic nature that allows alginate complexation to these cations [2+ crosslinking [Sodium alginate is a linear anionic binary polysaccharide that consists of α-L-guluronic acid (G units) and (1-4)-linked β-D-mannuronic acid (M units) segments. This biopolymer is mostly obtained from widely available seaweeds, which makes it a great candidate for a diverse range of tissue engineering applications . Consecu cations . This mo cations . A partislinking . Alginatslinking . PDGF an2) slightly slower than the random scaffold (0.76 μg/mm2) after seven days.An alternative to overcoming the challenges faced by composite biomaterials is the use of cellulose and other nature-derived polymers once vast manufacturing approaches and sources are available . CelluloChitin–chitosan is a nitrogen-containing polysaccharide-based biopolymer group derived from diverse natural raw materials such as fungi, crustaceans, and insects ,97. ChitRecent studies have suggested GAGs as potential biomaterials for tissue engineering application, as this biopolymer predominantly exists in the ECM, has low immunogenicity, and can perform strong interactions with GFs . The strA large number of techniques have been presented and employed to manage the release kinetics of GFs entrapped in scaffolds. A majority of successful methods is based on encapsulating GFs in a degradable polymeric network , which cPhysical entrapping processes for the incorporation of bioactive molecules in polymer networks can also strongly affect the performance of these systems. Different techniques are available to entrap drug molecules in the structure of scaffolds, which facilitate their contact with migrating cells and regulate cell behavior . SurfaceFrom a technical point of view, physical adsorption can be considered the most straightforward method for embedding biomolecules into polymer scaffolds . PhysicaGF adsorption on the defect site has to be steady and localized, and a GF–receptor interaction must occur to activate signaling cascades, inducing osteoblast proliferation, to effectively allow tissue regeneration . AccordiSurface functionalization via physical adsorption has the advantage of being a simple and gentle procedure accompanied by limited damage to fragile structures and biomolecules. However, biomolecule binding to scaffold surfaces can be relatively weak . The scaA brushite/PLGA composite system to control the release of PDGF, TGF-β1, and VEGF was designed to promote bone remodeling . PDGF anChemical conjugation, or covalent bonding, offers prolonged and more stable drug molecule presentation than the physical adsorption method ,143. ForControlled and sustained release of BMP-2 and VEGF built-in silk fibroin/nanoHA scaffolds via chemical and physical covalent bonding, respectively, was observed . VEGF prBiochemical gradients in the cellular microenvironment are known to drive a variety of physiological processes including bone repair . The majOne of the strategies for sequential GF delivery assumes the incorporation of various nanoparticles with encapsulated growth factors into polymeric scaffolds   and bone morphogenic proteins embedded within an alginate/polycaprolactone scaffold. By binding BMP-2 to HMPs, the authors reduced the rate of biomolecule diffusion of BMP-2 by generating its long-term gradient and by controlling spatial localization . In anotRecent technological advances in biomanufacturing have enabled the biofabrication of biomaterials with differentially arranged growth factor gradients. These advanced techniques include 3D bioprinting, microfluidics, layer-by-layer scaffolding, and techniques that utilize magnetic or electrical fields to distribute biomolecules within scaffolds C 166,16. Layer-bAll currently used strategies for engineering and fabrication of graded tissue scaffolds for bone regeneration are guided by the same principles: (1) to mimic native bone tissues and to follow the ordered sequence of bone remodeling, (2) to generate complex multifunctional gradients, (3) to control the spatiotemporal distribution and kinetics of biological cues, and (4) to be easily generated by accessible and reproducible techniques.Growth factors have shown great potential in bone regeneration. However, their clinical applications are limited due to the following reasons: short biological life in physiological conditions due to rapid degradation and deactivation, high cost, and side effects . There aThe use of appropriate delivery systems can considerably enhance the safety and efficacy of GF therapies. When GFs are used for bone repair, the materials which are prepared for the delivery system must be nontoxic and biodegradable . The maiBesides the side effects, the cost-effectiveness of GFs for bone regeneration applications is also under debate. The translation of GFs is narrowed by their delivery issues, side effects , and lowThe bioactivity of GFs plays a vital role in bone regeneration. Even after several in vivo and in vitro studies, the ideal dosage of GFs applied for bone regeneration remains uncertain . When adTo meet the requirements of GF delivery, several scaffold-based approaches such as physical entrapment of GFs within the scaffold, covalent or noncovalent binding of the GFs to the scaffold, and the use of micro or nanoparticles as GF reservoirs have been developed . CovalenHydrogels are a common GF delivery strategy as they can act as a scaffold or as protein releasing matrices . StudiesIn this review paper, recent developments in fabricating scaffolds for GF delivery in bone tissue regeneration were discussed. Despite progress covered in this paper, more work is required to develop biomaterials that are porous and mechanically strong, that can present controlled degradation, and that match the rate of new bone formation. Well-known side effects of direct GF injection lead to the clinical need for developing delivery systems with controlled GF delivery. Among the different available strategies, GF encapsulation in the structure of scaffolds can be considered a promising method to control the release kinetics of GFs and to fabricate scaffolds with improved characteristics. The GF/scaffold release system should mimic the coordinated fracture repair pathway in practical applications. Moreover, delivery systems with the capability of delivering multiple GFs in a targeted manner could promote the inflammation, angiogenesis, and osteogenesis phases of bone formation.


