During public health emergencies, people with opioid use disorder (PWOUD) may be particularly impacted. Emergent disasters such as the COVID-19 pandemic disrupt already-strained harm reduction efforts and treatment availability. This study aims to answer three research questions. How do public health emergencies impact PWOUD? How can health systems respond to novel public health emergencies to serve PWOUD? How can the results of this scoping review be contextualized to the province of Alberta to inform local stakeholder responses to the pandemic?We conducted a scoping review using the 6-stage Arksey and O’Malley framework to analyse early-pandemic and pre-pandemic disaster literature. The results of the scoping review were contextualized to the local pandemic response, through a Nominal Group Technique (NGT) process with frontline providers and stakeholders in Alberta, Canada.n = 7) represented voices from urban services, and the other (n = 4) Indigenous contexts. Stakeholders suggested that simultaneous attention to multiple crises, with adequate resources to allow attention to both social and health systems issues, can prepare a system to serve PWOUD during disasters.Sixty one scientific journal articles and 72 grey literature resources were included after full-text screening. Forty sources pertained to early COVID-19 responses, and 21 focused on OUD treatment during other disasters. PWOUD may be more impacted than the general population by common COVID-19 stressors including loss of income, isolation, lack of rewarding activities, housing instability, as well as fear and anxiety. They may also face unique challenges including threats to drug supplies, stigma, difficulty accessing clean substance use supplies, and closure of substance use treatment centres. All of these impacts put PWOUD at risk of negative outcomes including fatal overdose. Two NGT groups were held. One group (This scoping review and NGT study uncovers how disasters impact PWOUD and offers suggestions for better serving PWOUD.The online version contains supplementary material available at 10.1186/s12889-021-11495-0. When COVID-19 was declared a global public health emergency by the World Health Organization in March 2020, the opioid crisis was already impacting communities across Canada. Early public health documents highlighted that PWOUD may have more severe outcomes if infected with COVID-19, due to poorer baseline health and increased risk of transmission due to social inequities . This stWe conducted a scoping review using Arksey and O’Malley’s six stage scoping review methodology  to: i) iWe identified our research questions in partnership with operational leaders and service providers in emergency and addiction care in the context of rising opioid deaths in Alberta early in the COVID-19 pandemic. In addition to our scholarly, knowledge-generating purpose, we had the knowledge translating purpose of contextualizing findings to Alberta. Following scoping review methods, our questions were refined throughout the course of the study as we gained familiarity with the literature, until they reached the final form reported in our introduction.To identify scientific literature, a librarian (AL) and research assistant searched 10 electronic databases in May/June 2020 with search terms related to: disease outbreaks or disasters; opioid and substance use disorder; health care services and access , a librarian (AL) completed Google searches using six search strings in June 2020, limiting results to the COVID-19 pandemic, Canadian sources, and the top 100 results of each search string for manageability , country, health service, disaster examined, disaster impacts (including affected populations and service disruptions), service adaptations, as well as resource type for grey literature . Three reviewers were involved in data extraction, with 100% of scientific literature and 20% of grey literature extraction cross-checked by a second reviewer to enhance relevance and comprehensiveness of extracted information.Literature review results were thematically outlined in information sheets in which analysts recorded common issues and ideas that appeared within sources, arranging these in bullet points with citations to relevant sources. Draft information sheets formed the basis for team discussion and organization of bullets under thematic headings. The information sheets formed an interim stage in analysis and were provided to stakeholders during Nominal Group Technique (NGT) sessions (discussed below) as a basis for co-interpretation of findings. Key themes presented to the stakeholders were: 1) increased risks during disasters for PWOUD, 2) models of care adaptations, and 3) cross-systems implications. Final tables of results, as they appear in this manuscript, were created based on stakeholder input. Our development of extraction forms, information sheets and final manuscript tables are in keeping with Levac, Colquhoun and O’Brien’s elaboration upon the Arksey and O’Malley framework, and their explanation that creation of data reporting products is part of the analysis work within qualitatively driven scoping reviews . OverallReconsidering our results with stakeholders who work in the field of interest is an effort to enhance the rigour of our analysis through triangulation of perspectives, as well as a strategy for optimizing relevance of findings for health practitioners. Contextualizing scoping review results to the local pandemic response in Alberta is also a key component of this project, as universal or generalized approaches to care are not effective across diverse settings and populations.A NGT was selected for its collaborative approach to building consensus between diverse, multi-sectoral knowledge users, for its attention to context, and as an effective knowledge mobilization method, as the engaged stakeholders can apply the evidence within their spheres of influence. This approach supports a broad view of the healthcare system integrated with Indigenous ways of knowing, including attention to community, relationships and healing the whole person (see ). The NGNGT groups were composed of care providers, systems-level decision-makers, and patient advocates from Alberta, Canada, as well as representation from First Nations stakeholders. Stakeholders were invited to identify where the literature was reflective of their own experiences in service settings during the early days of the COVID-19 pandemic and where the literature could better inform and support their practices. They contributed ideas and engaged in moderated discussions to prioritize core insights about the literature data and its limitations [n = 42, 60%) (Table n = 19). Forty sources pertained to early COVID-19 responses, 12 focused on OUD treatment during hurricanes and nine focused on OUD treatment during other disasters . We also synthesize COVID-specific scientific and grey literature below. Seventy-two grey literature results met inclusion criteria  represented voices from urban services, and the second (n = 4) represented voices from Indigenous contexts including First Nations reserve settings. Stakeholders elaborated upon ideas present within the literature with issues faced in local contexts, as presented in Table The first of the two NGT groups held  during a disaster.Providers emphasized that their regulated professional bodies require them to respond to the needs of their clientele and maintain high standards of practice. Yet they also outlined gaps in their ability to provide care without systems-level support. This undermines accountability to patients and to providers, who are susceptible to burnout without the resources necessary to support their patients.Providers further noted that the neighboring province of British Columbia had early access to data and practice guidelines. They felt British Columbia seemed to engage in evidence-informed decision-making that took into account both social and health systems issues. Many providers reported turning to sources from British Columbia to guide their practice and understanding of the needs of PWOUD in their care. Stakeholders described uneven political responses within distinct jurisdictions, noting that health authorities in British Columbia increased capacity for risk mitigation opioid prescribing.Emergent disasters increase burden on PWOUD trying to meet basic needs , and aggravate risk behaviour by intensifying reliance on informal economies, and more frequent (and dangerous) substance use in isolation. Disaster literature pre-COVID-19 shows that the intensification of adversities faced by PWOUD during disasters is predictable. Public health has little reason not to anticipate the unique consequences of emergent disasters for medically underserved or socially vulnerable groups. Preparation for how disasters will impact vulnerable populations, including PWOUD, should involve nurturing relationships between providers that patients access across complex health and social services systems . As shown by our review of grey literature, early COVID-19 public health guidelines generally did not attend to the social realities of PWOUD. In future, public health should anticipate negative effects of public health measures and new hazards for populations at risk for catastrophic results of combined crises, rather than focusing attention on one crisis at a time.Early public health responses to the pandemic identified COVID-19 as the primary threat to life, yet local outcomes raise questions about this assumption. An Alberta Health report on opioid deaths from Spring 2020 reported the highest ever number of opioid-related deaths in a single three-month period in Alberta . In MarcWhile the COVID-19 death rate would almost certainly have been higher without the public health measures, avoiding COVID-19 deaths and preventing overdose deaths need not be understood as goals in opposition to one another. The dual pubic health crises could be equally addressed through evidence-informed measures that anticipate and address patient needs. This review highlights that systems that are more attentive to social determinants of health and that prioritize contextually-tailored care are better prepared for disruptions as they emerge.COVID-19 adaptations to OAT access have focused on flexibility measures  that may have helped many, but have largely relied on individual patient and provider adaptations, without systemic supports. This lack of system and service accountability to address emergent patient needs early during disruptive events is avoidable and puts unnecessary burdens on patients and providers. We present recommendations for system and service accountability in OUD care during disasters in Table Predicting the needs of diverse populations and their providers could prevent systems from becoming overwhelmed. Systems can be supported and funded to be more ready and less reactionary when the unexpected happens. Funded supports might include clinical and office space, as well as staff such as implementation leads, policy writers, planners, case managers, and social workers. Such funded supports can provide for informed approaches to both social realities and health systems issues, and examples of successful models exist. At the service level, health system navigation and case management for chronically ill and unstably housed patients has shown promise in addressing social determinants of health . At the Such approaches, and the kinds of public health approaches requested by NGT stakeholders, are united by the concepts of harm reduction and contextually tailored care . Hyshka The COVID-19 pandemic is now over a year old, and an important consideration for interpreting this review is our focus on novel public health disasters and early systems responses. COVID-19 has become a long-term event that is distinct from such disasters as hurricanes or terrorism events and more like the opioid crisis itself. Future research could examine COVID-19 literature to understand how health system approaches change over the course of such longer term disasters. Readers may judge for themselves the degree to which strained healthcare settings and pandemic-focused