Prescription drug misuse and abuse is an established public health challenge, and young adults are particularly affected. There is a striking lack of real-time, naturalistic data collection assessing intentions to misuse and other precipitating factors at the time of actual misuse, leaving the conditions under which individuals are most likely to misuse prescription medications unknown. Ecological momentary assessment (EMA) apps and protocols designed to capture this information would accelerate and expand the knowledge base and could directly contribute to prevention and treatment efforts.The objectives of this study are to describe the development and administration of a mobile app and the EMA protocol designed to collect real-time factors associated with college students’ prescription drug misuse intentions and behaviors in daily life; present completion rates, compliance, acceptability, and reactivity associated with the EMA protocol for participants who endorsed recent prescription drug misuse at screening  and those who did not ; and establish initial construct validity by linking the reports of misuse behaviors in daily life collected via the EMA app to prescription drug misuse reported on a standard survey.An EMA data collection app and protocol were designed specifically to capture hypothesized contextual factors along with prescription drug misuse intentions and behaviors in daily life. Using this protocol, young adult college students (N=352) completed signal- and event-contingent reports over a 28-day period. When the intention to misuse a prescription drug was endorsed, a brief follow-up prompt was sent 15 min later to collect participants’ indications of whether or not misuse had occurred.P<.001), to complete one or more follow-up reports (P<.001), and to endorse any prescription drug misuse behavior in daily life on the follow-ups (P<.001). Overall, participants demonstrated consistent engagement with the EMA procedures and returned an average of 74.5  reports. Participants in the risk and nonrisk groups did not differ in the number of reports they completed (P=.12), the number of their reporting days (P=.32), or their average completion rates (P=.14). The results indicated some evidence of reactivity to the momentary reporting procedure. Participants reported uniformly positive experiences and remained highly engaged throughout the reporting protocol and broader study.Risk-group participants were significantly more likely than nonrisk counterparts to endorse any prescription drug misuse intentions in daily life (The novel EMA app and protocol provide an effective way to assess real-time factors associated with prescription drug misuse intentions and behaviors in daily life. The resulting investigations offer the potential to provide highly translatable information for research and prevention efforts. Prescription drug misuse is an established public health concern in the United States and beyond ,2. AlthoPrescription drug misuse itself is potentially harmful and poses additional costs to individuals and society for its established links with illicit drug use, alcohol abuse, mental health problems, risky sexual behaviors, and overdose-related deaths -14. CollThe status quo in the literature on college students’ serious medication practices has been largely cross-sectional and based on retrospective surveys of prescription drug misuse over an extended time frame. For example, the available research predominantly involves participants indicating whether they have misused any prescription drugs over the past 12 months or their lifetime -26. ThisPreliminary findings have demonstrated the utility of collecting young adults’ momentary prescription drug misuse reports in real-world settings. We obtained data from 49 mixed-sex dating couples reporting 3 times per day for 10 days . The appBuilding on our preliminary work, the objectives in this study are to (1) describe the development and administration of a mobile app that was designed to collect real-time factors associated with college students’ prescription drug misuse in daily life; (2) present completion rates, compliance, acceptability, and reactivity associated with the EMA method for both risk group  and nonrisk group  participants; and (3) establish initial construct validity by linking the reports of misuse behaviors in daily life collected via the EMA app to prescription drug misuse reported on a standard survey.Participants attended two laboratory sessions that were scheduled an average of 35 days apart, with the EMA protocol implemented in between these laboratory sessions. Before this study, university institutional review board approval and the National Institutes of Health Certificate of Confidentiality were obtained. Participants completed questionnaires in each laboratory session, including an EMA feedback form to describe their reporting experiences.Between September 2017 and September 2019, participants at a large university in the Midwestern United States were continuously enrolled into an ongoing longitudinal study on daily behaviors and health in college life. This study is drawn from the baseline phase of a broader study . ParticiThe web-based screening obtained prospective participants’ indications that they were in a private location and agreed to complete the screening questions, which asked about potentially sensitive day-to-day behaviors. Participants were instructed to think back over the past 3 months and indicate whether they used the medications listed in any way a doctor did not intend, such as use without a prescription, increased amounts, more often, or longer than directed. The screener presented 4 prescription medication classes adapted from the 2015 National Survey on Drug Use and Health (NSDUH) : pain reGiven the main objective of capturing prescription drug misuse in daily life during this study, we oversampled for participants who endorsed recent prescription drug misuse . This study included 300 participants from the risk group and 55 participants who did not endorse recent prescription drug misuse in the screener . To minimize any differences in recruitment between groups, the nonrisk participants were enrolled simultaneously with the risk participants throughout the recruitment period. Participants completed informed consent procedures at the start of their first laboratory session. The typical EMA reporting period was scheduled for 28 days following the first laboratory session. The length of the reporting period was adjusted for some participants due to scheduling conflicts  or device issues. During the second laboratory session, participants returned their devices and completed additional measures. Participants received their choice of electronic or check payments; compensation included US $75 for session 1, US $84 for reporting in daily life , US $55 for session 2, and a US $36 bonus for maintaining compliance across the planned reporting period.The app was developed in collaboration with the university’s technology division and was installed on an Apple sixth-generation iPod Touch device. Before the initiation of this study, the research team met with developers several times to provide feedback on the app features and interface design. The survey was administered through an app presented across multiple screens, with question completion automatically advancing the participant to the next screen. The questions allowed for different user inputs . Throughout the course of this study, the developers provided technical assistance related to software updates and other questions that arose. Developers also provided training to the research team on how to program the app and device for individual participants.Given the sensitive nature of the data, security was a primary concern. We maximized the security of the data in several ways. First, the app was installed locally on laboratory-owned devices, which were provided to all participants for the duration of their reporting periods. Second, the data collected from the app were stored directly on the devices; the resulting data files were downloaded directly from the device to a secure server when participants returned to the laboratory . Third, all data were recorded using a study identifier that contained no personally identifying information. Fourth, devices were also placed in restricted mode, which prevented participants from accessing a completed report or using other device functions. In addition, wireless transfer of data to and from the device was prevented. Finally, responses were all numeric and stored on the device with nondescript variable names. The list of variables was kept separate and accessible only by laboratory members. In the unlikely event someone gained access to the completed surveys, there would be no indication of what the data represented.Measures). Signal- and event-based EMA report items were identical. Reports took approximately 2 min to complete. To reduce participant burden, a signal-contingent prompt was not sent within 2 h after a report had been completed. These EMA reports included items about intentions to misuse prescription drugs. If misuse intention of one or more of the medication classes was endorsed, participants were then sent a brief follow-up report 15 min later to assess misuse behaviors that might have occurred since the completion of the associated report; previous EMA protocols [Consistent with numerous EMA protocols on addictions -33, bothrotocols  have capprescription drug misuse was not used. Participants were trained to provide both signal- and event-contingent reports and the follow-up reports. They were also instructed to contact the laboratory staff if they experienced any problems with the device. Participants completed a practice report during their laboratory session and were instructed to carry the device at all times.During the first session, participants were thoroughly trained to complete the app reporting procedures. A comprehensive definition of the focal behavior was provided ; the term During the first laboratory session, participants reported on their gender, race, and ethnicity. In addition, the participants reported fraternity or sorority affiliation and medication prescriptions they had received in their lifetime.EMA reports captured prescription drug misuse intentions. Participants were asked “Are you about to take a medication listed here in any way a doctor did not direct you to use it?” . Four medication classes and examples were provided . Reports also collected contextual information about the current location of participants and who they were with as well as hypothesized predictors of prescription drug misuse, including other substance use, mood, pain, fatigue, and stressful events. To strengthen the predictive design, reports assessed current feelings and situations; substance use covered behaviors in the past 15 min. Items were selected from brief, validated scales used in previous research with college-based populations when possible. The number of items on each report could vary because questions used conditional answer choices  whenever possible. Follow-up reports presented the following question for each of the 4 medication classes: “Have you recently taken a medication listed here, in any way a doctor did not direct you to use it?” . Additional questions about current mood states and recent physical activities were included in the follow-up reports.. Participants rated their broader study experiences with the following: “If I contacted the lab, my questions were addressed in a helpful and timely manner”  and “Would you recommend this research opportunity to your friends?” .During their second laboratory session, participants reported on their experiences with the app and device procedures. Participants rated the extent to which “the reporting occurred during a period that reflects my typical daily life”  and the extent to which “the iPod touch device was user-friendly” During the baseline visit, participants completed a series of questions about the use of prescription medications following the 2015 NSDUH . For thit tests and chi-square analyses to compare demographic information, completion rates, and acceptability indicators of participants in the risk and nonrisk groups. Throughout this study, we report Fisher exact test when any single cell was based on <5 participants. Reactivity was tested by correlating indicators of time and length  with the outcomes of interest . Initial construct validity was examined by relating past-year survey reports of prescription drug misuse assessed during the study baseline to misuse behavior on the EMA. Given that signal- and event-based EMA report items were identical (and thus were indistinguishable in the resultant data files), both types of reports were included in the completion rates and construct validity results.We employed independent samples Participants in the risk and nonrisk groups did not differ with respect to the demographic characteristics of age or class standing . The risA total of 28,701 EMA reports were completed by 352 of the 355 participants; data were not available from 3 risk-group participants (2 did not return their data collection device and data from 1 participant’s device was not retrievable due to a password error). An important feature of the EMA protocol was collecting self-initiated reports; therefore, the app did not restrict time between reporting and allowed participants to complete several surveys within a short time frame. Upon examination of the collected data, the study team deemed it reasonable for 2 reports to be completed within 5 min of each other. Any reports beyond the second that were completed within 5 min  were removed. This resulted in the removal of 1.1%  of the obtained reports that we did not plan to include in any analysis stemming from the broader project.Some report submissions did not adhere to the EMA protocol. Given our goal of documenting the utility of the protocol, we focus only on the reports that were obtained during the scheduled reporting periods and only the follow-ups that were completed within the instructed time frame. Although the typical reporting period was scheduled for the 28 days following the first laboratory session, some participants continued reporting until they returned for the second laboratory session. We removed a total of 2147 reports that were completed outside of the participants’ designated reporting days. The resulting 91.4%  of obtained reports were associated with 439 completed follow-ups; 64.0% (281/439) of the follow-ups were completed within 15 min of being sent (following the associated report) and were retained in this analysis.P<.001). Risk participants  were also significantly more likely than nonrisk participants  to complete one or more follow-up reports according to the study protocol (Fisher exact P<.001). In addition, risk participants  were more likely than nonrisk participants  to endorse any prescription drug misuse behavior in daily life on the follow-ups (Fisher exact P<.001).In line with our sampling strategy, risk-group participants  were significantly more likely than the nonrisk counterparts  to endorse any prescription drug misuse intentions in daily life across the four medication classes . Participants in the risk versus nonrisk groups reported similar experiences with procedures and uniformly positive experiences. As shown in r=–0.01; P=.04) and number of reports completed . In contrast, when prescription drug misuse behavior occurred in daily life, reactivity in the opposite direction was found, with misuse behavior endorsed on the follow-up increasing as a function of reporting day  and report number , that is, we found evidence that prescription drug misuse intentions decreased over time (or were less likely to be endorsed as participants completed more days of reporting and more reports), whereas actual prescription misuse behavior increased over time (or was more likely to be endorsed as participants completed more reporting days and more reports). It should be noted that the magnitude of reactivity effect sizes for intentions was negligible and that for behavior was small [We examined reactivity by testing whether prescription drug misuse intentions and behavior were more or less likely to be endorsed by participants over time, in terms of their reporting length (the number of days) and reporting amount (the number of reports). Specifically, reactivity was documented with prescription drug misuse intentions decreasing as a function of the number of reporting days . Examination by medication class further revealed reliable consistency in participants reporting prescription misuse behavior across the methods. Specifically, the results indicated that the proportion of participants who endorsed misuse of a medication class in daily life according to the study protocol was significantly higher among those who had reported past-year misuse of that medication class than among those who did not for all 4 medication classes: pain medication , tranquilizers , stimulants , and sedatives or barbiturates .There was a robust association between participants engaging in prescription drug misuse in the past year at the baseline assessment and during the daily life procedure: Reporting misuse behavior (of one or more medication classes) in daily life on the EMA follow-up was significantly more likely among participants who had indicated any past-year prescription drug misuse  than among those who did not . In addition, we presented the initial construct validity of our EMA approach by linking reports of misuse behavior in daily life to prescription drug misuse reported on a standard survey. Together, the results of this study provide early support for the feasibility and value of collecting young adults’ reports of prescription drug misuse intentions and behavior in daily life.more particular risk and protective factors associated with behaviors that hold important consequences for health and adjustment. This protocol is indeed highly novel for collecting hypothesized predictors and prescription drug misuse intentions before the follow-up reports of the behavior. The resulting responses can allow many contextually based and temporally precise investigations across levels  to identify direct and interactive situations that increase college students’ likelihood of endorsing prescription drug misuse in daily life. Findings from our investigations, therefore, can provide highly translatable information for modifiable prevention and treatment targets.Although EMA still relies on self-report, this method offers improvement over more traditional assessments by minimizing reliance on memory and collecting situational factors at the time of the behavior of interest. Accordingly, these designs can provide specific momentary information to identify Our sample was highly committed to the overall study and was particularly engaged with reporting in daily life. From the start of recruitment, interest in the research was high. We maintained continual interest from potential participants. Positive engagement was further reflected by nearly all participants returning to the second laboratory session with their device and saying they would recommend the experience to others. We obtained average completion rates that were similar to a pooled compliance rate on the basis of many EMA protocols that examined different substances . We inteThe results indicated some evidence of reactivity to the momentary reporting procedure, as has been found in reporting other substances using EMA approaches  . It is pIn testing adherence to the method here, we conservatively focused on reports that matched protocol training time frames. The reports we plan to include in future studies with this data set will be preregistered on the Open Science Framework and will depend on particular research questions. When we investigate momentary experiences as predictors of prescription misuse behaviors in daily life, we plan to restrict analysis of the focal misuse behavior outcomes to those that were reported on follow-ups completed within 15 min of being sent (consistent with protocol training). Alternatively, studies that are designed to test background factors, or other person-level characteristics, associated with risk of engaging in prescription drug misuse in daily life would not need to be bound by these temporal restrictions. Indeed, for certain research aims, having more instances of the behavior available would result in more representative findings and more powerful statistical tests. These trade-offs should be considered and justified by the purpose of this study.after the behavior occurred would no longer represent contextual triggers of misuse. Instead, we focused on thoroughly training participants to respond to the prompts and self-initiate as described in the protocol. Studies focused on other research questions and aims should consider whether the addition of retrospective endorsement of the behavior would bolster their design. Consistent with the development of any rigorous study, careful planning of an EMA design that matches the research question is critical.We note that our EMA reports were identical regardless of whether participants were responding to a prompt or self-initiating a report. Thus, we could not determine the completion rates for signal-based versus event-based reporting. We were also unable to establish how many participants completed event-based assessments (or the associated follow-ups). Another protocol development decision was that we did not have participants indicate retrospectively whether prescription drug misuse had occurred since their last report, that is, a coverage strategy . There iOur sample was representative of the college campus from which it was drawn but is not necessarily reflective of students at other types of higher education institutions or of nontraditional students. Additional work will be needed to understand the applicability of this method to individuals across different contexts and locations. A practical limitation of using a separate device for EMA data collection is that the approach may be more burdensome for participants and requires greater investment in study materials. We expect that these considerations were offset by the anonymity and protection of the participant data provided by a separate device. Although reported behavior instances might be lower if the device was not readily available to the participant, the responses obtained may be more forthright due to security and privacy assurances. Nevertheless, in light of the recent successful apps on young adults’ own smartphones to assess drinking in daily life ,39, thisEstablishing real-time mood states, pain symptoms, stressors, and other substance behaviors as predictors of college students’ prescription drug misuse is critical for obtaining a more precise understanding of the behavior in daily life situations. In line with the social ecological perspective ,40, our