Given the frequency of natural hazards in Haiti, disaster risk reduction is crucial. However, evidence suggests that many people exposed to prior disasters do not engage in disaster preparedness, even when they receive training and have adequate resources. This may be partially explained by a link between mental health symptoms and preparedness; however, these components are typically not integrated in intervention.N = 480), across three time points, using a randomized controlled trial design. The 3-day community-based intervention was culturally-adapted, facilitated by trained Haitian lay mental health workers, and focused on enhancing disaster preparedness, reducing mental health symptoms, and fostering community cohesion.The current study assesses effectiveness of an integrated mental health and disaster preparedness intervention. This group-based model was tested in three earthquake-exposed and flood-prone communities (Consistent with hypotheses, the intervention increased disaster preparedness, reduced symptoms associated with depression, post-traumatic stress disorder, anxiety, and functional impairment, and increased peer-based help-giving and help-seeking. Mediation models indicated support for the underlying theoretical model, such that the effect of the intervention on preparedness was mediated by mental health, and that effects on mental health were likewise mediated by preparedness.The community-based mental health-integrated disaster preparedness intervention is effective in improving mental health and preparedness among community members in Haiti vulnerable to natural hazards. This brief intervention has the potential to be scaled up for use with other communities vulnerable to earthquakes, seasonal flooding, and other natural hazards. In recent years, the UN's head of disaster planning has warned that a global failure to plan for future natural disasters will have ‘inconceivably bad’ consequences, especially as climate change leads to increase in chronically-occurring natural disasters and the humanitarian crises that often result Jones, . The UN et al., et al., et al., et al., et al., In chronically disaster-exposed contexts, engagement in individual and community-level disaster preparedness is critical. Even in resource-poor settings, low-cost options exist, such as planning evacuation routes, identifying community vulnerabilities and resources, practicing safe storage of documents, creating preparedness kits comprised of basic supplies, and teaching children about disaster response strategies. Yet, despite substantial efforts to train disaster-prone communities in disaster risk reduction strategies, growing evidence suggests that many people do not engage in preparedness activities, even when they possess sufficient resources, receive related education, and/or have a history of disaster exposure . Findings from research with survivors of the 2010 earthquake in Haiti indicate high rates of PTSD, depression and other forms of distress, yet access to effective treatments is rare  design.In chronically disaster-prone contexts, it is important to focus on promoting recovery from past disasters and encouraging preparedness for future disasters. We hypothesize that a brief intervention that integrates disaster preparedness content with mental health psychoeducation and coping skills, as well as a framework that encourages social cohesion and peer support, is likely to be especially effective in such settings. To this end, we developed a 3-day This study tested the following hypothesized intervention effects: (1) increase in engagement in disaster preparedness activities; (2) decrease in depression, PTSD , and generalized anxietyIn addition to investigating intervention outcomes, the current study aims to elucidate support for the underlying theoretical model. Specifically, we investigate the premise that prior disaster exposure can be associated with reduced preparedness activities, particularly among those suffering from mental health symptoms. We hypothesize correlations among prior disaster exposure and disaster preparedness and mental health symptoms at baseline (Time 1), such that greater disaster exposure is associated with (6) reduced preparedness and (7) increased mental health symptoms. Further, we predict (8) mediated effects (at Time 1) such that the relationship between disaster exposure and reduced preparedness is explained by heightened mental health symptoms.Finally, we investigate potential change pathways for intervention effects. We hypothesize correlations among disaster preparedness, mental health symptoms, and social cohesion at Time 1, such that (9) less severe mental health symptoms are associated with increased preparedness, and (10) social cohesion is positively associated with both mental health and preparedness. Further, we hypothesize mediated intervention effects such that (11) a change in mental health  will explain the impact of the intervention on preparedness. As little work has been done in this area, we will also test a reversed model to determine if (12) a change in preparedness will explain the impact of the intervention on mental health. Finally, we predict (13) a change in social cohesion will explain the impact of the intervention on preparedness and on