public health responses emphasizing physical distancing, which do not adequately support PWOUD, remain salient in light of successive COVID-19 “waves” and emergence of new coronavirus variants of concern.This scoping review and NGT study uncovers how disasters impact PWOUD and offers suggestions for better serving PWOUD. Our contextualization of findings to Alberta may be useful as a guide for those considering contextualization of literature evidence to their own contexts. Informed approaches to addressing social determinants of health and patient needs are required for greater accountability to PWOUD early during emergent disasters. As a component of disaster preparedness, healthcare systems need to engage in planning for key patient populations such as PWOUD to ensure their care can be continued concurrent with the response to the disaster. Stakeholder contextualization of the literature to Alberta highlights gaps in multi-risk management, data and decision-making, and public health organizing to respond to heightened adversities for PWOUD early during the pandemic. Simultaneous attention to multiple crises, with adequate resources to allow attention for both social and health systems issues, can prepare a system to serve PWOUD during disasters.Additional file 1. Supplementary Methods; this document provides the full search strategy including search termsAdditional file 2. Supplementary Results of Scientific Literature; this document provides a full list of the included scientific literature and brief overview of sources in a table with descriptions of pertinent findingsAdditional file 3. Supplementary Results of Grey Literature, this document provides a brief summary of the grey literature, highlighting critical documentsAdditional file 4. Literature Summary Sheet, this document provides infographics synthesizing the academic and grey literature according to three thematic areas  that guided the NGT discussions


It has always seemed likely that viruses originated early in the history of life. However, until the identification of endogenous viral elements (EVEs), there was little if any direct evidence for most virus groups ever having existed in the distant past . EVEs arBornaviridae) are a poorly understood group of single-stranded negative sense RNA viruses  that infect vertebrates. Until 2015 the family contained only one genus (Orthobornavirus); however, recent progress in sampling viral diversity has led to the establishment of two novel genera (Carbovirus and Curtervirus) (Bornaviruses (family ervirus) . The proervirus) . The asservirus) ] of zoonervirus) .A zoonotic transfer event involving a novel bornavirus was recently recorded in Germany following the deaths of three men from progressive encephalitis. Strikingly, all three were also breeders of exotic squirrels, and this epidemiological connection soon led investigators to identify the pathogen responsible—variegated squirrel bornavirus 1 (VSBV-1) . RetrospInformation archived in EVE sequences can reveal the long-term evolutionary history of viruses, leading to a more complete understanding of their biology. The distribution and diversity of bornavirus EVEs have been investigated previously, but no previous study approaches the scale of that performed by Kawasaki et al. They searched the genome sequences of 969 eukaryotic species for bornaviral EVEs and identified hundreds of unique loci. Most of these bornavirus “fossils” are highly degraded and fragmentary, making it difficult to do meaningful comparative analyses. However, by artfully combining high-throughput computational approaches with careful human oversight, Kawasaki et al. tease out the evolutionary connections between contemporary bornaviruses and the extinct “paleoviruses” represented by EVE sequences, thereby reconstructing a detailed picture of bornavirus evolution . NumerouOf all the kinds of inferences that can be extracted from EVE sequence data, age calibrations based on the detection of orthologous EVEs in related species are among the most reliable, since they are derived in part from an orthogonal data source (the fossil record of host species) . HoweverFor an EVE sequence to arise, DNA derived from a virus genome has to become integrated into the genome of an infected germline cell. Current evidence indicates that in the case of bornaviruses and other mononegaviruses, integration usually occurs via LINE1-mediated retrotransposition: Viral messenger RNA (mRNA) becomes associated with a LINE1 protein complex, which leads to it being reverse-transcribed into DNA and integrated into the nuclear genome . A telltThe frequency with which bornavirus EVEs occur in certain vertebrate groups raises questions about their potential functional roles as co-opted or exapted host genes. Previous work has found evidence that some have antiviral functions, and conceivably bornaviral EVEs could be involved in other biological processes –15. PracThe PNAS study illustrates some of the limitations of nonretroviral EVE data. Even though bornavirus-derived EVEs are quite common in some groups, they are sparsely and patchily distributed overall. This can make it quite difficult to interpret the relationship between EVE distribution and diversity and long-term virus–host interactions. For example, the absence of EVEs from some species groups might superficially seem to imply the historical absence of virus, but other factors impacting the distribution of EVEs need to be considered. These are not restricted to the biological aspects of virus replication that increase the likelihood of germline integration —all of which could potentially vary greatly among virus strains and host species—but also encompass a broad range of factors influencing the frequency with which integrated EVE sequences become fixed in the germline. Straightforward population genetics laws dictate that the vast majority of EVEs will be eliminated from the gene pool within a few host generations. Consequently, factors impacting the likelihood of fixation  could potentially have played an important role in shaping EVE distributions, and this may distort our impressions.While EVE-based studies have limitations, there is no denying their fundamental impact on our understanding of virus evolution. Increasingly, this is being reflected in studies of contemporary viruses as date calibrations allow species diversity and biological properties to be placed into evolutionary context , 16. In


Knowledge regarding symptom clusters may inform targeted interventions. The current study investigated symptom clusters among cancer survivors, using machine learning techniques on a large data set.Data consisted of self-reports of cancer survivors who used a fully automated online application ‘Oncokompas’ that supports them in their self-management. This is done by 1) monitoring their symptoms through patient reported outcome measures (PROMs); and 2) providing a personalized overview of supportive care options tailored to their scores, aiming to reduce symptom burden and improve health-related quality of life. In the present study, data on 26 generic symptoms  were used. Results of the PROM of each symptom are presented to the user as a no well-being risk, moderate well-being risk, or high well-being risk score. Data of 1032 cancer survivors were analysed using Hierarchical Density-Based Spatial Clustering of Applications with Noise (HDBSCAN) on high risk scores and moderate-to-high risk scores separately.When analyzing the high risk scores, seven clusters were extracted: one main cluster which contained most frequently occurring physical and psychosocial symptoms, and six subclusters with different combinations of these symptoms. When analyzing moderate-to-high risk scores, three clusters were extracted: two main clusters were identified, which separated physical symptoms (and their consequences) and psycho-social symptoms, and one subcluster with only body weight issues.There appears to be an inherent difference on the co-occurrence of symptoms dependent on symptom severity. Among survivors with high risk scores, the data showed a clustering of more connections between physical and psycho-social symptoms in separate subclusters. Among survivors with moderate-to-high risk scores, we observed less connections in the clustering between physical and psycho-social symptoms. Cancer survivors experience a myriad of symptoms rooted in physiology caused by the disease itself or caused by the treatment thereof . ProblemSuch interrelated symptoms are referred to as symptom clusters, and knowledge regarding such symptom clusters may inform targeted interventions . Some stAnother systematic review was performed for studies up to 2016 which focused on cancer patients receiving primary or adjuvant chemotherapy . NineteeIn 2015 an international expert panel regarding “Advancing Symptom Science Through Symptom Cluster Research” was formed . This paCurrently, we are in limbo of the explorative phase regarding symptom cluster research, where universal symptom clusters require identification. The inconsistencies in symptom clusters as identified by the aforementioned systematic reviews , 11 and Previous symptom cluster research is predominated by methods focused on latent variable (mixture) modeling. This method classifies individuals into unobserved clusters with (more) homogenous patterns within the clusters. Models based on homogeneity have the drawback that if most individuals are relatively alike, very large clusters will be identified. The previous literature , 11 indiOne further limitation to previous symptom cluster research is the limited breadth of symptoms studied . ResearcThe aim of this study is to investigate symptom clusters among cancer patients and survivors, by analyzing a large dataset of a broad range of self-reported symptoms using HDBSCAN . The resThe study sample consisted of users of the eHealth application Oncokompas . These uOncokompas is a fully automated online application that supports cancer survivors in their self-management by 1) monitoring their health-related quality of life (HRQOL) and (cancer-generic and tumour-specific) symptoms and health determinants; and 2) providing tailored feedback on their scores with a personalized overview of supportive care options, with the aim to reduce symptom burden and improve HRQOL . OncokomData of cancer survivors who used Oncokompas up to April 29th 2019 were used. Users can fill in Oncokompas more than once. Unfortunately, not enough users filled in Oncokompas more than once to be able to analyze trends over time. To remove within-user variance, when users had filled in Oncokompas more than once, one random time point was selected of the user for the current study. Sensitivity analyses were run by repeating the analysis a number of times using different random seeds for row selection. The results did not differ regarding the main results, and will not further be discussed. All analyses were run in R version 3.5.3 , or in PFor the cluster analyses, HDBSCAN  was perfThe network analyses were performed using the tidygraph  and ggraThe choice was made to perform the clustering based on the presence of a symptom or health determinant, instead of a continuous score of severity. Methodologically, clustering based on a dichotomous presence of a symptom or health determinant results in clusters that are more easily interpretable  than clusters based on symptom severity (i.e. “these patients share a similar range of symptom severity”). The benefit of a more easily interpretable cluster is two-fold. Firstly, it will hopefully lead to more easily replicable symptom and health determinant clusters in follow-up confirmatory research. Secondly, the knowledge regarding identified symptom and health determinant clusters it will hopefully be more useful in practice. We theorize it is easier for clinicians to relate the presence of symptoms and health-related issues to one another, than it is to relate the severity of such symptoms and health-related issues to one another. For example, it appears easier to identify the probability of insomnia being present when a patient has been diagnosed with depression, rather