The recent influx of machine learning centered investigations in the spine surgery literature has led to increased enthusiasm as to the prospect of using artificial intelligence to create clinical decision support tools, optimize postoperative outcomes, and improve technologies used in the operating room. However, the methodology underlying machine learning in spine research is often overlooked as the subject matter is quite novel and may be foreign to practicing spine surgeons. Improper application of machine learning is a significant bioethics challenge, given the potential consequences of over- or underestimating the results of such studies for clinical decision-making processes. Proper peer review of these publications requires a baseline familiarity of the language associated with machine learning, and how it differs from classical statistical analyses. This narrative review first introduces the overall field of machine learning and its role in artificial intelligence, and defines basic terminology. In addition, common modalities for applying machine learning, including classification and regression decision trees, support vector machines, and artificial neural networks are examined in the context of examples gathered from the spine literature. Lastly, the ethical challenges associated with adapting machine learning for research related to patient care, as well as future perspectives on the potential use of machine learning in spine surgery, are discussed specifically. In clinical medicine, the rise of machine learning applications represents a new era of solving healthcare problems. This is particularly true in spine surgery where algorithmic decision support tools, computer assisted navigation, and surgical robots are already being used in the clinic and operating room. While the appetite for machine learning and its role in artificial intelligence has grown amongst spine surgeons, very little discussion has revolved around how to evaluate these applications and their contributions to patient care. In 2019 alone, 82 publications (more than twice the previous year) were PubMed indexed when searching for the terms “machine,” “learning,” and “spine” together. A core component of proper peer-review requires familiarity with machine learning methodology among clinicians. Until this can be achieved, machine learning in the spine literature will either foster skepticism or flawed enthusiasm. The intricacies and real patient-safety concerns when dealing with the spine necessitates that clinicians familiarize themselves with the terminology and guiding principles of machine learning. This review will introduce the origins of the artificial intelligence field and provide an organic discussion on how to practically synthesize machine learning modalities in spine surgery. A glossary of key terms in this review can be referred to in 21 bytes) (19 bytes)  originated back in the summer of 1956 when Dr. John McCarthy and contemporaries gathered at Dartmouth College. They “proceeded on the basis of the conjecture that every aspect of learning or any other feature of intelligence could in principle be so precisely described that a machine could be made to simulate it , 11.” Wh1 bytes) , 13. Thi9 bytes) . At an i9 bytes) . Experts9 bytes) . This ma9 bytes) . This inMachine Learning (ML) is a subset of AI that focuses on developing automated computer systems (learners) that predict outputs through algorithms and mathematics (discrimination (probability of predicting outcomes accurately) and calibration (degree of over- or under-estimating the predicted vs. true outcome) (hematics . The outoutcome) . Exampleoutcome) –20], preoutcome) –25, amonSupervised learning entails labeled data based on a grounded truth (training dataset) is analyzed to build a model that synthesizes the pattern between independent variables  and dependent variables (presence or absence of pathology). Individual radiograph pixels in this example are known as feature values or vectors (untrained dataset) are fed to the machine, which is then assessed based on its ability to accurately predict a fracture or otherwise. As such, supervised learning excels in exercises of linear classification (where the outputs are discretely defined categories) or linear regression .The two major forms of ML are supervised and unsupervised learning. ed truth , 5. For  vectors , 5. The Unsupervised learning, on the other hand, involves the analysis of unlabeled datasets, and stems from neuropsychology research conducted by Dr. Donald Olding Hebb   , 35. Whiinfers relationships between variables, while ML attempts to predict these relationships  decision tree learning, (2) support vector machines, and (3) artificial neural networks. It is important to consider that while the following descriptions attempt to neatly categorize each model, they are flexible and can be adapted according to their needs. For example, support vector machines are often described as supervised models for linear classification, but there are many examples of them being used for unsupervised learning and non-linear classification exercises.Decision tree learning, or more specifically, Classification and Regression Trees (CART) is one of the more straightforward modalities because it is better appreciated visually, rather than mathematically  represent ends of the tree where an output is finalized. The figure depicted is simplistic, and in a real-world application would only represent a branch of a much larger CART. But decision trees have a habit of becoming unnecessarily deep or involving too many layers of complexity. Trees with excessive internal nodes sub-divide data into too many small clusters, such that the outcomes are grouped in a way that are practically meaningless. A CART is a fundamentally greedy algorithm because it naturally satisfies the condition at each node, rather than optimizing conditions across the length of the tree . On the other hand, an overfitting model attempts to observe the smallest of associations making the model relevant only to the training dataset, and by consequence, poorly generalizable   . In otheclusters that respond differently to treatment. As show in Tee et al. application of decision tree learning for optimizing patient risk stratification after spinal cord injury provides a framework for understanding this modality . They coThe need for substantial patient datasets in spine surgery is particularly noticeable when exploring ML applications for predicting patient-reported outcomes. Exploratory investigations using decision tree learning have been pursued in spine research. Khan et al. utilized seven different supervised learners to predict improvement in SF-36 (PCS/MCS) scores after surgery for degenerative cervical myelopathy . The arc3) permutations of these variables were analyzed using four separate models to determine pullout failure (<650 Newtons of force) or success (≥650 Newtons of force)   . Varghesigure 6) . Their banalysis . As the hyperplane (maximum-margin hyperplane (kernel functions are needed to transform the data into a higher dimension (*y). This is also known as a linear kernel. The byproduct of an SVM for an otherwise linearly inseparable dataset is shown in Support vector machines (SVMs) are also a commonly encountered ML modality in clinical literature. SVMs are intuitive and best appreciated graphically as shown in perplane , 44. Forperplane . But as Delving into the literature, SVMs are popular for classifying and detecting the presence of spine pathology on imaging. For example, a common problem when managing patients with osteoporosis arises from missed fractures on routine DEXA  scans . Given sAnother example of an image classification task achieved through SVMs was conducted by Seoud et al. The investigators attempted to determine scoliosis curve based on a modified Lenke classification system  for adolescents by analyzing surface topography data captured by multiple cameras . As a leregressions involve hyperplanes that minimize the distance between variables because the goal is to predict a continuous variable rather than a discrete one. Hoffman and colleagues also constrained the model to three input variables in order to curtail overfitting, which included preoperative ODI, symptom duration, and handgrip pressure. When compared to a traditional multiple linear regression, they achieved a higher goodness-of-fit or R2 of 0.93 via the SVM , modified Japanese Orthopedic Association scale (mJOA), and handgrip pressure . Their m the SVM . While thigh dimensional data where the number of features far exceeds the number of observations or samples  because tuning kernels allows for clinicians to assign appropriate weights according to their knowledge in that field . SVMs ar samples . Common  samples .Artificial neural networks (ANNs) are of particular interest because they are associated with deep learning, which has been traditionally unsupervised . Manually extracting features is near impossible because no two photos of dogs are the same. Practically, humans recognize dogs in photos despite variations in their pose, environment, lighting or orientation of the photo, among others. However, machines can only interpret pixels in an image, none of which are specific to a dog. Convolutional neural networks (CNNs), with the help of multiple hidden layers, are particularly adept at computer vision tasks and can be visualized graphically in convolves or filters the native input, extracting the “important” information and generates a feature map (a representation of the input). Subsequent max-pooling reduces complexity and minimizes overfitting by creating a more abstract form of the previous feature map and thus more applicable to generic pictures of dogs. This process can be repeated for the desired number of hidden layers. Once all feature maps have been considered, the images are flattened and the desired output (dog or otherwise) can be generated. In many ways, CNNs are more so learning to identify small arrangements or motifs that resemble dogs. This concept is known as local connectivity, meaning two neighboring pixels are considered more relevant than two distant pixels  as experiencing better outcomes relative to the general population that is treated more conservatively. This harkens back to the point previously discussed regarding the importance of understanding exactly which question the model is being asked to answer. Beyond the black box, other ethical and logistical obstacles in machine learning in medicine include distributional shift (training datasets that may be biased toward race or socioeconomic status or simply outdated), insensitivity to impact , and reward hacking (the machine learns unexpected means of achieving an outcome that cheat the system)  in addition to their technical knowledge (easy to explain). And the solution to this problem may involve a combination of (1) accepting the black box of machine learning, and (2) testing them rigorously against multiple patient cohorts (While the challenge of explaining machine learning's method for reasoning persists, it draws some similarities to the way clinical medicine is practiced in the present. Physicians, much like deep learners, often treat patients using some component of their clinical experience or  cohorts . AltogetMC and NP were responsible for reviewing publications for inclusion in the review, drafting of the manuscript, and creating table and figures for the manuscript. JC and KN were responsible for critical revision of the manuscript for important intellectual content related to machine learning methodology and spine surgery research. AV was responsible for the conception of the review, supervision, and critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most amphibians have a complex life cycle with an aquatic larval and an adult (semi-) terrestrial stage. However, studies concerning spatial behaviour and orientation mainly focus on either the aquatic larvae or the adult animals on land. Consequently, behavioural changes that happen during metamorphosis and the consequences for emigration and population distribution are less understood. This paper aims to summarize the knowledge concerning specific topics of early amphibian life history stages and proposes several testable hypotheses within the following fields of research: larval and juvenile orientation, influences of environmental and genetic factors on juvenile emigration, their habitat choice later in life as well as population biology. I argue that studying larval and juvenile amphibian spatial behaviour is an understudied field of research, however, could considerably improve our understanding of amphibian ecology. Amphibians are among the most threatened taxa in the world, about 40% of the species in this group are at risk of extinction . HabitatInherent to such life cycles are breeding migrations, which in many cases require amphibians to navigate surprisingly long distances. Migration distances up to 15 km have been reported . Adult mThere are several obvious reasons why spatial behaviour of adult amphibians is much better studied than that of juveniles: adults are usually larger and therefore it is possible to use tracking devices such as radiotelemetry; their survival rate