In low-income and middle-income countries, most patients with febrile illnesses present to peripheral levels of the health system where diagnostic capacity is very limited. In these contexts, accurate risk stratification can be particularly impactful, helping to guide allocation of scarce resources to ensure timely and tailored care. However, reporting of prognostic research is often imprecise and few prognostic tests or algorithms are translated into clinical practice.Here, we review the often-conflated concepts of prognosis and diagnosis, with a focus on patients with febrile illnesses. Drawing on a recent global stakeholder consultation, we apply these concepts to propose three use-cases for prognostic tools in the management of febrile illnesses in resource-limited settings: (1) guiding referrals from the community to higher-level care; (2) informing resource allocation for patients admitted to hospital and (3) identifying patients who may benefit from closer follow-up post-hospital discharge. We explore the practical implications for new technologies and reflect on the challenges and knowledge gaps that must be addressed before this approach could be incorporated into routine care settings.Our intention is that these use-cases, alongside other recent initiatives, will help to promote a harmonised yet contextualised approach for prognostic research in febrile illness. We argue that this is especially important given the heterogeneous settings in which care is often provided for patients with febrile illnesses living in low-income and middle-income countries. Prognostic tools can improve the efficiency and utility of management algorithms for patients with febrile illnesses; a recent multinational stakeholder consultation identified that they could be particularly impactful in settings where diagnostic capacity is most limited.Clearly defined use-cases can help to focus efforts of researchers, product developers and policy makers to ensure that the proposed solutions are appropriate and relevant for the targeted contexts.Novel prognostic tools should improve recognition of impending serious illness in patients who lack clinical signs of severity as determined by existing algorithms, be subject to robust cost–benefit assessments, and be developed in partnership with end-users to ensure they function within the limited human and material resources available at the peripheral levels of most low-income and middle-income country health systemsGuidance to standardise measurement of candidate predictors and harmonise outcome assessments has recently been developed and should be used to contextualise results, facilitate data sharing and maximise comparability of findings from disparate studiesGlobally, febrile illnesses are among the most common reasons to seek healthcare.3Understanding the underlying causes of the main febrile syndromes is critical to successful treatment of febrile illnesses. Several recent initiatives have addressed this topic.is my patient’s condition likely to progress and require a higher level of care? A prognostic tool that could reliably risk stratify patients would have immense potential for benefit, through timely identification of patients at risk of deterioration and guiding appropriate use of scarce resources.Most febrile patients in LMICs are managed by community health workers and healthcare providers working at primary or district level. These practitioners often have limited training and inadequate access to the necessary supervision and diagnostic testing to support their clinical decision-making. In such contexts, in addition to assessing the cause of a patient’s illness, an equally pertinent question is: diagnostic test which determines whether a specific disease or health state is present at the moment the test is performed, a prognostic test provides information on the likelihood of a particular outcome occurring in the future.In contrast to a Common pathophysiological pathways to severe febrile illness exist across a range of microbial aetiologies.Unlike diagnosis, prognosis is inherently context-dependent: a patient’s eventual outcome is inextricably influenced by the available resources and quality of care. Hence, in order to advance the conversation around prognostic testing in febrile illnesses, specific use-cases must be defined. Each use-case should detail the clinical problem and consider the resources available to treat febrile illnesses in that setting , in order to contextualise the outcomes against which a candidate prognostic test or algorithm is to be assessed.In this paper, we first review the concepts of prognosis and diagnosis, with a focus on assessment of the severity of febrile illness. We then apply these concepts to define three potential use-cases for prognostic tools in the management of febrile illnesses in resource-limited settings: (1) guiding referrals from the community to higher-level care; (2) informing resource allocation for patients admitted to hospital and (3) identifying patients who may benefit from closer follow-up post-hospital discharge. For each use-case, we explore practical implications for new technologies, with an emphasis on the requirements for putative tests to measure biochemical biomarkers within various healthcare settings in LMICs. We conclude by reflecting on the challenges and knowledge gaps that must be addressed before prognostic tools could be incorporated into routine care settings, drawing on the findings from multiple recent stakeholder consultations.14Healthcare providers regularly integrate multiple sources of data  to determine the ‘true’ underlying disease or health state of their patient. Depending on the temporal relationship between these baseline data (predictors) and the disease or health state in question (the outcome), these predictions