than the probability of insomnia being present based on a severity scale of depression.One analysis was run on only high risk (red) scores as the definition of a symptom or health determinants being present, as these scores are based on cut-off scores with most empirical evidence. Three symptoms on which a high risk scores was not possible  clusters were found in our high risk score analysis, but not in the moderate-to-high risk score analysis. There may be an inherent difference on the co-occurence of symptoms and health determinants dependent on severity. For patients with higher severity, we observed more connections between the physical and psycho-social symptoms and health determinants, compared to patients with lower severity. This provokes the question of causality: do patients with higher severity of physical issues develop higher severity of psycho-social issues, vice versa, or are both higher severities developed in tandem due to a third causal force? The statistical methods we used are associative, intending to identify clusters of co-occuring symptoms and health determinants which do not necessarily share the same etiology . As suchIt has been suggested that cluster symptoms are not the same across different cancer diagnoses . The curcommunities in network terminology) are defined through the WalkTrap [While many studies researching symptom clusters pre-dominantly use variations of exploratory and confirmatory factor analyses , 11, innWalkTrap  or randoWalkTrap  algorithAs these are two distinctly different ways of defining what a cluster entails, it is of interest to see whether cluster machine learning algorithms and advanced network analyses reveal similar clusters. A comparison between the results of the current study and the aforementioned studies shows some overlap in the identified clusters. The study using PMRF found six main clusters: psychological symptoms, hormonal symptoms, respiratory symptoms, nutritional symptoms, CTX-related symptoms, and pain and abdominal symptoms . ParticuWhether this is due to the analytical method, the measurement method of symptoms, differences in population, or other factors is impossible to determine at this time. It would be of great interest for future research to investigate how resulting clusters differ between various machine learning clustering algorithms , various network analyses , and various more traditional clustering analyses . Research focused on comparing analytical methodology can use data simulation methods to validate which methods reproduce the most accurate clusters as the investigators directly control the existence of clusters. A data simulation approach can be supplemented with a comparison of results from real data to provide a holistic view of the results of differing clustering methods. Such a comparison can inform the previously identified objective of identifying robust “new analytic strategies for symptom cluster research” by the 2015 expert panel .One strength, but simultaneously a limitation, of this study is the use of different measurement tools for each separate symptom and health determinant. It has been argued that a standardization of how to measure symptoms for use in classifying symptom clusters is necessary for reproducible and valid interpretations . HoweverThere are three further limitations in regards to the way data was analyzed. First, while HDBSCAN was particularly suited for the current research question; due to the explorative nature of the analysis there is a distinct lack of easily interpretable fit metrics , 20. WhiThe research line of symptom clusters may eventually lead to practical applications in patient care. Currently we find ourselves in the explorative phase where plausible clusters need to be identified through the use of robust explorative analysis techniques. To move towards a confirmatory phase of research, the identified symptom and health determinant clusters need to be replicated in samples using other measurement techniques as well as using confirmatory analysis techniques. Stable and replicable symptom clusters need to be identified. As such, it is of interest to see whether the symptom and health determinant cluster found in the current study can be replicated in other research.. A form of standardization on both measurement techniques and (confirmatory) analysis techniques has been argued , but whiTo move beyond the confirmatory phase, and towards a phase of practical implementation, causal modelling techniques could be used to investigate possible etiological connections between symptoms, as well as by using subgroup analyses for differing tumor types. In particular, an investigation of how these clusters may change over time is of interest for such causal analyses. Furthermore, implementation of risk and protective factors in future models can help identify which symptoms may be best targeted as well as how care for individual patients should be tailored .Knowledge regarding symptom clusters may inform targeted interventions . While t


Electrical spinal cord stimulation (SCS) has been gaining momentum as a potential therapy for motor paralysis in consequence of spinal cord injury (SCI). Specifically, recent studies combining SCS with activity-based training have reported unprecedented improvements in motor function in people with chronic SCI that persist even without stimulation. In this work, we first provide an overview of the critical scientific advancements that have led to the current uses of SCS in neurorehabilitation: e.g. the understanding that SCS activates dormant spinal circuits below the lesion by recruiting large-to-medium diameter sensory afferents within the posterior roots. We discuss how this led to the standardization of implant position which resulted in consistent observations by independent clinical studies that SCS in combination with physical training promotes improvements in motor performance and neurorecovery. While all reported participants were able to move previously paralyzed limbs from day 1, recovery of more complex motor functions was gradual, and the timeframe for first observations was proportional to the task complexity. Interestingly, individuals with SCI classified as AIS B and C regained motor function in paralyzed joints even without stimulation, but not individuals with motor and sensory complete SCI (AIS A). Experiments in animal models of SCI investigating the potential mechanisms underpinning this neurorecovery suggest a synaptic reorganization of cortico-reticulo-spinal circuits that correlate with improvements in voluntary motor control. Future experiments in humans and animal models of paralysis will be critical to understand the potential and limits for functional improvements in people with different types, levels, timeframes, and severities of SCI. Recent studies combining spinal cord stimulation (SCS) with activity-based training have reported lasting improvements in motor function that were historically thought impossible in the chronic stage of spinal cord injury (SCI). In 2018, three independent groups demonstrated, for the first time, that participants with chronic motor-complete SCI could achieve overground walking with SCS  muscles  C who improved their ability for treadmill and overground walking after training with SCS in addition to weight-assisted training, further than what could be achieved by weight-assisted training alone  and a right lateral hemisection below at T10  in trained rats that were active during walking, suggesting these neurons may play a pivotal role in restoring voluntary locomotion. Although the SCI interrupted 98% of corticospinal tract axons, overground-trained rats recovered ~ 45% or pre-lesion fiber density in the inter-lesion dorsolateral funiculus, bypassing the T7 over-hemisection, branching into the gray matter, and recrossing the midline to develop bouton-like swellings, suggesting regenerative sprouting , a subregion of the medullary reticular formation. Silencing of glutamatergic neurons in the vGi blocked the cortical control of locomotion that had been previously enabled by optogenetic stimulation of the motor cortex and electrochemical stimulation of the spinal cord. Moreover, viral tracing and inactivation of these circuits in rats with contusion SCI revealed a reorganization triggered by neurorehabilitation with electrochemical SCS, and that regaining of volitional movements after training was contingent on these projections.Several animal studies in rats (Ballermann and Fouad The combined results from the three main clinical studies included in this review suggest recovery outcomes are directly related to lesion severity Fig.  which weWhile we believe that residual supra-spinal input may determine the upper-bounds for potential clinical outcomes, it is important to note that anatomical Kakulas  and neurTo properly address the topic of responsiveness to SCS, future clinical studies should perform objective evaluations of the integrity of residual descending projections that go further than clinical function and impairment scores. In this case, initial quantifications of spared neural circuits could dictate the types of rehabilitation exercises that could maximize an individual’s potential for SCS-mediated neurorecovery. In addition, SCI research will greatly benefit from studies on large numbers of participants to determine other factors that may affect responsiveness to SCS, such as sex, race, genetics, and economic backgrounds and generate evidence-based knowledge that appropriately represents target populations to addresses their specific needs.The studies presented in this work make clear that volitional effort from the participant is essential. When stimulation is provided alone, as done initially five decades ago, improvements in motor performance were not consistently observed. Similarly, other rehabilitation methods like robotic training are more effective when requiring volitional effort from the participants than when full assistance is provided such that stepping is produced regardless of participant effort (Field-Fote and Roach In retrospect, this should not be surprising given the interpretation of SCS as potentiating residual descending inputs (Minassian et al. Because some arm function is essential to use assistive devices like walkers and crutches in locomotor training, the inclusion of individuals with injuries above C5 in locomotor-type rehabilitation would present significant challenges. Nevertheless, novel rehabilitation strategies using wearable sensors (Seanez-Gonzalez et al. How should be exercises be diversified? We believe that the way to approach this problem is tightly connected to the diversity of function of the different components of brain-spinal projections. For example, corticospinal projections contribute to dexterous tasks in humans (Bunday et al. Improvements in motor function mediated by SCS have been observed after high-intensity rehabilitation protocols that require daily practice and months-length durations. In contrast, rehabilitation protocols covered by insurance in the early and intermediate phases of SCI are low intensity and short in duration. Moreover, clinical SCI rehabilitation protocols usually end when a patient reaches a plateau in performance (Behrman et al. Transcutaneous SCS was developed (Minassian et al. Severe injuries above T6 commonly lead to hemodynamic instability and cardiovascular dysfunction that cause repeated hypotensive episodes known as autonomic dysreflexia (Weaver et al. Although SCS-facilitated therapies have shown unprecedented improvements in motor function that persist even without stimulation, increases in strength production, range of motion, and even clinical outcomes have not been shown to have a significant, positive impact on the quality of life of people with SCI. Therefore, rehabilitation strategies should be aligned to the health and life priorities of individuals with SCI (Simpson et al. The prosthetic effect of SCS on motor function below the injury can enable the delivery of activity-based interventions that lead to unprecedented functional improvements in the chronic stage of paralysis (Angeli et al.