is higher, this means capture-recapture studies are more suitable; while adult migrations are often tightly timed and happen over defined periods, juvenile movements may appear more stochastic and timing of such are not well understood . NeverthFrom a conservation standpoint, understanding juvenile emigration is at least as important, as the reoccurring migration of adults, many life history decisions related to space use  happen at this stage . In thisDuring the larval stages, most amphibians suffer from high predation risk , which lIn order to describe this general phenomenon Y-axis orientation behaviour is easily trained and elicited and therefore has been used in many sensory studies, that were concerned with cues that amphibians use for compass orientation. In such orientation assay, animals are held for several days in tanks with an inclined bottom, leading to a deep and shallow side of the holding tank e.g., . TypicalAmphibian larvae and juveniles tested in such assays, have been shown to use a variety of orientation cues. The earlier work focused on the well-developed sun compass response of amphibians . FurtherInterestingly, the behavioural responses of larval amphibians are often axial, this means animals are orienting on the expected axis, but appear to be unable to distinguish between the direction of the shallow and deep end . HoweverOnce the larvae completed metamorphosis, the juveniles usually stay close to or in the aquatic habitat. At this stage their orientation behaviour is comparable to adult amphibians at the breeding habitat  . JuvenilAn important question for conservation management is the timing of emigration/dispersal, in order to organize roadkill mitigation efforts, which are most often temporary . DispersIn addition to environmental factors, juveniles also have certain emigration windows each year which might depend on the time of parental breeding. There are striking interspecific differences between such emigration windows. For example, wood frogs and marbled salamanders have tightly synchronized emigration movements – over few days – in contrast, yellow-spotted salamanders and red-spotted newts disperse over the course of many weeks . In addiThe general environmental factors and morphological constraints and therefore the timing of emigration might be similar between sexes, but how is it about the propensity of dispersal? In many amphibians, sex-dependency has been shown, however, there does not seem to be an overall trend towards male-biased or female-biased dispersal and in some species a sex difference is missing completely . Juvenile migratory birds, such as blackcaps, manage to find their winter habitats without learning a direction and exhibit the seasonal appropriate responses  even when they are reared in the laboratory. It has been shown that crossing of blackcaps from two populations with different winter habitats and therefore different dispersal directions, leads to offspring with an intermediate orientation response . These eTo the best of my knowledge, only Such directionality of emigration  could have important consequences for the spatial distribution of a population. For instance, if there were directional tendencies of amphibians emigrating from their breeding habitat, which may translate in a re-occurring migratory route, this would change their distribution in the landscape. Essentially, part of the surrounding area of a breeding habitat might be ‘avoided’ by the animals, due to the initial – potentially inherited – emigration direction, independent of and adding onto other known factors . Therefore, demographic estimates capturing adults at the breeding sites), such as population size, would in reality be unequally distributed around the breeding habitat while modelling approaches would assume equal distribution . This coBombina variegata population analyses  a predator avoidance behaviour, perturbation of such orientation system will increase larval predation.There is an inherited component of juvenile emigration directions, which functions in an interplay with learned behavioural responses and passive factors such as physical barriers, to shape the spatial distribution of adult (pond breeding) amphibians.Such (inherited and learned) directional factors can be incorporated in population models and will increase the explanatory value of such models in terms of predicting spatial distribution.Amphibians optimize the timing of their individual emigration based on a fitness-trade-off between internal factors  and dispersal risk.Cues of the natal habitat influence the general migration behaviour of amphibians, i.e., high quality habitats correlate with lower dispersal propensity and higher site fidelity.There exist a disconnect between our understanding of the larvae and juvenile amphibian spatial behaviour in their natal habitat and its influence on emigration. Furthermore, the processes shaping the emigration direction are poorly understood, despite its importance. I propose five research hypotheses which could provide valuable insights in amphibian population and migration biology: I hope that this short paper can spark some interest of fellow researchers to follow up some of these rather specific but important issues in amphibian ecology. I am confident that this will not only improve our understanding, but also help conservation efforts around the world.


Although great advancements in evidence-based therapies, chronic suicidal patients with borderline personality disorder (BPD) still challenge our mental health system. While BPD patients continue suffering from distress and aversive emotions, therapists and relatives feel often stunned and helpless when confronted with suicidality resulting in interruption of therapies, repeated presentations to emergency rooms and referrals to hospitals. Reviewing the current knowledge of the functions and background of non-suicidal self-injury, we learned that reinforcement mechanisms play an important role to understand why individuals act in deliberate self-mutilation. While individual motives for non-suicidal self-injury and suicidal behavior including suicidal ideations can differ, the principle mechanisms appear to be transferrable. Elucidating the individual motives and function of suicidal behavior is an important therapeutic step, giving us access to very central maladaptive schemes and false believes that we need to address in order to reduce chronic suicidality in BPD patients. This Perspective article aims to give a better idea of what is behind and what are the differences between non-suicidal self-injury, suicidal ideations and suicide attempts. It further integrates recent developments of behavioral science in a reinforcement model of suicidality that can provide therapists a practical armamentarium in their work with chronic suicidal clients. Suicidality is one of the most alerting and urgent symptomatology in mental health. It summarizes a subset of psychopathological phenomena ranging from suicidal ideations  to ambivalent suicide attempt, suicide attempt and suicide , 2. AcroHence, what is behind chronic suicidality? What are the psychological mechanisms that maintain its chronicity? Can we apply concepts that helped us understanding and treating NSSI also for suicidal behavior? Can or should we do therapy in suicidal patients at all? And if yes, what are useful interventions?NSSI  is a world-wide phenomenon that occurs not only in BPD . Thanks These observations correlate nicely with recent neurobiological findings of NSSI, so that we can now retrace why patients repeatedly harm themselves - accepting necessity of surgical intervention, subsequent conflicts with their relatives, and even stigmatizing scars on their skin. In particular, NSSI reduces the activity of the amygdala while functional connectivity to the superior frontal gyrus is normalized in resting-state functional magnetic resonance imaging . ClinicaClinically, we can use this background knowledge in therapy. For example, behavioral analysis like the stimulus-organismic-response-contingencies (SORKC or SORC) model of reinforcement-consequence  can illuBrown, Comtois, and Linehan  were amoNevertheless, suicide attempts are just one symptom of suicidality and patients not conducting SA may still have frequent suicidal ideations or occupy with death and suicide in the internet and exchange suicide methods in social media . Indeed,When we work with behavioral analysis in psychotherapy, we usually focus on (mostly) dysfunctional behavior, i.e., things that we have done, that have some kind of positive or negative consequences. As seen above, these consequences can reinforce me to act similarly the next time: Cutting in a stressful situation, e.g., will immediately reduce aversive tension (negative reinforcement) and may even give me the feeling of control over my emotions (positive reinforcement). But, is being absorbed to suicidal ideations really that different if these suicidal thoughts give me a kind of perspective, relief or just the idea that the current aversive situation will end? Chiles & Strosahl report that adolescents that experience intense emotional pain in response to internal  or external stimuli can feel a kind of relief from the emotional distress when thinking about suicide . SimilarIt is frequently observed (for example in acute psychiatric settings) that BPD patients adopt dysfunctional behavior from other BPD patients  indicating that the behavior must have a quite strong (at least short-term) positive consequence (they find out what helps best or they feel connected to peers that understand their need) , 38. OneWhen we look out for a new approach addressing suicidality in BPD, then working with the background and motives becomes vital in the proper sense. In the language of behavioral analysis, it is the organism variable that determines how we rate and react upon upcoming stimuli , 41. BesUsing behavioral analysis, the therapist may elucidate reinforcing contingencies within the vicious chain of cues, activated faulty believes and subsequent suicidal ideations. He further can look for more adaptive behavioral alternatives or identify possible obstacles that inhibit the application of functional behavior. Typical obstacles can be, e.g., intensive emotions of fear, shame or guilt, or faulty believes and assumptions  . The latSimilar to the reinforcer model of NSSI described above, we can speculate that the confirmation of central schemes together with the reinforcement of suicidal ideations stabilizes the dysfunctional system of chronic suicidality. Conversely, in NSSI on the other hand, the rapid dynamic of the next aversive emotional states directly after NSSI exemplified in the Houben study  acts as Addressing the motives and psychic function behind suicidal behavior depending on specific situations (or triggers) may be a first, but potentially very powerful step in the therapeutic work with suicidal BPD patients. They may feel a substantial validation by going through their individual behavioral analysis and by understanding their own reinforcement mechanisms , normaliWe have seen that similar to NSSI, suicidal ideations and behavior can have various motives and functions. Overall, these functions appear to have in common to significantly reduce mental pain, be it by reducing aversive tension, by giving an idea of a way out of the current situation, or by giving a sense of control of difficult emotions . Thus, it appears plausible that reinforcement mechanism as presented here are substantially involved not only in NSSI, but also in chronic suicidality. Intriguingly, a recent functional MRI study strongly substantiates this concept from a neurobiological perspective using autobiographic transcripts to recall patients' previous suicidal episodes: In this paradigm, mental pain triggering suicidal behavior is associated with decreased prefrontal activity whereas planning and acting out suicidal impulses (in mind) in response to mental pain is associated with increased activity in the medial prefrontal cortex, the anterior cingulate cortex, and the