are either diagnostic or prognostic.8diagnostic process, whereas predicting the probability that an individual may develop active tuberculosis (TB) within the next 2 years based on their demographics, medical history and latent TB infection test result (PERISKOPE-TB)prognosis rognosis .For predictors of severity these concepts can become blurred. A patient’s severity reflects their likelihood of a poor outcome and hence predictors of severity are inherently prognostic. However, certain predictors also indicate a patient’s ‘level of severity’ at the time of measurement  and in this sense, as well as providing prognostic information, can also be considered diagnostic of a patient’s severity at that moment. This is in contrast to other predictors that are primarily harbingers of future deterioration in patients who appear otherwise well .7 17 18Most predictors used for the assessment of severity fall into the first group, serving both diagnostic and prognostic purposes. For example, many guidelines and tools devised to inform the management of febrile illnesses in resource-limited settings use ‘Danger Signs’ to identify patients who are severely ill at the time of assessment and at high risk of mortality.14It is often poorly reported whether (and what proportion of) patients had met the predefined severity endpoint  at the time the baseline predictors were measured .Most patients with febrile illnesses present to peripheral levels of the health system.28Even with optimal deployment of existing algorithms, cases of serious illness can be missed and patients are inappropriately referred.31In settings where referral is not immediately feasible, accurate prognosis could guide provision of prereferral care, such as the first dose of parenteral antibiotics for suspected serious bacterial infections.Rather than being mutually exclusive, prognostic and diagnostic tests should be considered complementary: an algorithm integrating prognostic and diagnostic components can be envisioned as being highly useful in this context. First the algorithm could identify patients likely to benefit from referral for higher-level care, as described above, and second it could guide the further management of individuals who were identified as suitable for care at the community level .In many LMICs febrile illnesses remain the leading cause of hospitalisation.34Being able to predict the likely course of a patient’s illness given the resources available at a typical district-level hospital could enable better resource prioritisation—from simple measures such as facilitating early discharge or increased frequency of vital observations, to admission to restricted-capacity high dependency areas or informing timely transfer for tertiary-level care.At regional-level and tertiary-level hospitals, accurate risk stratification might help direct resources towards patients more likely to benefit, for example, early institution of high-cost therapies and adjunctive procedures. This may reduce the likelihood of prolonged admission and subsequent long-term morbidity, and the financial burden of this on patients and their families.Survivors of severe infections are at increased risk of morbidity and mortality but this risk is modifiable with post-discharge care.37Risk stratification of patients using data collected in the lead up to discharge would enable limited resources to be focused on more comprehensive follow-up of individuals at highest risk of post-discharge complications. Appropriate risk thresholds could be determined based on resources available for such a programme. Prognostic factors and algorithms that predict poor outcome following hospitalisation have been identified.39Prognostic tools could also improve management of febrile illnesses indirectly. Stratifying participant recruitment into trials of novel therapeutics by expected prognosis would ensure comparability between different sites, as well as selection of a study population in whom the attack rate is sufficiently high to adequately power the trial. Furthermore, if prognostic utility is verified, surrogate endpoints based on these markers could reduce the number of participants required, increasing the feasibility of conducting trials in more peripheral settings, allowing inclusion of populations more representative of those the proposed interventions are intended to benefit. Outside of clinical trials, accurate prognostication could help assess the impact of quality improvement initiatives, training programmes and organisational changes, as well as facilitating interunit comparisons and benchmarking.40Markers of common pathophysiological pathways can improve identification of febrile patients with a poor prognosis.For tests performed at the community level, simplicity is key. Tests will require the most robust features to address stability and transport stress across wide temperature and humidity ranges. Additionally, these tests need to be energy-independent and results must be straightforward to interpret. For prognostic biomarker tests this poses a specific design challenge as many emerging biomarkers are concentration dependent, requiring either quantitative or semiquantitative results.42For tests employed at the district hospital level or above more advanced infrastructure exists  to enable the use of robustly designed instrumented technologies that account for the varied conditions in laboratory settings common in LMICs. Instrumentation permits technologies capable of high sensitivity measurement and quantification, as well as more versatile throughput, sample processing and analyte detection methodologies.Results of a recent multinational stakeholder consultation indicate that better tools to guide community healthcare providers in their referral and admission decisions are urgently required, in particular