Cancer is a complex disease, driven by a combination of genetic and epigenetic alterations. DNA and RNA methylation modifications are the most common epigenetic events that play critical roles in cancer development and progression. Bisulfite converted sequencing is a widely used technique to detect base modifications in DNA methylation, but its main drawbacks lie in DNA degradation, lack of specificity, or short reads with low sequence diversity. The nanopore sequencing technology can directly detect base modifications in native DNA as well as RNA without harsh chemical treatment, compared to bisulfite sequencing. Furthermore, CRISPR/Cas9-targeted enrichment nanopore sequencing techniques are straightforward and cost-effective when targeting genomic regions are of interest. In this review, we mainly focus on DNA and RNA methylation modification detection in cancer with the current nanopore sequencing approaches. We also present the respective strengths, weaknesses of nanopore sequencing techniques, and their future translational applications in identification of epigenetic biomarkers for cancer detection and prognosis. Epigenetic alternations are important to different types of human cancers and are now known to link genetic changes to drive the initiation and progress of cancers. These changes can be observed as abnormal patterns of DNA methylation, disrupted patterns of histone modifications, and changes in chromatin components. More than 17 types of modification in DNA and 160 post-transcriptional modifications in RNA have been found, respectively , among wThe commonest epigenetic change in cancers is 5-methylcytosine (5mC) DNA modification. For the detection of 5mC in DNA, bisulfite converted sequencing is the most commonly applied method. Although bisulfite-based sequencing remains the gold-standard method for DNA epigenetic analysis, it has a number of limitations and is not easily applicable to resolve multiple cytosine modifications. A significant drawback of these methods is that bisulfite chemical treatment requires a high temperature and different pH conditions. It can cause significant degradation of the original DNA . Oxford DNA modifications have crucial roles in several biological processes, including cancer  and aginBecause of the different chemical properties corresponding to every modified type of nucleic acids, the methods for detecting each modification are also extremely diverse . Here, wAt present, the whole-genome bisulfite sequencing (WGBS)  mainly uUp to now, the methods of detecting m6A in RNA are various . The mosIt is worth noting that the methods listed previously basically require one of the three complex pre-treatments, namely, chemical conversion, enzyme recognition, and antibody enrichment . NanoporZNF14 gene occurred concomitant with increased epigenetic activity (unmethylated and accessible) and subnucleosomal footprints near transcription start sites (TSSs) in the cancer cells as compared to normal cells. Nanopore sequencing is also capable to analyze regions of differential methylation between parental alleles, which results in parent-of-origin effects on gene expression. These so-called imprinted regions exhibit differential methylation of CpG sites, and the long reads make it possible to accurately assess the haplotype of each read (“phasing”)  .Several bioinformatics approaches have been developed to call DNA modifications on the basis of the electric signal obtained during the sequencing run: Nanopolish , signalAMethods for detection of RNA modifications, including antibody immunoprecipitation   and chemArabidopsis . Among these loci, the nanopore-derived methylation patterns were compared against previously released WGBS data in GM12878 (r = 0.81) by directly comparing per-CpG methylation between nCATS quantitative methylation and WGBS at each locus. Additionally, they explored this approach to examine regions with differential methylation using data from breast cell lines, and then they identified one gene, the keratin family member gene KRT19, which showed differential methylation in breast cell lines. As previously reported, the expression of KRT19 is especially high in breast cancer, and KRT19 mRNA has been a suitable marker for identifying micrometastasis of breast cancer to lymph nodes and used for circulating tumor cell detection , which encodes a core protein component of the telomerase complex. Telomerase that acts to maintain the telomeric sequence to chromosomal ends is repressed in almost all somatic cells, although it is commonly expressed in the vast majority of cancer cells  gene mutation status and the methylation level of O6-methylguanine-DNA methyltransferase (MGMT) promoter in four human glioblastoma (GBM) cell lines and eight fresh human brain tumor samples. It has been known that the IDH mutation and MGMT promoter methylation status are commonly used as prognostic makers in patients with GBM (IDH1 and IDH2 mutations within 36 h, and the assessment of IDH mutational status was in agreement with Sanger and Illumina sequencing data. Using a methylated and unmethylated DNA standard, nCATS was able to provide high resolution of MGMT methylation pattern along the entire promoter region, exon 1, and a portion of intron 1 in both samples that was comparable to results generated with pyrosequencing assays. For MGMT methylation, they also applied nCATS to four well-characterized GBM cell lines and clinical samples and found that there was a significant positive correlation between the percent methylation of these four cell lines assayed by nCATS with the percent methylation returned by pyrosequencing. These MassARRAY® results they obtained also showed a similar trend with nCATS results over the same CpG sites. They further determined the relationship between the methylation level and MGMT expression for all GBM cell lines and tumors and observed a positive correlation between intronic CpG methylation and MGMT expression and a negative correlation between exonic CpG methylation with MGMT expression. Overall, these studies described previously illustrated that there is great potential to use nCATS as a clinical tool for cancer precision medicine. The nCATS requires only ∼3 μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of nanopore sequencing in research and in the clinic and will be a very active area of development, and it is possible for us to see many new exciting applications and protocols in the future.Moreover, the study by with GBM . They deIn this review, we highlighted the advances of nanopore sequencing techniques for detecting methylation modifications and biological discoveries with their application in the context of cancer. The emergence of nanopore sequencing analysis has enabled direct methylation readout throughout genome, transcriptome, and target region sequences, including previously inaccessible epigenomic regions, such as highly repetitive areas. With respect to the epigenetic features on DNA, nanopore sequencing makes it possible to evaluate differential methylation and chromatin accessibility from a single high-throughput experiment, providing excellent opportunities to pinpoint cancer gene regulation. Moreover, the nanopore sequencing peculiarity of being able to directly sequence RNA with high accuracy allows a better study of differential modifications and expression from a single high-throughput reaction. Furthermore, the sequencing depth granted by nCATS is extremely useful for analyzing heterogeneous samples typically obtained from pre-clinical and clinical samples, shedding new light on the epigenetic changes in the cancer onset and progression. However, nanopore sequencing as a technology still under development and frequent updates in chemistry and software currently challenge its clinical application and need to be addressed to allow standardized diagnosis across laboratories. Further development and improvement of nanopore sequencing accuracy are also required to thoroughly resolve and decode cancer epigenetic changes at a single-base level.SW and HL conceived the idea and revised the manuscript. JZ led the writing and produced the table and figure. SX wrote part of the manuscript. SW supervised the study and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Preservatives in multidose formulations of topical ophthalmic medications are crucial for maintaining sterility but can be toxic to the ocular surface. Benzalkonium chloride (BAK)—used in approximately 70% of ophthalmic formulations—is well known to cause cytotoxic damage to conjunctival and corneal epithelial cells, resulting in signs and symptoms of ocular surface disease (OSD) including ocular surface staining, increased tear break-up time, and higher OSD symptom scores. These adverse effects are more problematic with chronic exposure, as in lifetime therapy for glaucoma, but can also manifest after exposure as brief as seven days. Multiple strategies are available to minimize or eliminate BAK exposure, among them alternative preservatives, preservative-free formulations including sustained release drug delivery platforms, and non-pharmacological therapies for common eye diseases and conditions. In this paper, we review the cytotoxic and clinical effects of BAK on the ocular surface and discuss existing and emerging options for ocular disease management that can minimize or eliminate BAK exposure. Preservatives serve a critical role in the formulation of topical ophthalmic medications used to treat a wide variety of ocular conditions. Required in multidose formulations by most regulatory bodies since the 1970s . Their eMany different molecules have antimicrobial properties and have been incorporated into ophthalmic formulations. These have been extensively reviewed –7 and arOf these many molecules, Benzalkonium chloride (BAK) is used in approximately 70% of ophthalmic formulations , 9 and iThe monograph aims to review the adverse effects of acute and chronic exposure to BAK and to describe options for reducing or eliminating exposure to BAK and other preservatives in topical ophthalmic medical therapy.The cytotoxic effects of BAK on ocular tissue cells have been extensively documented Table . The thrThe underlying mechanism(s) by which BAK damages cells of the ocular tissues has not been fully elucidated. BAK is a quaternary ammonium compound and is thus cationic, so it is possible that BAK interacts with mitochondria, which are the only negatively charged intracellular compartment. Short-term exposure to cultured conjunctival epithelial cells stimulates the production of hydrogen peroxide, a reactive oxygen molecule, resulting in partial mitochondrial dysfunction . BAK hasMost of what it is known about chronic BAK exposure comes from patients with glaucoma receiving chronic therapy with one or more BAK-preserved medications dosed one or more times daily. Given the known cytotoxic effects of BAK on the ocular surface, one would expect a higher rate of ocular surface disease (OSD) in eyes with chronic BAK exposure. In fact, the prevalence of OSD among glaucoma patients has been reported to be 30–70% in various studies –42, whicAdditional insight in BAK toxicity can be drawn from patients with OSD and allergy. OSD therapies are also formulated with preservatives, including BAK , which aStaphylococcus epidermidis [S. aureus species including pathogenic methicillin-resistant S. aureus [Also, BAK-preserved artificial