mental health.This study was reviewed and approved by the Institutional Review Board at the University of Colorado, Boulder, as well as Enstite Travay Sosyal ak Syens Sosyal  in Port-au-Prince.Research included 480 randomly selected community members, drawn from three disaster-affected communities (160 participants in each) in metropolitan Port-au-Prince between July 2014 and April 2015. Identification of research communities and participant recruitment procedures are detailed in online Supplementary Appendix 1.Research utilized a RCT design. All participants  completed assessment interviews at baseline  including outcome measures, demographic factors, and indicators of vulnerability (see Measures section below for details). After all completed baseline interviews, half of participants from each community  were randomly selected to receive the 3-day intervention soon afterward (August 2014). Randomization occurred using a random number generator applied to participant lists. Due to staffing constraints, interviewers were not blind to condition, as team members served as both interviewers and intervention facilitators . Members of the research team observed intervention sessions, completing a fidelity checklist to ensure that key components were covered.Over the next several months, Port-au-Prince experienced a typical hurricane season with moderate associated flooding and other storm-related damage in the research communities. In December 2014 (3–4 months after the first assessment), Time 2 data collection was conducted for all (treatment and control group) participants using the same measures employed at baseline. In March 2015, all participants completed a third  assessment, again using a similar interview schedule. In August 2015, following the third data collection timepoint, wait-list control group participants were invited to participate in the intervention.mental health integrated disaster preparedness intervention utilizes an experiential approach, including facilitated discussion, space for sharing personal experiences and exchange of peer-support, establishing safety and practicing coping skills targeting disaster-related distress, and hands-on training in disaster preparedness and response techniques for use by participants in their own lives and to support other community members. Content was developed in collaboration with Haitian team members, using an approach consistent with best practice guidelines and recommendations from others working in Haiti  and had more children than the intervention group.Baseline data, collected at Time 1, for the full sample and for intervention and control groups, are presented in At baseline, the most frequently endorsed chronic stressors were problems with employment, food, and private life. Participants reported high levels of prior disaster exposure, with nearly all exposed to an earthquake and two-thirds exposed to flooding. Most participants (70%) reported significant disaster-related property damage and nearly 60% reported that they had been permanently or temporarily displaced by a prior disaster. Almost half of participants reported that a friend or family member had been killed in a disaster, and nearly a quarter reported that they had been personally injured. There were no significant differences between intervention or control groups in chronic stressors, disaster exposure, or in outcome variables at baseline.See Results were qualitatively similar for all models when disaster exposure was controlled for by inclusion as a covariate. Gender did not significantly moderate the impact of the intervention for any of our key dependent measures, suggesting that the intervention was equally effective for women and men.In baseline (Time 1) data, there was a trend-level negative correlation between disaster exposure and disaster preparedness (Hypothesis 6). Disaster exposure was positively correlated with mental health measures , such that increased exposure was associated with more mental health symptom severity (Hypothesis 7). See ab) (Hypothesis 8). Increased disaster exposure was associated with more severe mental health symptoms (a), which were in turn associated with poorer disaster preparedness (b). Depression, PTSD , anxiety, and functional impairment all acted as significant mediators, and in all cases, there was no direct effect of exposure on preparedness (c’), indicating full mediation. Social cohesion was not a significant mediator. Disaster preparedness did not mediate a relationship between disaster exposure and mental health symptoms. See Mediation models conducted with Time 1 data revealed that, as predicted, the relationship between disaster exposure and reduced disaster preparedness was mediated by mental health symptoms  were correlated with disaster preparedness, such that fewer mental health symptoms were associated with more preparedness (Hypothesis 9). Higher social cohesion was associated with better mental health (Hypothesis 10). See c’). Furthermore, as hypothesized (11), the effect of the intervention on disaster preparedness was partially mediated by changes in depression, PTSD  and anxiety (ab), such that the intervention decreased mental health symptoms (a), and this reduction in symptoms partially explained the increase in preparedness (b). There were no mediating effects of functional impairment. In a multiple mediator model involving all three significant mental health mediators acting in parallel, only anxiety remained a significant mediator of the effect of the intervention on preparedness .We then asked whether the intervention effect on disaster preparedness was mediated by changes in mental health symptoms and/or functional impairment. As expected based upon main intervention results, there was a direct effect of the intervention on preparedness (As hypothesized (12), intervention effects on depression, PTSD , and anxiety were likewise partially mediated by preparedness, such that the intervention increased preparedness, and heightened preparedness partially explained decreased mental health symptoms. Disaster preparedness did not mediate the intervention's effect on functional impairment. Social cohesion did not mediate effects of the intervention on preparedness or on mental health (Hypothesis 13).ab/c), indicated that mediated effects were greater in general for models in which disaster preparedness mediated the effect of the intervention on mental health, relative to models in which mental health mediated the effect of the intervention on preparedness. Moreover, in both cases, models involving anxiety exhibited the largest mediated effects. See A measure of the size of the mediated effect, calculated as the ratio of indirect effect to total effect . Intervention participants engaged in an average of four additional preparedness behaviors compared to those in the control group. The intervention also decreased symptoms of depression, PTSD , anxiety, and functional impairment, measured using culturally-adapted tools. Intervention participation also resulted in a trend-level increase in perceived social cohesion, and significant increases in help-giving intention, with regard to both mental health and disaster mitigation, and in disaster-related help-seeking. There were no significant effects on mental health related help-seeking, perhaps due to difficulty interpreting this single item; although we had intended to capture even informal help-seeking , it may have been interpreted as referencing formal mental health services, which are notoriously limited and hard to access in Haiti  were significant up to seven months after the intervention, suggesting relatively sustainable intervention effects.et al., et al., et al., In addition to assessing intervention outcomes, data were further analyzed to assess the validity of the underlying theoretical model. These analyses expand upon existing evidence regarding behavioral theories of disaster preparedness, which are primarily derived from developed, western countries . This is consistent with research suggesting that symptoms typically associated with depression and other mental health issues may interfere with preparedness (see Eisenman ab/c) was generally larger for models in which preparedness mediated the effect of the intervention on mental health compared to models in which mental health mediated the intervention's effect on preparedness, suggestive of the potential importance of this putative causal pathway. Moreover, in our work with earthquake-affected communities in Nepal using a similar intervention, mediation models indicated that the effect of intervention on mental health (depression) was partially explained by preparedness, whereas the inverse was not true  in this population.Work in highly impoverished, transient, disaster-prone communities invites inevitable challenges. Participant attrition was high, largely due to population transience and initial mistrust of service providers related to a history of low NGO follow-through in the intervention communities. Efforts were made to accommodate for this through use of conservative intent to treat (ITT) analyses, designed to more accurately reflect the impact of an intervention in practice by accounting for treatment non-compliance and dropout in an unbiased manner Gupta, .Additionally, logistical and resource challenges meant that local team members served as both facilitators and interviewers. It was therefore not feasible to have interviewers blind to condition, creating the potential for bias . Moreover, this study entailed comparison to a wait-list control group. Future studies should compare this mental health-integrated disaster preparedness model to disaster preparedness as usual and mental health as usual models, to determine whether the combination of elements is in fact more effective than each element administered alone.et al., Future work should also test adapted versions of this intervention in other cultural and disaster contexts. It is possible that integration of mental health and disaster preparedness interventions may be more critical in certain contexts, such as those in which disasters are chronic, mental health problems are prevalent and communities may feel especially hopeless, avoidant, or anxious about preparedness. Of note, two related studies were conducted in Nepal, using a parallel intervention adapted to flood- and earthquake-exposed communities. Whereas preparedness and social cohesion results are generally consistent, mental health results do appear to vary across contexts, with more robust results in earthquake-affected communities  psychosocial interventions; group-based interventions; and interventions using natural support systems, evaluated using scientifically rigorous methods (Blanchet