hippocampus suggesting that goal-directed suicidal behavior is associated with a reduction of mental pain .Nevertheless, there is still a big gap in the literature delineating the continuum from passive suicidal thoughts, ideations of dying or being death to suicide attempt preparation and definite suicidal acts, and the psychological function of each of these suicidal behaviors may be very different within and between individual subjects. Indeed, there is strong evidence from neurobiology, that NSSI, SI, and SA have very distinct (and sustained) effects on the regulation of the hypothalamic-pituitary-adrenal (HPA) hormonal axis, a system that is essentially important in adaptation to challenging situations in life. It has been shown, for example, that cortisol response in the combined dexamethasone suppression/corticotropin releasing hormone stimulation test is attenuated in both, past and recent suicide attempters compared to suicide ideators or non-suicidal patients in major depression . In anotFrom the clinical perspective too, a thorough assessment of all degrees of suicidality becomes crucial for the therapist when estimating the individual patient's risk to proceed from ideating to acting in suicide during treatment. In particular, he wants to know, which factors pushes the patient form ideation to suicidal action. Fearlessness about death and pain tolerance occurs in several suicide models as a factor differentiating suicide ideators from attempters , 52, 53,Although we have learned a lot about suicidality during last years and specific programs from different therapeutic schools including behavioral and psychodynamic approaches  helped mThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diarrheal disease remains a major cause of childhood mortality and morbidity causing poor health and economic outcomes. In low-resource settings, young children are exposed to numerous risk factors for enteric pathogen transmission within their dwellings, though the relative importance of different transmission pathways varies by pathogen species. The objective of this analysis was to model associations between five household-level risk factors—water, sanitation, flooring, caregiver education, and crowding—and infection status for endemic enteric pathogens in children in five surveillance studies. Data were combined from 22 sites in which a total of 58,000 stool samples were tested for 16 specific enteropathogens using qPCR. Risk ratios for pathogen- and taxon-specific infection status were modeled using generalized linear models along with hazard ratios for all-cause diarrhea in proportional hazard models, with the five household-level variables as primary exposures adjusting for covariates. Improved drinking water sources conferred a 17% reduction in diarrhea risk; however, the direction of its association with particular pathogens was inconsistent. Improved sanitation was associated with a 9% reduction in diarrhea risk with protective effects across pathogen species and taxa of around 10–20% risk reduction. A 9% reduction in diarrhea risk was observed in subjects with covered floors, which were also associated with decreases in risk for zoonotic enteropathogens. Caregiver education and household crowding showed more modest, inconclusive results. Combining data from diverse sites, this analysis quantified associations between five household-level exposures on risk of specific enteric infections, effects which differed by pathogen species but were broadly consistent with hypothesized transmission mechanisms. Such estimates may be used within expanded water, sanitation, and hygiene (WASH) programs to target interventions to the particular pathogen profiles of individual communities and prioritize resources. Despite considerable progress in recent years, diarrheal disease remains a major cause of mortality and morbidity in childhood ,2—responIn low-resource settings, infants and young children are exposed to numerous risk factors for enteric pathogen transmission within their homes, though the relative importance of different transmission pathways vary depending on the pathogen type and species . While fThe objective of the analysis reported here was to model the associations between five household-level risk factors—water, sanitation, floor material, caregiver education, and household crowding—and infection status for 16 common, high-burden enteric pathogens ascertained in children aged 0–5 years enrolled in five surveillance studies. The a priori hypothesis to be tested was that improved sanitation and water sources, finished floors, caregiver primary school completion, and lack of crowding are protective against enteric infection, but to an extent that differs by pathogen species and taxon.This analysis combined data from five studies. Studies were considered eligible for inclusion if they used polymerase chain reaction (PCR) to diagnose the same panel of 16 enteropathogens, if they had collected data relating to all 5 exposures and if they were willing to share data for the purposes of the analysis. The Etiology Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) project was a multi-site cohort study in which 2100 newborns (227–303 per site) were recruited from communities in eight different Low- and Middle-Income countries and were monitored continuously over their first 2 years of life . The StuAeromonas, Campylobacter, enteroaggregative Escherichia coli (E. coli) (EAEC), enteropathogenic E. coli, ((EPEC) typical and atypical), heat-labile enterotoxigenic E. coli (LT-ETEC), and heat-stable ETEC (ST-ETEC), Salmonella, Shigella/enteroinvasive E. coli (EIEC) (qPCR uses the same gene target for these two closely related pathogens), Cryptosporidium, and Giardia. Several important pathogens  were excluded from consideration due to there having been too few detections to fit the regression models, or due to not having been tested for in every study. To ensure that a single infection episode was not counted multiple times, Campylobacter- and norovirus-positive samples were excluded if they were collected within 30 days of a previous sample that was positive for the same pathogen strain without being separated by an intermediate negative sample [Cryptosporidium parvum or Cryptosporidium hominis) or assemblage  as a prior sample from the same subject were excluded unless separated by three negative samples in recognition of their association with chronic infections.In the MAL-ED, Novel Biomarkers, and SHINE studies, stool samples were collected from the subjects by locally recruited fieldworkers according to a predefined schedule  and upon reporting of a diarrheal episode by the child’s caregiver. In the other studies, locally recruited fieldworkers collected a single stool sample from each case and their matched control subject upon enrollment . Enteropathogen-specific infection status was ascertained using probe-based quantitative PCR (qPCR) assays on custom-developed TaqMan Array Cards   for all e sample . For allDrinking water: whether or not the subject resided in a household with access to an improved drinking water source  .Sanitation: whether or not the subject resided in a household with access to an improved, non-shared sanitation facility  .Flooring material: whether or not the subject resided in a household that had a covered (“improved”—rudimentary or finished) as opposed to natural (“unimproved”—earth or sand) floor .Caregiver education: a binary variable indicating whether or not the subject’s caregiver had completed primary education (≥6 completed years of schooling ).Household crowding: a binary variable indicating whether or not the subject resided in a household with 3 or more residents per bedroom .The primary exposures of interest were the following binary household-level factors, selected on the basis of their hypothesized association with enteric pathogen transmission and the comparability of their definitions across the contributing studies:Site: a categorical variable indicating at which of the 22 study sites the subject was enrolled, included to adjust both for between-site differences in background pathogen transmission levels and for potential confounding engendered by differences in surveillance methods between the 5 studies.Sample type: whether the stool sample was collected during a diarrheal episode  or while the subject was asymptomatic .Age: the subjects’ age in continuous months at the time of stool sample collection, modeled using linear, quadratic, and cubic terms to account for non-linearity of association with enteric pathogen presence.Feeding status: a categorical variable indicating whether the child was being exclusively breastfed, partially breastfed or had been fully weaned (no longer receiving any breastmilk) at the time of sample collection.Nutritional status: two binary variables indicating whether or not the child was moderately or severely stunted or underweight  to adjust for both the impact of nutritional status on susceptibility to infections  and poteDefinitions of these variables used by each study were mapped as closely as possible to those used in previous publications from the World Health Organization, the Demographic and Health Surveys and others, as shown in Missing covariate data were imputed using predictions from either multivariate normal regression, linear mixed effects models or Cox proportional hazards models, methods that are described in detail in the For all pathogen-specific associations, modified Poisson regression models were fitted to the binary outcome in the full database using generalized linear models with cluster-robust variance estimation to calculate adjusted risk ratios (RRs) for infection for each of the five risk factors in the presence of each other and the potential confounders . This anSalmonella spp. to 50.8%—EAEC.Aeromonas spp. by a moderately statistically significant 21% ) and EAEC by a slightly statistically significant 8% ).Aeromonas spp. These estimates were statistically significant at the α ≤ 0.05 level for two of the five virus species (astrovirus and norovirus), three of the bacterial infections  and for Cryptosporidium. Regarding the taxon-specific effects, improved sanitation afforded a moderately significant 12% reduction in risk of infection with any enteric virus ) and a slightly significant 9% for either protozoa ), and a highly statistically significant 19% reduction in the equivalent risk for any enteric bacteria ).Household access to an improved sanitation facility was associated with a slightly statistically significant 9% reduction in risk of diarrhea ). Protective effects of improved sanitation were observed and were similar in magnitude for all included species of enteric pathogens with the one exception of Campylobacter spp. ). One of the largest effects of this or any other variable was seen for infections with Giardia spp., a moderately statistically significant 16% reduction ).Living in a dwelling with a covered floor had the same association with the risk of diarrhea as sanitation—a moderately statistically significant 9% decrease ), but showed no significant associations with any enteric viral infections. Significant protective effects of improved flooring against numerous enteric bacteria species were observed ranging from a slightly statistically significant reduction in EAEC risk of 8% ) to a highly statistically significant 13% decreased risk of infection with Shigella spp./EIEC ), and any enteric bacteria ), moderately significant for Giardia spp. ), and any enteric protozoa ), and highly so for Campylobacter spp. ).Having a primary caregiver who had completed primary education was associated with a slightly statistically significant increase in the risk of all-cause diarrhea of 7% ). Most other pathogen- and taxon-specific models showed a small protective effect of this exposure, which was slightly statistically significant for astrovirus ), any enteric virus ), typical EPEC ), Campylobacter spp. ).Living in a household with fewer than three residents per bedroom was associated with a slightly statistically significant 7% lower risk