tears alter the bacterial flora of the nasal mucosa reducing the population of dermidis , which c. aureus , 72 as w. aureus . The eff. aureus .The diagnosis of BAK toxicity is clinical in nature. The clinical manifestations of BAK toxicity occur in a spectrum from subclinical to severe and are often overlooked. The onset can be insidious and appear weeks or months after starting therapy, or only after a threshold number of medications or drops per day is reached, confounding the causal relationship to therapy. Mild signs may be overlooked by busy clinicians who either do not recognize the findings or choose not to address them in non-complaining patients. Likewise, patients may not report symptoms, particularly if therapy is achieving therapeutic efficacy goals, choosing instead to accept redness, discomfort, or even blurred vision as a price to pay for satisfactory glaucoma control. For patients who do report bothersome symptoms, the well-described disconnect between the signs and symptoms of ocular surface disease (OSD) can result in a lack of objective findings , 76, faiGiven the frequency and severity of BAK-related ocular toxicity, it is worthwhile to review the therapeutic options that can ameliorate BAK toxicity or reduce or eliminate BAK exposure in patients requiring short-term or long-term topical ophthalmic medical therapy Table .Table 4SThe adverse effects of BAK can be ameliorated by adding preservative-free lubrication formulated with taurine to the BAK-preserved medical regimen, which has been shown to improve tear break-up time (TBUT), conjunctival goblet cell density, and Ocular Surface Disease Index (OSDI) and Glaucoma Symptom Scale (GSS) scores . For patBAK exposure can be eliminated by using therapies formulated with preservatives other than BAK, by using preservative-free formulations (including emerging sustained drug delivery platforms), or by utilizing non-medical therapies Table . Each ofA number of alternative preservatives have been developed for topical ophthalmic therapeutics that are collectively less harsh and better tolerated than BAK. The key drawback to these preservatives is that they are proprietary in nature and are thus limited to use—at least currently—in formulations produced by their manufacturer.SofZia  is an ionic buffered solution containing borate, sorbitol, propylene glycol and zinc  that crePolyquad (Alcon) is a hydrophilic cationic quaternary ammonium polymer  27 timesPurite  is a stabilized oxochloro complex (SOC) containing 99.5% chlorite, 0.5% chlorate, and trace chlorine dioxide that converts to sodium and chloride ions, oxygen, and water upon contact with the tear film . ChlorinSelect topical ophthalmic therapies for the treatment of glaucoma, OSD, and allergic eye disease, among others, are available in preservative-free formulations  at the intended therapeutic concentration for a sustained period of time, avoiding the peaks and troughs of pulsed topical dosing. There are numerous advantages and disadvantages to these drug delivery platforms. As for advantages, these options reduce or eliminate the need for patient self-dosing, which in turn reduces or eliminates nonadherence and therefore may be associated with improved clinical outcomes and quality of life. Sustained drug delivery can significantly reduce the treatment burden imposed by daily topical dosing of glaucoma medications or reduce the frequency of intravitreal injections for ocular inflammatory disease, for example. Drugs can be delivered directly to target tissues, which may include the ocular surface to treat OSD, both the ocular surface and the anterior segment to treat glaucoma and postoperative inflammation, and the posterior segment to treat retinal diseases. Intraocular drug delivery spares the ocular surface exposure to active and excipient ingredients. Both steady-state and tapering drug levels can be delivered by sustained drug delivery platforms over time periods ranging from 30 days to three years by altering design elements.Disadvantages of sustained drug delivery platforms include the need for administration by healthcare professionals in most cases, often via medical or surgical procedures that may incur procedure-related safety events in addition to drug-related side effects, although in some cases administration can be paired with planned surgical procedures to minimize added risk or exposure. Dose titration—strength/concentration, dosing schedule, etc.—is not possible as the daily dose delivered is a function of each platform’s design. For intraocular platforms, it can be difficult or impossible to discontinue therapy before the drug is fully depleted, such that drug-related side effects may be difficult to manage. Existing platforms are designed for single-drug delivery only, which limits the value of these options for patients who require multiple drugs to manage their ocular condition(s). There is the theoretical possibility of reduced efficacy compared to standard pulse dosing, for example in the case of receptor saturation and downregulation with sustained release of prostaglandins for glaucoma therapy , 106. InSustained release technology is not new to ocular therapeutics. More than 40 years ago the Ocusert was a sustained delivery system that resided on the ocular surface in the conjunctival fornix and delivered pilocarpine for IOP control for a week at a time . In the Durasert  is a small, injectable sustained delivery system that can deliver small molecules at therapeutic concentrations for up to three years without the need for preservatives. This technology is based on a permeable polyvinyl alcohol membrane positioned between the drug reservoir and the device’s aperture that controls the rate of drug elution. The platform is nonerodable and a capsule remains in the eye after complete drug elution. The system has been incorporated into several commercial products to treat a variety of chronic ocular conditions , includiThe NOVADUR system  is another notable sustained delivery system. In this system, the active drug is embedded in a polymer matrix of poly  (PLGA) that slowly degrades to lactic and glycolic acids which in turn degrade to water and carbon dioxide upon drug depletion, leaving no residue in the eye . Two bio® intracanalicular insert  is a preservative-free sustained-release formulation of dexamethasone 0.4 mg encapsulated in a hydrogel sustained delivery system indicated for control of inflammation and pain following ophthalmic surgery. As it dissolves within the canaliculus, the insert delivers a tapering dose of dexamethasone to the tear film up to 30 days. Hydrogel-based sustained release therapeutics are also being developed for the treatment of ocular surface diseases and intracameral delivery of travoprost for glaucoma as well as intravitreal delivery of tyrosine kinase inhibitors for retinal vascular disorders [Hydrogels are another promising sustained release drug delivery platform. Hydrogels are biocompatible hydrophilic crosslinked polymer networks that swell when exposed to water . Active isorders , 117.The Verisome platform (EyePoint) is a versatile sustained drug delivery platform that can deliver large or small molecules for periods ranging from weeks to months formulated into biodegradable solids, gels, or liquids . Dexycu At the extreme end of BAK avoidance is the eschewing of medication therapy altogether. While medication therapy is the preferred first-line approach for glaucoma treatment, laser therapy (selective laser trabeculoplasty) , minimalBAK is nearly ubiquitous in topical ophthalmic formulations and is associated with multiple adverse effects on ocular health that are aggravated with chronic exposure. As discussed above, there are many options for alternate therapies to treat a variety of conditions in patients for whom BAK avoidance is desired or necessary. Many of these, however, have limitations, including expense, availability, invasiveness, and a reduction or elimination of dose titration. There remains a significant unmet need for further development of widely available, and easy-to-administer BAK-free therapies for common ocular conditions. Innovation in bottle design might reduce preservative exposure at the point of dosing , 128. No


Every 3 minutes a parent is heartbroken by their child's cancer diagnosis .Over the last few decades, cutting-edge advances in cancer treatment have raised the 5-year cancer survival rate from 58 % in the mid-1970s to 84 % in 2019 . These Endocrinopathies like diabetes mellitus (hereafter abbreviated as diabetes) and metabolic syndrome are stated as the most common treatment-related complication with around 50 % of survivors having at least one hormonal disturbance . CardioMetabolic syndrome is a constellation of metabolic abnormalities including insulin resistance, systemic hypertension, central obesity, and dyslipidemia . The paThis letter aims to illustrate the pathophysiology of metabolic syndrome in childhood cancer survivors, learn about the different methods of screening, and early detection of metabolic syndrome, and also focus on the prevention strategies in order to increase the survival of childhood cancers. Metabolic syndrome as mentioned earlier is a cluster of symptoms, the pathogenesis of which is not completely known. Visceral adiposity has been shown to be the primary start point leading to the development of metabolic syndrome . The thDiabetes is described as hyperglycemia caused by either insufficient insulin secretion due to autoimmune destruction of pancreatic β cells or insulin resistance. In recent times it has been shown that the development of diabetes has increased in childhood survivors, especially if treated at a young age or exposed to abdominal or total body irradiation . AbdoHypertension is a consequence of arteriolar vasoconstriction leading to blood exerting extra force against the vessels resulting in heart strain. Therefore, hypertension is considered one of the greatest cardiovascular determinants for morbidity and mortality. St. Jude lifetime cohort study (SJLIFE) concluded that more than one third were found to have pre-hypertension, thus increasing lifetime risk for hypertension . Howevers2286615 in BAD gene is linked with obesity, insulin resistance and dyslipidemias  . Varianal., 2017). Likewial., 2017). Moreoval., 2017).A large number of children with cancers are treated with corticosteroids and cranial irradiation. There has been a significant increase in net body fat has been reported with patients receiving steroids . All thInnumberale chemotherapeutic agents have been linked with an enhanced inflammatory reaction in the adipose tissue, thus leading to obesity. This causal relationship has been extensively explored by Caubet Fernandez et al. (2019), who opLupo et al. (2019) studiedFoster et al. (2019) have coFurthermore, Lorenc et al.  identifAll childhood cancer survivors should be counseled annually on the importance of regular physical activity and a heart-healthy diet. In order to increase the free and overall cancer survival, weight management program and physical activity have been linked with beneficial outcomes . HoweveZhang et al. (2019) like FoAlthough existing guidelines can be utilized for primary prevention of cardiometabolic complications, Gibson et al. (2016) calls fWe searched three databases which highlight the fact that we might have missed some papers. It was noticed that since it