Proteomics is the large-scale study of the total protein content and their overall function within a cell through multiple facets of research. Advancements in proteomic methods have moved past the simple quantification of proteins to the identification of post-translational modifications (PTMs) and the ability to probe interactions between these proteins, spatially and temporally. Increased sensitivity and resolution of mass spectrometers and sample preparation protocols have drastically reduced the large amount of cells required and the experimental variability that had previously hindered its use in studying human neurological disorders. Proteomics offers a new perspective to study the altered molecular pathways and networks that are associated with autism spectrum disorders (ASD). The differences between the transcriptome and proteome, combined with the various types of post-translation modifications that regulate protein function and localization, highlight a novel level of research that has not been appropriately investigated. In this review, we will discuss strategies using proteomics to study ASD and other neurological disorders, with a focus on how these approaches can be combined with induced pluripotent stem cell (iPSC) studies. Proteomic analysis of iPSC-derived neurons have already been used to measure changes in the proteome caused by patient mutations, analyze changes in PTMs that resulted in altered biological pathways, and identify potential biomarkers. Further advancements in both proteomic techniques and human iPSC differentiation protocols will continue to push the field towards better understanding ASD disease pathophysiology. Proteomics using iPSC-derived neurons from individuals with ASD offers a window for observing the altered proteome, which is necessary in the future development of therapeutics against specific targets. Autism spectrum disorders (ASD) are a broad range of neurodevelopmental disorders, ranging in severity between individuals. It is defined by two core symptoms, impaired social communication and reciprocal interaction, and the presence of repetitive behaviors and restricted interests. Due to heterogeneity in ASD, treatments are currently focus on the associated symptoms of ASD, specifically irritability and aggression through either risperidone or aripiprazole, both originally prescribed as antipsychotics . HoweverLarge-scale genome sequencing studies have identified numerous ASD risk genes , which Studies of the transcriptome have been key for identifying disrupted networks , 8 revi. Very feASD-associated genes can also be categorized into many major gene networks including cytoskeleton, channels, post-synaptic density, chromatin, and intra-cellular signaling , 18. A rMass spectrometry (MS) is the popular choice to study proteomics due to its ability to handle complex protein samples with high resolution. It is highly versatile and can be applied to study protein abundance, PTMs, and protein interactions. Proteins from biological samples are ionized into fragments and then into precursor ions for detection by a specific mass analyzer. The two most common ionization tools are liquid chromatography-electron spray ionization (LC-ESI) and matrix-assisted laser desorption ionization (MALDI) . LC-MS iThere are two methods of quantitative proteomics, top-down and bottom-up Fig. a, d. TopShank3 mouse model of syndromic ASD [Label-free quantification (LFQ) is a MS-based proteomic method that determines the relative or absolute amount of proteins between biological samples. LFQ methods are able to achieve higher sequence coverage than label-based methods, allowing for highly reliable protein identification . Due to omic ASD . Howeveromic ASD .Label-based proteomics is achieved using chemical or metabolic tags. The most popular choice are the isobaric tags for relative and absolute quantification (iTRAQ) , 30. TheFmr1 KO mice cortical neurons showed decreases in synaptic proteins and mRNA transport [The most conventional metabolic labeling technique is stable isotope labeling with amino acids in cell culture (SILAC), which involves feeding cells media containing isotopic amino acids  and can ransport . Tang etransport . Furtherransport . While Sransport –43.Fmr1 KO mouse embryonic fibroblasts revealed multiple changes to the MAPK, mTOR, Wnt, and p53 signaling pathways [Shank3 mutant mice found large changes in the S-nitroso-proteome, which altered vesicle release at the presynaptic terminal [PTMs are important as they can alter a protein’s spatial-temporal function by changing its conformation, activity, or stability Fig. c. Proteopathways . Althougpathways . More reterminal . Interesterminal . Methylaterminal . TogetheProteomics studies can provide direct information about the altered protein levels in genetic mouse models of ASD; however, they cannot identify the networks that are directly regulated by the protein. The increasing number of genes identified in neurodevelopmental disorders highlight the obstacles in identifying relevant pathways , 36. TraTwo-hybrid (2H) screening