of all-cause diarrhea ), 5% for any enteric virus ), and 7% for any During early childhood a large proportion of time is spent within the family dwelling, and therefore characteristics of the household and caregivers contribute substantially to a child’s health status, especially with regard to diarrheal disease and its sequelae. Features of the household environment may serve to promote or interrupt the transmission of diarrhea-causing pathogens via the fecal–oral and other routes. Researching these effects can be challenging, however, because studies of diarrheal disease outcomes, particularly those that make comparisons across multiple populations, may be biased by differences in caregiver reporting and understanding of diarrhea symptomatology. Detections of specific enteric pathogens from stool samples can serve as more objective, generalizable morbidity metrics for evaluating the impact of specific factors on risk of contracting disease and are increasingly being reported by trials and observational studies . This waTo address these limitations this analysis combined results from several, rigorous population-based studies that each used highly sensitive, broad spectrum molecular diagnostics, and standardized exposure definitions. The resulting dataset—some 58,000 stool samples from 15,000 subjects in 22 locations—is of a size and diversity of epidemiologic contexts that is unparalleled in the study of enteric pathogens. While many of the findings appear to confirm prevailing assumptions about the transmission pathways of the analyzed pathogens, others are suggestive of hitherto novel hypotheses.Aeromonas spp. infection risk was particularly reduced by improved water source, a biologically plausible finding for a pathogen that lives freely in sea and surface waters, the clinical importance of that finding is unclear. Although evidence has been published of Aeromonas-associated morbidity in specific contexts [Household use of an improved water source had the largest protective effect against all-cause diarrhea of any of the five exposures. However, the direction of the association of this exposure with particular pathogens was inconsistent and, in almost all cases, non-significant. It has been suggested that sanitation interventions may be more likely to interrupt transmission of bacteria and parasites than viruses which have more environmentally mediated transmission routes . Howevercontexts  , it is contexts .Campylobacter and Giardia, given that many of the study sites contributing data to this analysis are in settings characterized by high levels of household-scale livestock husbandry, as well as children sharing the peridomestic space and even their sleeping quarters (the intradomiciliary space) with animals [Campylobacter spp. can be linked to a hypothesized decrease in exposure to avian feces once improved flooring is introduced [Campylobacter in non-soil surfaces that are easily cleaned and dried [Shigella and E. coli pathotypes) suggesting that improved flooring diminished transmission from human as well as animal sources.Having covered floors in the dwelling had the same effect on all-cause diarrhea as improved sanitation and to a higher level of statistical significance. Pathogen-specific estimates however show a distinct pattern of viruses being unaffected by this exposure, while consistent protective effects against bacterial and protozoal infections are evident. It is notable that the decrease in infection risk was most marked for the zoonotic enteropathogens  animals . We hypo animals  while altroduced , as wellnd dried ,47,48. ICampylobacter and Giardia. This too may be a marker of proximity to livestock, with children living in crowded households more likely to be forced to share their living or sleeping quarters with animals [Lack of crowding also showed a protective effect against the two zoonotic pathogens  animals ,49,50,51Another limitation inherent to the analysis of observational data, is that the role of residual confounding, in this case by socio-economic status, which can be expected to be associated with both the exposures and the outcomes, cannot be ruled out. By adjusting each of the household exposures for each other and for the two anthropometric indicators—childhood nutritional status being highly correlated with household socio-economic indicators —we hopedWhile systematic reviews of household-level interventions report pooled protective effects of 25–75% for diarrheal outcomes ,54, receCampylobacter, EAEC, Giardia, and Atypical EPEC, as has been reported elsewhere. Improvements in sanitation may reduce diarrheal disease by suppressing transmission of enteropathogens of all three taxa, while improvements in flooring may do so specifically through decreases in enteric bacteria and protozoa infection without interrupting virus transmission, with the reverse being the case for reducing household crowding. While drinking water source improvements also appear to reduce diarrhea risk, it is not clear from these findings which pathogen taxa mediate this association. By combining data collected at multiple epidemiologically and socioeconomically diverse sites, this analysis was able to quantify the associations between each of five household-level exposures on the risk of specific enteric infections, effects which differed by pathogen species and taxa but were broadly consistent with hypothesized transmission mechanisms. With further research, such estimates may be used within expanded WASH programs to target interventions to the particular pathogen profiles of individual communities and prioritize resources accordingly.In conclusion, a diverse array of enteropathogens are endemic and highly prevalent across distinct geographical and epidemiological settings, notably


Zika virus (ZIKV) emerged in Brazil during 2013–2014 causing an epidemic of previously unknown congenital abnormalities. The frequency of severe congenital abnormalities after maternal ZIKV infection revealed an unexplained geographic variability, especially between the Northeast and the rest of Brazil. Several reasons for this variability have been discussed. Prior immunity against Dengue virus (DENV) affecting ZIKV seems to be the most likely explanation. Here we summarise the current evidence regarding this prominent co-factor to potentially explain the geographic variability.This systematic review followed the PRISMA guidelines. The search was conducted up to May 15th, 2020, focussing on immunological interactions from Zika virus with previous Dengue virus infections as potential teratogenic effect for the foetus.Eight out of 339 screened studies reported on the association between ZIKV, prior DENV infection and microcephaly, mostly focusing on antibody-dependent enhancement (ADE) as potential pathomechanism. Prior DENV infection was associated with enhancement for ZIKV infection and increased neurovirulence in one included in vitro study only. Interestingly, the seven in vivo studies exhibited a heterogeneous picture with three studies showing a protective effect of prior DENV infections and others no effect at all. According to several studies, socio-economic factors are associated with increased risk for microcephaly.Very few studies addressed the question of unexplained variability of infection-related microcephaly. Many studies focussed on ADE as mechanism without measuring microcephaly as endpoint. Interestingly, three of the included studies reported a protective effect of prior DENV infection against microcephaly. This systematic review strengthens the hypothesis that immune priming after recent DENV infection is the crucial factor for determining protection or enhancement activity. It is of high importance that the currently ongoing prospective studies include a harmonised assessment of the potential candidate co-factors. Despite the large volume of Zika virus (ZIKV)-related research, the difference in the frequency of ZIKV-related severe congenital abnormalities between Northeastern Brazil in the rest of Brazil and other locations has not been adequately explained. This systematic review summarizes the existing evidence for the role of prior Dengue virus (DENV) infections as co-factor to explain the variability in the frequency of ZIKV-related severe congenital abnormalities. Results differed significantly according to study type (e.g. in vitro versus in vivo studies), and no clear picture emerged with regard to prior DENV infections causing ZIKV-related severe congenital abnormalities. However, evidence from additional studies not included in this systematic review that focus on immunological interactions between DENV and ZIKV infections imply the potential that severe ZIKV disease is more frequent in those with prior DENV infections. Therefore, an assessment of background immunity against closely related viruses (e.g. the flavivirus family) needs to be included in future and ongoing longitudinal studies on ZIKV-related congenital abnormalities. Zika virus (ZIKV) swept through most of Latin America and the Caribbean in 2015/16, but severe complications were mainly reported in the Northeast of Brazil and the urban centers bordering the Atlantic Coast of Brazil [When officially reported data across Brazil were assessed, an unexplained variability of the frequency of microcephaly over geography , and oveMajor cohort studies estimate the risk of congenital abnormalities in ZIKV infection of 4–10% . HoweverOfficially reported data in Brazil showed considerable heterogeneity between the first wave of ZIKV infections in 2015 with higher numbers of congenital complications  compared to the second wave in 2016 ,11. The Some researchers have argued that this variability may at least partly result from inconsistent terminology, difficulties in the accurate measurement of microcephaly and unreliability of secondary data sources ,10,18.Even though DENV is endemic all over Brazil, the transmission intensity, the timing of local outbreaks, and the mix of serotypes differs geographically. Antibody dependant enhancement (ADE) has been suggested to explain a higher risk of severe Dengue in secondary infections caused by a heterologous serotype compared to primary infections . BecauseWe conducted this systematic literature review following a pre-defined research protocol registered in the Open Science Framework database and in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . We aimeth of May 2020. A broad search was carried out with the key terms “Zika” AND “Dengue” AND either “microcephaly” or “congenital abnormalities”. We used the COVIDENCE web-based software platform as a literature management tool. Titles and abstracts of the identified references were screened by two authors against the inclusion/exclusion criteria. Any discrepancies between the screening determinations were resolved between the reviewers by consensus.We searched Cochrane, Google scholar, LILACS and PubMed online databases until the 15Study quality assessment  was applied, using a modified tool of the Joanna Briggs approach . We usedWe identified 339 non-duplicate articles . When apOf the eight included studies that focused on prior DENV infections, seven were in vivo and one in vitro study. Interestingly, within the in vivo studies, three papers described a protective effect of prior DENV infections against symptomatic ZIVK infection. The remaining in vivo papers did not detect an association between prior DENV antibody status and ZIKV infection, severity or congenital abnormalities. The one in vitro study described an enhancement effect of prior DENV infections and ZIKV infection with microcephaly.Three case-control studies carried out in Salvador de Bahia, Brazil, were included. In one of the case-control studies, Moreira-Soto et al. found that a prior DENV infection was not associated with higher ZIKV-specific plaque reduction neutralization test (PRNT) titers in cases–on the contrary, the presence of anti-ZIKV antibodies was associated with an absence of microcephaly . The autHalai et al.  studied Castanha et al.  exploredCampos et al.  analysedA second ecological study was included. Carvalho et al.  studied Contrary to the findings of the in vivo studies—which detected no or rather a protective effect, the in vitro study detected and described enhancement activity between ZIKV and DENV. Rathore et al.  used a mThis systematic review yielded eight studies that addressed Dengue as potential co-factor for ZIKV infection related congenital abnormalities. Most studies focused on either antibody-dependent enhancement (ADE) as potential pathomechanism, or on cross-protective mechanisms induced by prior DENV infections. The findings differ significantly between in vivo and in vitro data. Enhancement after prior DENV infection was only observed in in vitro studies. Interestingly, in vivo studies showed a mixed picture with some in vivo studies reporting a protective effect of prior DENV infections while others report no effect at all.