takes decades for cardiometabolic complications, many studies did not follow the subjects for a longer period of time to prove the causal link. Metabolic syndrome presents as diverse disease in different ethnicities thus an in-depth analysis of confounding factors is required. Various genetic biomarkers have been identified to be early predictors for cardiometabolic complications, however dearth of evidence on prevention of these complications calls for large scale studies.We focused on the association of metabolic syndrome in childhood cancer survivors as this has become a concern in recent times. Treatment advances and other risk factors have increased the prevalence of metabolic syndrome and thereby cardiovascular disease in these survivors. Treatments like total body irradiation and exogenous steroids have been shown to promote insulin resistance, weight gain and inflammatory response in the childhood cancer survivors.Additionally, chemotherapeutic agents have been demonstrated to enhance the adipose tissue inflammation and alter weight parameters. In addition, several novel predictive biomarkers have been collected, indicating early diagnosis and intervention. In conclusion, preventive mechanisms such as regular monitoring of HbA1c, blood pressure, and lipid profile are very important in survivors of childhood cancer through adulthood. Educating families and spreading awareness about the long-term effects of treatments with enhanced follow-up of these patients is pivotal in preventing cardiovascular morbidity in the pediatric cancer population. However, our analysis calls for further studies to demonstrate how particular treatment modification can help reduce risk of cardiometabolic complications. How a particular biomarker can predict long-term complications, the timing of intervention, what factors affect the outcomes of particular treatments, and when and how long to follow-up require further large-scale and comprehensive studies. We also hope that our survey will motivate future researchers to discover new treatment strategies, especially for childhood malignancies which have less long-term side effects on growing children.


Introduction.Landscape changes disrupt environmental, social and biological systems, altering pathogen spillover and transmission risks. This study aims to quantify the impact of specific land management practices on spillover and transmission rates of zoonotic and vector-borne diseases within Malaysian Borneo. This protocol describes a cohort study with integrated ecological sampling to assess how deforestation and agricultural practices impact pathogen flow from wildlife and vector populations to human infection and detection by health facilities. This will focus on malaria, dengue and emerging arboviruses (Chikungunya and Zika), vector-borne diseases with varying contributions of simian reservoirs within this setting.Methods. A prospective longitudinal observational cohort study will be established in communities residing or working within the vicinity of the Stability of Altered Forest Ecosystems (SAFE) Project, a landscape gradient within Malaysian Borneo encompassing different plantation and forest types. The primary outcome of this study will be transmission intensity of selected zoonotic and vector-borne diseases, as quantified by changes in pathogen-specific antibody levels. Exposure will be measured using paired population-based serological surveys conducted at the beginning and end of the two-year cohort study. Secondary outcomes will include the distribution and infection rates ofAedes andAnopheles mosquito vectors, human risk behaviours and clinical cases reported to health facilities. Longitudinal data on human behaviour, contact with wildlife and GPS tracking of mobility patterns will be collected throughout the study period. This will be integrated with entomological surveillance to monitor densities and pathogen infection rates ofAedes andAnopheles mosquitoes relative to land cover. Within surrounding health clinics, continuous health facility surveillance will be used to monitor reported infections and febrile illnesses. Models will be developed to assess spillover and transmission rates relative to specific land management practices and evaluate abilities of surveillance systems to capture these risks. Landscape changes, including land use and land cover changes such as deforestation and agricultural development, alter the socio-ecological systems in which pathogens, disease hosts and insect vectors operate and interact. Such change can either increase or decrease disease risks in different settings, with the potential to undermine control and elimination efforts and lead to outbreaks with devastating human health and economic consequences. These rapidly changing environments challenge existing disease surveillance systems to detect constantly evolving geographic distributions of infection caused by changing human and environmental systems. New approaches are needed to quantify the resulting pathogen exposure caused by specific land management practices to better target surveillance activities. Zoonotic and vector-borne diseases (ZVBD) are major causes of morbidity and mortality globally. The processes driving ZVBD spillover and transmission risks are determined by interactions between human and natural systems occurring across spatial and temporal scales. The impacts of these phenomena are increasingly evident in Malaysia; a country with one of the highest rates of landscape change globally with 34% of the land area of Borneo deforested in the past four decades. Historically, Malaysia has had a highly malaria effective control programme, culminating in the near elimination of nonzoonotic malaria species by 2020. However, deforestation has triggered the recent emergence of the zoonotic malariaPlasmodium knowlesi in humans. Since 2004, incidence of reportedP. knowlesi cases in Malaysia increased over 50-fold, andP. knowlesi is now the main cause of human malaria, with cases highly concentrated in Malaysian Borneo. Malaysia is also experiencing exponential increases in mosquito-borne arboviruses, with landscape change hypothesised as the main driver of recent expansion. For example, while dengue is commonly associated with increased urbanisation, the incidence of dengue in rural areas of Malaysia is now approaching levels observed in urban centres. This may be attributed to the importance of sylvatic dengue reservoirs in Malaysia. For example, pilot studies identified high rates of dengue seropositivity in macaques  andAedes mosquitoes in rural areas, suggesting transmission may be partially driven by zoonotic spillover . Similarly, both chikungunya and Zika viruses have been isolated in non-human primates in Borneo and sporadic outbreaks are reported in rural populations in Malaysia. This suggests landscape changes could alter the relative importance of sylvatic transmission with potential to generate contradictory effects on human risks. For example, development can lead to higher rates of human-to-human transmission but reduced risk from zoonotic spillover. Together, these data indicate landscape changes are substantially altering ZVBD epidemiology within this region and highlight the need to understand how specific land management practices impact infectious disease risks.Globally, land use change is one of the major drivers of changes in species distribution and pathogen spillover. Coupled human-environment systems, also called socio-ecological systems, refer to systems with feedbacks between environmental and societal dimensions. For example, while human activities shape landscapes, these environments in turn influence human mobility, behaviour and economic status. Assessing impacts of these environmental changes requires integrating data on human behaviour, ecology and pathogen transmission. This may be further challenged by the limited detection of infections within human populations due to variations in health-seeking behaviour, misdiagnosis and frequent asymptomatic infections. Serological surveillance provides new opportunities to measure previously undetected exposure to multiple pathogens simultaneously. By measuring long-lived antibody responses, these methods substantially increase the probability of detecting rare events and can be used to estimate transmission intensity over time or across different ecological types. Integrating serological data with detailed field studies can lead to new insights on how land management impacts transmission.Despite extensive research identifying environmental risk factors and landscape change impacts on vector populations e.g.–19, ZVBSAFE Project) The SAFE project uses an experimentally fragmented landscape (100km2) and sampling within conservation areas (1020km2) and oil palm plantations (30km2) to monitor the effects of tropical forest modification on biodiversity and ecological processes across varying spatial and temporal scales . This study will build on this experimental ecosystem to examine how specific forest management practices and agricultural developments influence ZVBD spillover and transmission risks.This study will investigate the effects of land management on ZVBD risks within large-scale experimental land use study, the Stability of Altered Forest Ecosystems (SAFE) project in Malaysian Borneo  in Malaysian Borneo. This will quantify how specific land management patterns impact socio-ecological systems to drive pathogen spillover and transmission risks. The primary outcome of this study will be the change in magnitude in antibody responses in individuals working and residing within forests, plantations, farms and rural villages surrounding the SAFE Project. Exposure will be measured by the presence of antibodies to pathogen-specific antigens. As secondary objectives, this study will collect data on distribution and infection levels of mosquito vectors, assess human behaviours and measure the incidence of malaria, dengue and arbovirus infections from clinically reported cases. Specific objectives include:1)To determine the incidence and serological exposure to malaria, dengue and selected arboviruses in human populations residing and working within in the region surrounding the SAFE Project;2)To characterise human behaviour and movement patterns influencing pathogen exposure risks;3)Anopheles andAedes mosquito vectors relative to land type and wildlife reservoir abundance; andTo describe the spatiotemporal distribution of4)To estimate human exposure to malaria, dengue and arboviruses associated with specific land management practices.Anopheles andAedes mosquito vectors relative to habitat. The full study design is outlined inThis study will be a prospective longitudinal cohort study to evaluate exposure to malaria, dengue and other arbovirus infections in human populations living and working in the vicinity of the SAFE Project in Tawau District, Sabah, Malaysia. Exposure to selected ZVBDs will be assessed using paired cross-sectional serological surveys at the start and end of the two-year study period. During this period, continuous health facility surveillance will record reported febrile illnesses and household locations. Throughout the study, randomly selected individuals residing in the study area will be asked to participate in questionnaire surveys and GPS tracking studies. Longitudinal entomological sampling will be conducted to evaluate the density