is used as a confirmation tool to determine if two proteins are in close proximity or interacting directly . This meSLC9A9) alters the interaction with caveolae-mediated endocytosis and MAP2K2-mediated signaling [Affinity pull-down or co-immunoprecipitation (Co-IP) coupled with mass spectrometry is an approach to identify interacting proteins Fig. . After cignaling . Finallyignaling . TogetheUbe3a mouse model [Technical limitations of Co-IP include the strength of the interaction after cell lysis, quality of the antibody, and the use of appropriate controls. However, techniques such as chemical cross-linking mass spectrometry have been used to identify more transient interactors in the synaptic compartment . Co-IP wse model . AlthougProximity-based protein labeling coupled to MS has been a ground-breaking method for identifying protein-protein interactions (PPIs) . It alloThere are two major methods, engineered ascorbate peroxidase (APEX) or the promiscuous BirA* biotin ligase (BioID) , 78. BirAlthough proximity labeling is a powerful approach, users should be aware of limitations. APEX requires the addition of hydrogen peroxide and cannot be used in vivo due to toxicity. Tyrosine residues are among the least abundant amino acids in and therefore could result in missed labeling . AlternaBioID has not yet been reported to be used in iPSC-derived neurons or brain organoids where it will be very useful to identify distinct and shared human signaling networks. It is important to remember that endogenously biotinylated proteins are always present and could create a background of biotinylated proteins. The proper controls are therefore required to compare the experimental condition to a control . HoweverStudying neurological disease in the human brain is difficult due to the inability to acquire live samples. Post-mortem brains offer a way to study the human brain directly and have been used in a limited fashion to study ASD –98 Fig.. RNA seqThe limitations in obtaining the human brain historically has forced studies to use easily accessible samples, including urine, blood, and saliva. Analysis of urine identified changes in metabolites, toxins, and proteins as potential markers associated with ASD, but the relevance remains in question because the samples contain salts, which may skew mass spectrometry results. Blood offers a live biological samples for mass spectrometry, where MALDI-TOF was used to identify proteins that are changed in ASD (or subtypes of ASD) in several studies –107. MorHuman iPSCs allow the study of human neurological disorders with a human genetic and biological background, which is not possible in animal models , 118. HuTargeted mass spectrometry of neurons differentiated from iPSCs identified increased expression of pre- and post-synaptic proteins , suggesting that major neuron-specific proteins can be identified . In an iMAPT (which encodes for the tau protein) [Mass spectrometry to identify PTMs of proteins has also been applied to neurodegenerative models. PTMs in the tau protein were found using iPSCs generated from individuals diagnosed with frontomporal dementia and possessing the A152T mutation in protein) . They chprotein) . Furtherprotein) . This stThe same methodology can be used to track the changes in the proteomic profile of other differentiation protocols. Varderidou et al. used two iPSC to neuron differentiation protocols, expression of NGN2 to generate excitatory neurons and small molecules to generate motor neurons . InteresMass spectrometry has also been used to study the proteome of non-neuronal brain cells. Differentiation of iPSCs from patients with Costello syndrome, a neurodevelopmental disorder, casued by heterozygous mutations in RAS (HRASG12S), revealed an increased differentiation and maturation into astrocytes. To identify the extracellular proteins produced by mutant astrocytes, shotgun mass spectrometry was used to identify an enrichment of extracellular matrix remodeling proteins and proteoglycans, which have an important role during critical periods of maturation and synaptic plasticity . FinallyAdvancements in neural differentiation of iPSCs has allowed for the generation of 3D human neuron cultures, known as organoids –140, whiMass spectrometry requires the mapping of peptides and proteins, based on their spectral signature, to annotated protein databases . HVariability remains a major challenge in proteomic studies of neurological disorders. In fact, a meta-analysis of 87 synaptic proteomic studies found that only 6% of all dysregulated proteins were the same across studies. However, when looking at proteins similarly affected across experiments, proteomic signatures could be identified for multiple neurological disorders . AnotherThe majority of iPSC studies on ASD are focused on genomic sequencing; however, there is much less research done at the protein level. The coupling of fractionation with mass spectrometry has drastically increased the resolution between proteins and the accuracy of quantification; however, future improvements to mass spectrometer technology will allow for reduced amounts of required sample. In this review, we provided multiple examples of how proteomics can be applied to study neurological disorders with iPSCs, as shown in Fig.