γ-receptor mediated activity which is essential for the transplacental transfer of IgG.In general, the large body of literature about in vitro ADE does not include microcephaly or congenital abnormalities as outcomes, but operates under the assumption that enhancement is associated with disease severity. For this reason, this literature is not formally included in this review—however it does constitute a potential link between epidemiological and in vitro data—firstly, because of its role as assumed underlying pathomechanism for more severe course of disease via higher viral load; secondly, because of the Fc Examples of in vitro studies not included in this systematic review because of our inclusion and exclusion criteria include Londono-Renteria et al. , DejinirThe enhancement between subsequent infections has long been described between heterogenous DENV serotypes. Katzelnick et al.  studied Castanha et al. also described differences in enhancement mechanisms depending on the interval between either primary or secondary DENV infection and the subsequent ZIKV infection. Enhancement was not observed during the acute phase of the ZIKV infection after primary DENV infection, but at later time points. In addition, the enhancement effect for subsequent ZIKV infection was observed to be differential according to prior DENV serotype . In lineAn underlying assumption in the studies about ADE between DENV and ZIKV is that an increase in ZIKV loads is associated with severity of disease, and subsequently with the risk of severe abnormalities . AlthougInterestingly, the two ecological studies, selected through this review, agreed that prior DENV infection should not have been the only cause for the variability in microcephaly incidence. However, ecological studies are not the best study design for epidemiological analysis of causal associations. This highlights the need for prospective studies to acquire an in-depth understanding of the role of previous DENV exposure on ZIKV infection.Some researchers refuted the need for co-factors altogether to explain the variability of the burden of microcephaly. They suggested for example that the different frequency of severe complications between regions of Brazil can be explained by a higher transmission intensity translating to an increased case load of ZIKV infected cases in the Northeast of Brazil . If trueAedes aegypti populations and thus higher transmission rates in poor neighbourhoods. Studies reported a high frequency of ZIKV positivity among DENV-immune mothers which might reflect the high risk of this population to the exposure to Aedes aegypti and arthropod-borne virus infection [Although not formally included as it is likely a composite of more than one risk factor, socio-economic status (SES) or poverty was implicated with severe abnormalities in several studies. The Northeast of Brazil is one of the poorest regions of Brazil and had the highest microcephaly rates. Poverty increases the risk of malnutrition, which goes with poor general health. Possible mechanisms for SES to mediate severe abnormalities include higher prior exposure rates to the other co-factors  or reduced access to abortion services. The accumulation of garbage and rainwater storage could have led to an increase of nfection ,45.This systematic review focused on prior DENV infection as a co-factor to explain the variability of severe abnormalities in newborns after maternal ZIKV infection during pregnancy.Results differed significantly according to study type (e.g. in vitro versus in vivo studies). Prior DENV infection was associated with enhancement for ZIKV infection and increased neurovirulence in one included in vitro study only—however, the seven in vivo studies exhibited a heterogenous picture with three in vivo studies showing a protective effect of prior DENV infections and others no effect at all. Evidence from additional studies that focus on immunological interactions between DENV and ZIKV infections imply the potential of enhancement being associated with more severe disease, but did not link enhancement to congenital abnormalities in ZIKV infections. If present, this association is time-dependent with a window of cross-protection and subsequently increased risk of enhancement, depending on the time interval and DENV antibody levels. This systematic review also highlights, that the variability of microcephaly and other neurological abnormalities after ZIKV infection is not well understood and needs to be further addressed. In the absence of other explanations for the variability of severe abnormalities across geography and over time, immunological interactions between DENV and ZIKV are still worth to be investigated in future studies . An asseS1 PRISMA Checklist(DOC)Click here for additional data file.S1 Quality Assessment Table(DOCX)Click here for additional data file.


Aging is the single largest risk factor for many common diseases that burden public health. This is especially true in the prostate; as men age, the prostate undergoes a prototypical aging change, fibrosis. The aged fibrotic prostate causes urinary symptoms which will afflict nearly every man if they live long enough. However, the molecular mechanisms responsible for the aging-dependent promotion of fibrosis are largely unknown and understudied. In this study, we sought to reveal the contribution of mitochondrial dysfunction to fibrosis in the aging prostate, ultimately leading to prostate dysfunction, urinary symptoms, and overall poor health. The demise of mitochondrial function is well established in other aging-associated diseases but has not been investigated in the prostate. Our analysis revealed an increase in cellular senescence and mitochondrial dysfunction in BPH patient tissue compared to normal prostates. Furthermore, selective inhibition of mitochondrial complex I by rotenone in cultured human prostate fibroblasts led to myofibroblast phenoconversion as characterized by increased expression of Col1a1, Col3a1, and ACTA2. To determine the contribution of mitochondrial dysfunction on fibrosis and lower urinary tract dysfunction (LUTD), we examined the prostate lobes in both aging and steroid-induced LUTD in mice. These models have been extensively characterized and exhibit an age-mediated increase in LUTD and fibrosis. We observed a decrease in mitochondrial function and an increase in cellular senescence corresponding to an increase in fibrosis and LUTD. This suggests that normal aging-dependent reductions in mitochondrial complex I function in the prostate may promote fibrosis and contribute to urinary dysfunction.


Telephone-based digital triage is widely used by services that provide urgent care. This involves a call handler or clinician using a digital triage tool to generate algorithm-based care advice, based on a patient’s symptoms. Advice typically takes the form of signposting within defined levels of urgency to specific services or self-care advice. Despite wide adoption, there is limited evaluation of its impact on service user experience, service use and clinical outcomes; no previous systematic reviews have focussed on services that utilise digital triage, and its impact on these outcome areas within urgent care. This review aims to address this need, particularly now that telephone-based digital triage is well established in healthcare delivery.Studies assessing the impact of telephone-based digital triage on service user experience, health care service use and clinical outcomes will be identified through searches conducted in Medline, Embase, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Web of Science and Scopus. Search terms using words relating to digital triage and urgent care settings  will be used. The review will include all original study types including qualitative, quantitative and mixed methods studies; studies published in the last 20 years and studies published in English. Quality assessment of studies will be conducted using the Mixed Methods Appraisal Tool (MMAT); a narrative synthesis approach will be used to analyse and summarise findings.This is the first systematic review to evaluate service user experience, service use and clinical outcomes related to the use of telephone-based digital triage in urgent care settings. It will evaluate evidence from studies of wide-ranging designs. The narrative synthesis approach will enable the integration of findings to provide new insights on service delivery. Models of urgent care continue to evolve rapidly, with the emergence of self-triage tools and national help lines. Findings from this review will be presented in a practical format that can feed into the design of digital triage tools, future service design and healthcare policy.CRD42020178500).This systematic review is registered on the international database of prospectively registered systematic reviews in health and social care (PROSPERO 2020 The online version contains supplementary material available at 10.1186/s13643-021-01576-x. Telephone-based digital triage has been widely used by services that provide urgent care over the last several decades , 2. UrgeDigital triage within these services involves a care service staff member using a digital triage tool to generate algorithm-based care advice, based on a patient’s symptoms. Advice typically takes the form of signposting within defined levels of urgency to specific services, such as an emergency department (ED), out-of-hours centre, general practice (GP) appointment or self-care advice.In part, these services have been implemented in response to increasing demand on primary care and hospital-based EDs over the last several decades . They ofSystematic reviews in this research area were conducted several years ago (between 2005 and 2012) , 14–16. Despite wide adoption of digital triage within urgent care, previous reviews have not focussed on the digital triage element of services. In older literature, digital triage is often referred to as the use of computerised ‘clinical decision support systems’ (CDSS) in the context of telephone triage or consultation, as they were previously known . InsteadThese previous reviews show mixed results in terms of service user experience, clinical and service use outcomes, which likely result from varying contextual factors, including whether services use digital triage, the type of service, care setting, levels of clinical supervision, types of staff conducting triage and level of staff training. Compared to previous reviews, this review addresses a more narrow review question, which is focussed on services that utilise digital triage in the provision of out-of-hours urgent care. We are excluding ‘in hours’ care as to date digital triage has not been widely adopted in these settings during usual business opening hours.This review additionally addresses the need to evaluate more recent studies that have analysed large routine triage and patient outcomes datasets that have become more readily available in recent years . PreviouHow does telephone-based digital triage affect service user experience, clinical outcomes and health care service use in patients using out-of-hours urgent care services?To describe characteristics of patients accessing these services and