of. This includes a network of camera traps and extensive data onAedes andAnopheles mosquito ecology  of the SAFE Project in Tawau, Sabah, Malaysia. To define this population, a complete census will be conducted to record basic demographic data and household GPS coordinates for all individuals residing within this area. Throughout the study period, this census will be updated monthly to reflect individuals moving into or out of this area.Individuals will be eligible for this study if they reside within the defined study area. Residence is defined as spending more than 30 continuous days within the study area prior to the survey activity. For example, individuals moving into the study area will be eligible for inclusion into GPS tracking or serological survey activities once they have resided within the study site for 30 days.Individuals will be excluded from this study if they do not reside in the area, do not consent or are under the age of 3 months. For GPS tracking studies, individuals will be excluded if they are under the age of 8 years old.Subjects can choose to withdraw at any time. If the subject chooses to withdraw from the study after previously providing consent, their specimens will be destroyed and data will not be used for analysis unless permission is explicitly given. A subject can withdraw consent at any time by contacting the study team .P. knowlesi, 3% to dengue within rural Sabah, Malaysia, expected low incidence of other arbovirus infections  toections  and 5% pThis study will consist of a two-year cohort study with parallel ecological monitoring of environmental variables and mosquito densities. The study activities and proposed timelines are outlined inPrior to commencing any activities, meetings will be held with community leaders and members to introduce the study and answer any questions. Additional meetings and community sensitisation activities will be conducted with the SAFE Project staff and key stakeholders, such as plantation and forestry workers. Based on these meetings, a study area will be defined with a complete list of kampungs (villages). Kampung lists will include villages as well as plantation and forestry camps. Following initial meetings, all households within the study areas will be enumerated and assigned a unique identification code linked to the household GPS coordinates. If no household members are present at the time of enumeration, fieldworkers will consult with the head of the village and may revisit the household or attempt to contact residents by phone.Results from this census will be employed to identify the sample population using a two-stage sampling design. At the first stage, kampungs (the primary sampling unit) will be selected from updated kampung lists obtained from the census. Kampung selection will be stratified based on indicators of land cover into three strata: forest areas, plantation camps and residential villages. At the second stage, a fixed number of households will be randomly selected from each kampung using a random selection function in R statistical software to select household prior to survey activities. All individuals above the age of 3 months in these selected households will be asked to participate in the cohort study, with a total sample size of 870 individuals (approximately 220 households). For children under 18, permission will be requested from parents or guardians . Two cross-sectional serological surveys will be conducted at the start and end of the cohort study.Open Data Kit or similar software and data will be collected using encrypted electronic tablets. A combination of village meetings and call-backs will be used to trace non-responders.Selected households will be visited and all individuals who have resided in the household for the previous month will be asked to participate in the serological surveys. The study will be explained by a trained research fieldworker and written informed consent will be obtained during a household visit. Consenting individuals will be interviewed on demographic characteristics, movement patterns, travel history, health seeking behaviour and land use practices. Electronic forms of questionnaires will be designed using. All blood samples will be processed and stored for serological analysis of exposure to malaria, dengue and arboviruses. EDTA blood samples will be centrifuged and plasma separated for serological assays with the cellular fraction retained for molecular analysis of infection. Blood smears for malaria parasites or malaria rapid diagnostic tests (RDTs) will also be prepared for all consenting individuals. Febrile individuals or individuals with a history of fever within the past 24 hours will have results assessed within 48 hours to ensure any infected individual receives appropriate treatment. Subjects with positive tests will be referred to the local district referral hospital  where they have free access to treatment and management as per standard Malaysian Ministry of Health guidelines. All other participants will be provided with their study code and contact information for the study team if they have any questions about their results.Samples will only be collected during the two serological surveys. A trained fieldworker will perform finger prick blood sampling to collect a maximum of 500μl of whole blood using an EDTA micro-container and three blood spots (20μl each) on filter paper. Plasma will also be assayed to assess exposure to other infectious diseases of interest to the Malaysian Ministry of Health. Assays will be conducted in Malaysia with a subset of samples run for quality control at the London School of Hygiene and Tropical Medicine in London, UK. Specimens will be stored at -20°C, following human tissue storage procedures established by the Universiti Malaysia Sabah.Sero-epidemiological methods will be used to detect human antibodies to pathogen-specific antigens. These antibodies reflect exposure to previous infection and can be used to characterise disease transmission e.g.,32,33.. During this period, a research assistant will regularly visit to check the device and change batteries. These data will be used to develop predictive spatiotemporal maps of human space use relative to habitat.Throughout the two-year study, rolling cross-sectional surveys of individuals residing within selected kampungs will be conducted to characterise movement patterns and disease prevention practices. Consenting, randomly selected individuals will be interviewed on occupational activities, mobility, travel history and health behaviours. Individuals aged 8 years and over will be asked to participate in GPS tracking studies. These individuals will be selected from the communities included within serological surveys, with final sample sizes to be confirmed based on demographic distribution of study participants. During these studies, participants will be given a GPS tracking device (e.g. QStarz BT-Q1000XT or a mobile phone) and asked to wear the GPS device at all times outside of the house, including on trips overnight. The device will be programmed to record a GPS location every minute for a minimum sampling period of two weeks, with participants informed about GPS tracking data, interviewed about the use of GPS tracking devices and data downloaded and manually checked by study personnel. These data will be collected at clinics by health facility staff using encrypted tablets with offline satellite maps to report and geolocate the residences and workplaces of health facility attendees seeking treatment for febrile illnesses. Other explanatory variables, including demographic information and occupational activities, will be collected using online questionnaires. Data will be integrated into models of infection rates and used to assess the sensitivity of passive surveillance systems.Additionally, to assess health seeking behaviour throughout this two-year period, continuous health facility monitoring will be established at health facilities serving the study area. This will capture anonymised data on the numbers of febrile patients attending the clinic daily, number of diagnosed infections and the residence locations of these cases. These data are routinely captured as part of Malaysia’s vector-borne disease surveillance systems. Additionally, consenting health facility attendees will be asked to provide more detailed information on their household location and movement patternsAnopheles andAedes mosquito vectors across the study site. The primary outcome of the entomological surveillance will be the abundance of host-seeking vectors, which will be used to develop predictive spatiotemporal models of vector density. Additionally, mosquitoes will be assayed for pathogen presence with molecular methods. Locally validated methods of trapping host-seeking vectors using a Mosquito Magnet trap with an attractant, carbon dioxide and counterflow technology to attract and capture mosquitoes  will be used to collectAedes andAnopheles vectors; this method has shown strong correlation with human landing catches in this region. ForAedes species, sticky traps will be used to increase the probability of detecting infected mosquitoes. No human landing catches will be used to collect mosquitoes due to ethical considerations.Ecological studies of the surrounding area will be conducted in parallel to cohort activities. Detailed data on land cover, topography and the distribution of wildlife populations will be obtained from the SAFE Project. Throughout the study period, longitudinal entomological sampling will be conducted to evaluate the density of. Entomological sampling will be conducted at eight separate locations for two days/nights at monthly intervals throughout the study. To determine the locations of entomological sampling sites, we will use a simulation-based approach designed to optimise habitat fragmentation studies. This approach extends commonly used geostatistical survey designs, in which survey locations are positioned to maximise predictive abilities and minimise survey effort by leveraging spatial autocorrelation between survey points. Geostatistical sampling designs will be adapted to maximise habitat differences between sampling locations by integrating metrics on land cover, fragment size and distance from forest edges. Previously collected mosquito sampling data from the SAFE Project . Models will explore methods of accounting for observation biases and spatial and temporal autocorrelation when relevant , healthcare coverage  andelevant . Models . Hierarchical Bayesian modelling approaches will be implemented in open-source software such as Integrated Nested Laplace Approximation (INLA) using R statistical programming language.All data analysis will be performed using R statistical softwareThis study will be conducted in compliance with ethical principles outlined in the Declaration of Helsinki and Malaysian Good Clinical Practice Guidelines. All procedures will require approval from the Malaysian National Ethics Committees and relevant Institutional Review Boards. Additionally, permission will be required from the Sabah Biodiversity Council to conduct entomological sampling and access environmental data. Access to the SAFE Project site will require protocol review and permission from the SAFE research network through an established review process.For human studies, written informed consent or assent will be obtained from all study