Understanding how building blocks of life contribute to physiology is greatly aided by protein identification and cellular localization. The two main labeling approaches developed over the past decades are labeling with antibodies such as immunoglobulin G (IgGs) or use of genetically encoded tags such as fluorescent proteins. However, IgGs are large proteins (150 kDa), which limits penetration depth and uncertainty of target position caused by up to ∼25 nm distance of the label created by the chosen targeting approach. Additionally, IgGs cannot be easily recombinantly modulated and engineered as part of fusion proteins because they consist of multiple independent translated chains. In the last decade single domain antigen binding proteins are being explored in bioscience as a tool in revealing molecular identity and localization to overcome limitations by IgGs. These nanobodies have several potential benefits over routine applications. Because of their small size (15 kDa), nanobodies better penetrate during labeling procedures and improve resolution. Moreover, nanobodies cDNA can easily be fused with other cDNA. Multidomain proteins can thus be easily engineered consisting of domains for targeting (nanobodies) and visualization by fluorescence microscopy (fluorescent proteins) or electron microscopy (based on certain enzymes). Additional modules for e.g., purification are also easily added. These nanobody-based probes can be applied in cells for live-cell endogenous protein detection or may be purified prior to use on molecules, cells or tissues. Here, we present the current state of nanobody-based probes and their implementation in microscopy, including pitfalls and potential future opportunities. Defining protein identity and visualizing protein localization is fundamental in biology. Uncovering dynamics of protein localization and function were boosted when green fluorescent protein (GFP) and other fluorescent proteins (FPs) were developed and used to tag proteins of interest . AdvantaCamelidae species (Nanobodies are single variable domains of heavy-chain only antibodies(hcAB) derived from  species , but do  species . Nanobod species , because species  and can  species . This cD species . Here, aNanobodies see . The impSynonyms: single domain antibody (sdAB), variable domain of heavy-chain only antibody (VhH), nAbs. Small antigen binding protein, derived from heavy-chain only antibodies (hcAB). These are produced in cell culture or in bacteria. Intrabody The cDNA of this probe is expressed in the cells for intracellular antigen targeting. ChromobodySynonyms: Fluobody, fluorescent nanobody. Genetic fusion of intrabody and fluorescent protein. Direct visualization with microscopy is possible. Labeled nanobodiesSynonyms: fluobody, fluorescent nanobody. Nanobodies protein purified and tagged in vitro with e.g., a chemical dye.The first nanobody-based visualization of intracellular targets was achieved by the fusion to FPs : a circular permutated bacterial dihydrofolate reductase (cpDHFR) linked to the nanobody is in such a conformation that it recognizes and binds to the nanobody target. In the presence of cell permeable DHFR inhibitors, the conformation changes precluding the antigen binding site of the nanobody binding the target, and thereby loss of association of nanobody and target. This process can be reversed to activate the nanobody binding .The light-dependent nanobody, termed photobody, uses a genetic photocaged tyrosine variant that results in the inactivation of the antigen-binding site . The phoNanobodies can also be generated in cellular systems, subsequently purified and/or modified and then used in bioassays. Typically, these are secreted by mammalian cells or produced in high yields by bacteria . After pThe extracellular domain of peripheral membrane proteins in cultured cells is well accessible to ectopic added reagents and therefore straightforward to target in live-cell imaging. Nanobodies that target extracellular receptors may trigger receptor specific endocytosis, which can be important for e.g., drug delivery. Endocytosis can be triggered via binding with the human epidermal growth factor receptor 2 (HER2). Anti-HER2 nanobodies  coupled Super resolution localization of GFP surface-exposed by cells has been achieved while studing dynamic changes at the plasma membrane: The glycosylphosphatidylinositol (GPI)-anchored GFP reporter was further probed with Alexa647-conjugated nanobodies to enable SRM based on the blinking of the Alexa-dye. This resulted in higher resolution imaging of dynamic changes and detection of protein enrichments in the plasma membrane . IndirecThe plasma membrane is a physical barrier for the nanobodies to target intracellular proteins in live-cells. Therefore, custom delivery methods are needed to target endogenous proteins in (living) cells without permeabilizing the plasma membrane. If nanobody expression is not an option because it first needs chemical modification or the concentration should be well-controlled, a purified nanobody may be delivered to cells. Fluorescent nanobodies