the triage advice they receiveTo explore service user (patient or carer) experience of triageTo evaluate patient health care service use following triage, including hospital admissions, ED attendance and GP attendance.To evaluate patient clinical outcomes, including hospitalisations and mortalityThis review will explore the objectives below in out-of-hours urgent care services that utilise telephone-based digital triage:A completed Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) checklist  showing Eligibility criteria have been developed using the population, interventions, comparators, outcomes and study designs (PICOS) principle  and willThe review will include studies that evaluate the use of triage in the general population or within particular sub-groups of the general population (for example children or older people).Studies that assess the use of telephone-based digital triage in out-of-hours services that provide urgent care; these may include national or regional call centre-based urgent care telephone services, out-of-hours and urgent care centres and ambulance services. Services that only operate during ‘in-hours’  will not be includedStudies assessing the use of digital triage by the general population for any symptoms (not condition specific)Studies assessing the use of digital triage that results in signposting  and/or providing self-care adviceThe following eligibility criteria relating to the digital triage intervention will be applied to include:Characteristics of patients and triage adviceService user (patient or carer) experiencesHealth care service use following triage: including hospital admissions, ED attendance and GP attendancePatient clinical outcomes, including hospitalisations  and mortalityStudies that assess outcomes relating to at least one of the following outcomes will be included:All study types will be included: qualitative , quantitative  and mixed methods studies.Additionally, only studies published in the English Language in the last 20 years will be included (studies conducted from 2000 to 2020): this time period has been chosen to identify changes in outcomes over time in relation to changing models of service delivery, for example changes in workforce mix . Full inResearch databases will be searched using a search strategy and key words that have been developed with input from a librarian.Search terms will include variations of terms relating to ‘urgent care’, ‘triage’ and ‘digital’. Full search terms can be found in The search will be restricted to include studies published between the years 2000 and 2020, studies published in English, and studies electronically published (Epub) ahead of print.References identified in the searches will be managed in Covidence systematic review management software; identified references will be imported into Covidence and de-duplicated.In the first screening stage, titles and abstracts will be screened against the inclusion and exclusion criteria by two reviewers independently. References that meet the inclusion criteria will be screened again for inclusion at full-text level, by two reviewers independently. For any full-text articles that are excluded, exclusion reasons will be documented using Covidence.Any discrepancies on studies to be included at both screening stages will be resolved through discussion between the two reviewers. If a consensus is not reached, a third reviewer will be consulted. At the end of the two screening stages, a final set of studies to be included will be identified. The study selection process will be described through a Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart .Reviewers will independently extract relevant data from the included studies which will be recorded on a custom pre-defined data extraction form. The following information will be extracted and entered into the data extraction form: author, publication year, country, study design, care setting, participants, intervention details, type of care service staff conducting triage (doctor/nurse/paramedic/non-clinician), comparator, outcomes, effect of intervention and contextual factors . Data extraction discrepancies will be resolved through discussion between the reviewers, and a third reviewer will be consulted if necessary. Study authors may be contacted during the screening or data extraction where eligibility is unclear.References of included studies will be screened by hand to identify any other eligible studies. Different reports that relate to the same study will be identified and labelled to indicate that they refer to the same study.Quality assessment will be conducted for all full-text peer-reviewed publications that fit the inclusion criteria, using the Mixed Methods Appraisal Tool version 2018 (MMAT) , which iIf the reviewers disagree in their assessment of bias in a study, this will be resolved though discussion. Quality assessment will not be used to exclude studies from the review but will be taken into account in the synthesised findings.Different types of biases which may be present in each study will be considered and presented in a risk of bias table. If missing data or selective reporting of outcomes is apparent in a study, the study author will be contacted to obtain information on the reasons behind the missing data and to assess the risk of any systematic differences in missing data. Studies of equal quality as determined through assessment with the MMAT and risk of bias assessment will be considered to have similar weighting, and this will feed into the data synthesis to ensure trustworthiness of synthesis, serving to minimise bias.Additionally, for quantitative studies, the occurrence of reporting (non-publication) bias will be evaluated by conducting checks of study registers  to identify the completeness of the published literature included in the review; these findings will feed into the overall evaluation of the available evidence.A narrative synthesis approach will be used, which is a ‘synthesis of findings from multiple studies that relies primarily on the use of words and text to summarise and explain the findings’ . This stNarrative synthesis will be conducted to analyse, integrate and summarise the evidence identified through data extraction and the findings from quality assessment. An iterative approach will be followed, based on four main elements: (1) theory development, (2) preliminary synthesis, (3) exploring relationships between evidence from studies and (4) assessing robustness of the synthesis conducted . Key subThis review seeks to evaluate the impact of telephone-based digital triage by urgent care services on service user experience, and patients’ clinical and service use outcomes. This is the first systematic review to evaluate these outcomes in relation to digital triage in the urgent care setting. In addition, this review includes mixed studies, enabling the integration of evidence from studies of wide-ranging design. It will be possible to investigate how findings have changed over time, by comparing results of studies carried out early in the implementation of these services as well more recent studies conducted in well-established services, and how other contextual factors influence findings. Urgent care delivery continues to develop rapidly; findings from this review will have potential to inform policy and practice related to the design and delivery of urgent care service delivery and should also highlight gaps in the evidence that require further investigation.This review is registered on the international database of prospectively registered systematic reviews in health and social care (PROSPERO 2020 CRD42020178500). Amendments to the protocol will be amended on the registration.Additional file 1. PRISMA-P checklist.


Cardiac fibrosis begins as an intrinsic response to injury or ageing that functions to preserve the tissue from further damage. Fibrosis results from activated cardiac myofibroblasts, which secrete extracellular matrix (ECM) proteins in an effort to replace damaged tissue; however, excessive ECM deposition leads to pathological fibrotic remodeling. At this extent, fibrosis gravely disturbs myocardial compliance, and ultimately leads to adverse outcomes like heart failure with heightened mortality. As such, understanding the complexity behind fibrotic remodeling has been a focal point of cardiac research in recent years. Resident cardiac fibroblasts and activated myofibroblasts have been proven integral to the fibrotic response; however, several findings point to additional cell types that may contribute to the development of pathological fibrosis. For one, leukocytes expand in number after injury and exhibit high plasticity, thus their distinct role(s) in cardiac fibrosis is an ongoing and controversial field of study. This review summarizes current findings, focusing on both direct and indirect leukocyte-mediated mechanisms of fibrosis, which may provide novel targeted strategies against fibrotic remodeling. The healthy heart is supported by a distinct network of extracellular matrix (ECM) proteins, which function in part to preserve chamber structure and aid in cardiac cell communication . Fibroblde novo expression of α smooth muscle actin (αSMA) can contract to support wound healing  are recruited to promote transition to injury repair through secretion of pro-repair factors , peaking around 7 days post-injury  mice has suggested that macrophages can indeed transition toward fibroblast-like cells in the heart following MI with respect to their expression profile of fibroblast markers over time  showed negligible contribution of monocytes and macrophages to myofibroblasts post-MI, which suggest that myofibroblasts derive largely from proliferating and transdifferentiating resident cardiac fibroblasts  are most recognized for their function in regulating allergic responses; however, years of thorough investigation has revealed their much more considerable and widespread involvement in the immune system . These sTo date, a plethora of compelling results suggest roles for MCs in cardiac fibrosis . For oneEosinophils are a subset of circulating leukocytes that contribute to the clearance of injury primarily through their hydrolase rich granules . Recent Dendritic cells (DC) are key immune regulators that function to initiate adaptive responses through antigen processing and presentation . They ca+ cDCs to the heart following ischemic injury that increase starting 1 day post-MI that peak around 5–7 days post-MI  in cardiac fibrosis. These adaptive immune cells are found in small numbers in the steady state heart, but are known to rise post-injury . The fun+ T cells accumulate in the heart post-MI and -TAC, and are associated with enhanced cardiac fibrosis  subsets in fibrosis, and have mainly shown their role in protecting against fibrosis . Early sike TNFα . Additioin vivo will require consideration of numerous other factors which influence cardiac fibrosis. For one, several leukocyte subsets are implicated in the development of fibrosis. Currently, their explicit contribution to cardiac fibrosis, which appears context-dependent, is still being unraveled, as is the practicality of targeting specific subtypes at particular phases of post-injury remodeling of HF development. For instance, the presence of dendritic cells may be beneficial toward stable scar formation in MI patients, however, their exact role in the regulation of fibrotic remodeling and how to support their activity will need to be understood to effectively translate this knowledge toward therapeutic application (+ T cells; given the number of animal studies showcasing their association with fibrosis, antagonizing these lymphocytes could serve as a feasible therapeutic avenue against pathological fibrosis during HF (Fibrotic remodeling is a normal physiological response to injury in the heart, however, when it becomes excessive, fibrosis leaves the heart at a great detriment in that it leads to tissue stiffness and dysfunction . As suchlication . Furtheruring HF . To concAO and DT wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.