participants. The English versions of information sheets and consent forms will be translated to Bahasa Malaysia and the back translations will be submitted to the Malaysian Research Ethics Committee. Written informed consent will be obtained from all eligible adult participants and the parents or guardians of child participants (< 18 years old) prior to the collection of information. In order to explain the study, the information and consent forms will be read out to the participant or the participant’s guardian in Bahasa Malaysia or English and they will be invited to ask questions. If they agree, the participant or the guardian will sign the consent form. If the participant is illiterate, thumbprints will be used for the consent form. Assent will also be gained on a separate form for appropriate adolescents under the age of 18 using the same signature and thumbprint methods. If the potential participant does not give consent or does not meet the study inclusion criteria then the reason for non-participation will be documented in the recruitment log. Separate consent will be obtained for serological sampling for the cohort study and GPS tracking activities.The risks associated with participating in this study are minimal. Serological surveys will involve finger prick blood sampling (<500μl) taken from all consenting participants. While mild discomfort may be felt during and after the finger prick, this presents minimal risks to participants. Blood will be collected by trained personnel to ensure safety. All participants will benefit from knowing whether they have malaria infections. Participants with positive tests will be referred to local hospital clinics where they will be treated free of charge. Additionally, this study will provide a better understanding of the diseases being assessed to inform future surveillance and control activities.A primary ethical consideration for this study is maintaining data privacy and confidentiality. Study codes will be used to identify all participants and households. Electronic forms of questionnaires will be designed using ODK or a similar software program. Data will be collected using encrypted tablets and transferred to a central data repository. Data will be password protected and encrypted and all information on participants will remain confidential. Secured network file systems, which will be institutionally maintained with regular automated backups, will be utilised to store electronic data. All data involving human participants will be stored on secure servers accessible only to study personnel. Data will be collated after securing informed consent and destroyed if consent is withdrawn. Data will be anonymised as much as possible, coding and removing personal identifiers. GPS locations of households and GPS tracking data from individuals will be treated as identifiable information and access will be limited to designated study personnel who will be required to maintain confidentiality. No personal information will be shared with third parties and any maps or spatial data published will be modified so it cannot be linked to participants.All electronic and paper data access will be restricted to authorised study personnel only. Data, including laboratory notebooks, will be archived according to Universiti Malaysia Sabah policies and kept for a minimum of ten years. All electronic data, including spatial data, will be stored on secured servers for a minimum of ten years. Data collection, documentation, storage and preservation will follow the University of Glasgow Good Management of Research Data Policy and will be stored on systems compliant with the ISO27001 Information Security management standards. Details of blood samples will be entered into existing computerised databases on supported network file systems complying with the UK Human Tissue Act.All study results will be published in open access journals and presented to the Ministry of Health and other relevant organisations in Sabah, Malaysia. Summaries of research findings will be presented to local stakeholders and communities involved within this study. While ethical considerations preclude sharing identifiable data on human populations, other datasets (e.g. mosquito data) will be shared through publicly available research data repositories maintained by the University of Glasgow and SAFE Project. All statistical code developed during this project will be shared through GitHub repositories and published as supplements to publications to ensure reproducibility. Research outputs and tools will also be shared to wider audiences through networks such as the Health GeoLab Collaborative and Asia Pacific Malaria Elimination Network.This study will be the first to describe disease transmission and exposure within a large-scale experimental landscape. This will examine how specific land management practices impact zoonotic and vector-borne disease dynamics, developing methods for characterising health impacts of environmental changes in socio-ecological systems. This study should provide new insights into mechanisms driving disease emergence in changing landscapes and identify future surveillance and control priorities.This study is currently under review with relevant ethics committees, with study activities planned to commence in September 2022, conditional on COVID restrictions.No underlying data are associated with this article.https://doi.org/10.5281/zenodo.3697804Zenodo: SAFE Sampling area borders.Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the In plain text, authors will test 645 human subjects in given period and then two years later to obtain health status variation as 0, no change; 1, change to risk; -1, change to protection per zoonotic and vector-borne disease agent . They will adjust regression model having status variation as the response variable in function of explanatory variables: (1) human space use; (2) healthcare coverage; (3) mosquito vector abundance; (4) environmental covariates. It’s therefore a strong study design.Aedes andAnopheles will be tested in the Abstract; however, this method (rather complex) is not contained in this protocol of study. My only concern is with authors stating infections inIs the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Disease Vector EcologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is a well-written and intriguing protocol of public health relevance today. The unique opportunity afforded by an experimental landscape is a major benefit and the authors created a comprehensive, multi-pronged approach to analysis. This protocol submission describes a longitudinal observation cohort over two years that will incorporate a serosurvey in ~870 people for NVBD pathogens at beginning and end (plus demographic and risk factor data), monthly entomological collections, subset of mobility studies for both humans and macaques, and collection of population-level health records recording case counts of febrile illness, etc.et al., 2020, it highly likely that most of the population has developed multitype immunity. Meaning that of the 870 persons sampled, unless they are all children under age 10, they will likely not be arboviral-naïve and it will make it much more difficult to follow their antibody responses and the study would be drastically underpowered for arboviruses. However, this would depend to some extent on the planned assay but I cannot tell from the draft what multiplex bead array will be used as the references are all forPlasmodium infection - not arboviruses. However, with only 500ul of whole blood collected - this won't render enough sera to do serotype-specific neutralization assays that would allow for quantitative titers. I presume the serological analysis is a pan-dengue assay (not serotype-specific) for IgM or IgG. This approach may work for Zika or Chik since most of the population may be naïve, but I think you will be hard pressed to calculate an FOI from serological data from 870 people, the vast majority may be immune to at least one serotype. I agree this work with your naïve population. My questions are:What percent of your enrolled population will be naïve?Is the bead-based assay serotype specific for IgG and IgM and how do you account for the cross-reactivity from multiple endemic flaviviruses?How will JEV vaccination status or timing of Sabah impact either the baseline survey or following up magnitude of change in antibodies?If you could detect a serotype-agnostic change in antibody magnitude in a person who initially dengue-monotypic with your assay, is 24 months too large of time window and antibodies from a heterotypic infection will have decreased back to resting state?  My only other comment is that two days of mosquito trapping per month for two years at eight sites may result in a very low number of infectedAedes mosquitos. We find it very rare to actually find infected pools even when collecting in the midst of dengue epidemics . I would temper any expectations that this will yield much data in terms of infection, but may certainly help with abundance measures although we know that this can be wildly inconsistent and variable (but even better - can you assess host-vector contact via serology). This is well-thought out and I have minor comments on study design that I believe are valid for following antibody responses to arboviruses. The authors plan to sample at the beginning of Year 1 and the end of Year 2 (a 24-month time frame) and they estimate a seroconversion rate of 2.5% annual increase. Given the increasing dengue burden in Sabah as laid out by Murphy The mobility studies and ecological studies are fascinating, and I certainly don't have the expertise to comment on them. The continuous health facility monitoring - we have challenging making more granular conclusions from these data sources - but I agree it is important to capture. Overall, I believe this is an excellent protocol with a few details to be provided regarding arboviral Sero-epidemiology.Is the study design appropriate for the research question?PartlyIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:Dengue, Aedes, mosquito saliva, Sero-epidemiology, metagenomicsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This protocol presented by Dr Kimberly Fornace and an interdisciplinary and highly experienced team describes a planned observational cohort study to explore possible exposure to selected zoonotic pathogens and changes the antibody response over a one year of a human population residing in a tropical forest production landscape in Malaysian Borneo. Health facility monitoring data will be used to report febrile illness incidences. In addition, mosquito vectors will be collected in the field in order to describe spatiotemporal changes in their occurrence and infection status.https://www.safeproject.net), a study area of ~7,200 ha of forest fragments and a Virgin Jungle Reserve logged forest of Ulu Segama Forest Reserve. The study is proposed to investigate the human population near the Stability of Altered Forest Ecosystems project area .Details on the data origin and sampling design for camera trap and “macaque populations” data as mentioned in Figure 4. Some clarifications might include:Is the study design appropriate for the research question?PartlyIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Biodiversity, disease ecology, BorneoI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.