can enter cells via endocytosis, when they formed non-covalent complexes with oligomers  or they To prevent cellular uptake via endocytosis, cell-permeable nanobodies were generated by the addition of a cyclic cell-penetrating peptide . cCPPs Generating temporary permeable plasma membranes is another approach to artificially deliver cargo inside cells. Different methods to temporary permeabilize the membrane have been developed: (i) Electroporation to deliver nanobodies linked to fluorescent quantum dots (QDs) into cells . The QDsGenetic fusion with FPs cDNA is the widely used technique for protein visualization in living systems, but sometimes smaller peptide tags are preferred. Currently, there are no nanobodies available against common generic peptide tags . Hence, An array of peptides fused to the protein of interest can amplify the fluorescent signal, and thus increase the signal-to-noise ratio. A peptide-repeat called MoonTag  was crea2O2. Affinity-based probes include electron dense nanoparticles like nanogold and QDs , the ultrastructure is revealed, but localizing the protein of interest within this structure also requires probes. EM-visualization of targets benefits from the nanobody-technology because the probe is small and thus penetrates better. Therefore, milder permeabilization is needed, better preserving the ultrastructure. Moreover, the small size improves the resolution compared to traditional immuno-EM because the target and identifiable tag are in close proximity. Also in EM proteins can be specifically identified using genetically encoded or affinity-based probes. Genetically encoded probes may be based on peroxidases that creates black precipitates in the presence of diaminobenzidine (DAB) and H and QDs . Of courIntracellular nanobodies fused with soybean ascorbate peroxidase (APEX2)  can targPurified nanobodies can be applied for affinity-based immunolabeling, and thereby replace conventional IgGs. Immunolabeling with unconjugated nanobodies was used to label HER2 in breast cancer cells, followed by a secondary anti-nanobody IgG conjugated with nanogold . AlthougReagents used in CLEM that are readily detectable both in the FLM and EM, like nanogold or QDs, are relatively large and/or bulky in comparison to 15–30 kDa range FPs or even smaller fluorescent dyes like FITC. These inorganic nanoparticles whether or not conjugated to nanobodies, will have limited penetration . TherefoIn conventional EM glutaraldehyde is used to fix the samples for ultrastructural preservation . However+ stimulation, pHluorin-VAMP2 with bound nanobody is endocytosed, and new synaptic vesicles are formed. After EM preparation, the newly formed synaptic vesicles were stained black, as a result of the peroxidase attached to the nanobody, whereas older synaptic vesicles remained unstained. Thus, the endocytic route of specific proteins of interest can be visualized.Alternatively to expressing nanobody-based probes in the cellular system of interest, they also can first be purified and then applied to cells. An elegant example of this approach has been used to visualize the formation of synaptic vesicles in living cells . UltrastPurified nanobodies, fused to a FP and a peroxidase, were used to analyze the retrograde transport system in live-cells  or withiA) structure in membranes-like structures in presence and absence of natural ligands and antagonists  have a small diameter resulting in better resolution and better penetration; (ii) can visualize endogenous proteins in live-cell imaging; (iii) are encoded by a one cDNA, which enables easy molecular cloning; (iv) allow researchers to create and produce custom multi-modal probes. Potential artifacts specific for nanobody technology in microscopy include signal from chromobodies independent of target binding and modified localization of the protein target, as detailed above.Camelidae immunization, a route that may be a (seemingly) hurdle for scientists. However, these days the generation of nanobodies is both economically and time-wise competitive with generation of newly synthesized rabbit IgG against specific targets. The novice user obviously will benefit of expertise and accessibility to the needed infrastructure by more experienced users mentioned throughout this review. In addition, a fast method based on a yeast display platform to select nanobodies in vitro has been established . The major limitation in the use of nanobodies in general is the sparse availability of high affinity nanobodies for specific targets. Increasing the availability for different targets using immunization and/or micro-organism-based libraries will increase the variation in the decade to come. When more targets can be studied with nanobodies these will become a common tool in the lab and share the top-3 podium with genetically encoded tags as well as IgGs since they share benefits of both approaches to identify and visualize targets of interest in dynamic systems and with high precision.MB performed the experiment and made the figures. MB and BG wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.