to prevent under-referral .14 CruciThe ideal prognostic test or algorithm would have sufficient accuracy and also be generalisable across similar settings. Determining generalisability necessitates comparisons between different studies. Although clearly defined use-cases can harmonise study designs and promote consistency of reporting, standardised assessment methods are essential. Tools to support standardised measurement of baseline predictors have been proposed,One possibility is to adjust for the differing treatments and interventions a patient receives when data from disparate studies are synthesised. However, treatments and interventions are often applied imprecisely and adjustment for mediating variables is difficult and can introduce selection bias. An alternate solution is to use a standardised tool to assess the overall ‘level of care’ available in different healthcare settings .Additional measures of disease severity, such as need for hospital-level care, must be included alongside mortality. Although surrogate outcomes have important limitations , a comprehensive outcome set that includes, but is not limited to these surrogates, is important to consider. Mortality, although a ‘hard’ outcome, occurs infrequently in community settings and predicting death may be of limited utility, compared with predicting severe (and in many instances treatable) illnesses.Many studies evaluate clinical and biochemical prognostic factors in febrile patients.49Few studies consider the additional information that trends in different parameters may provide.Finally, once promising prognostic markers are identified, simple, affordable and reliable tests to measure them must be manufactured and supported by robust supply chains to ensure equitable access. Ideally, tests and algorithms would be quantitative, providing a mechanism to adjust cut-offs to achieve the desired NPV or PPV for a particular setting. Candidate predictors already routinely collected for other purposes should be prioritised for evaluation. The growing use of electronic health records may make this more feasible. For clinical features this could include increasing access to technologies such as pulse oximetry and other vital sign devices.Prognostic tools that improve risk stratification of patients with febrile illness would have enormous potential to improve patient outcomes and allocation of scarce resources. Each proposed technology requires careful cost-benefit assessment and must be developed in partnership with the healthcare providers working within the targeted contexts. Defining essential product design requirements in consultation with users is essential to ensure usability and promote understanding, acceptance and trust of these technologies. Importantly, donors and implementers must embrace integrated community care and move away from vertical disease-specific models, as the settings where prognostic tools could have greatest impact are precisely the contexts in which diagnosis remains most challenging.


Furthermore, recent evidence has shown that an innovative patient-centered approach taking into consideration patient characteristics, treatment preferences and accessibility to treatment is recommended to optimize clinical management of COMISA patients. However, in this complex mosaic, many other sleep disorders may overlap with COMISA, so there is an urgent need for further research to fully understand the impact of these therapies on outcomes for OSA patients with comorbidity. In light of this need, this review focuses on the major sleep disorders comorbid with OSA and the recent advances in the management of these insomniac patients.Obstructive sleep apnea (OSA) and insomnia are the two most common sleep disorders among the general population, and they may often coexist in patients with sleep-disordered breathing (SDB). The higher prevalence of insomnia symptoms in patients with OSA (40–60%) compared to that observed in the general population has thus led researchers to identify a new disorder named comorbid insomnia and OSA (COMISA), whose true burden has been so far largely underestimated. The combined treatment of COMISA patients with positive-airway pressure ventilation (PAP) with cognitive behavioral therapy for insomnia (CBT On the other hand, in those patients complaining of the first symptoms of insomnia, the rate of sleep apnea co-occurrence was from 7% to 69% . In addiOne study by Mundt et al., to examine the contributions of OSA and insomnia to chronic pain, found that 80% of participants reported chronic pain, with the most common types of pain being musculoskeletal (28.57%) and headaches (24.76%). A post-hoc comparison showed that individuals with comorbid OSA/insomnia reported an average pain intensity that was 20 points (out of 100) higher than individuals with insomnia or no diagnosis and 28 points higher than those with OSA. They concluded that chronic pain was highly prevalent in this sleep clinic population, with the most severe pain reported by individuals with co-occurring OSA/insomnia with important clinical implications, as both chronic pain and insomnia have been shown to predict poorer adherence to positive airway pressure treatment of OSA . AnotherFurthermore, numerous studies have analyzed a higher risk to develop cardiovascular comorbidities  among patients presenting comorbid OSA plus insomnia or other sleep disorders ,20,21,22For the aforementioned reasons, it is important to identify all comorbid disorders, diagnose them as separated ones and adequately treat each one of them to avoid long-term sequelae .Recent research has focused on symptom scores evoking the existence of distinct clinical phenotypes among OSA patients. Among these phenotypes, most patients present sleep disturbances with typical characteristics of chronic insomniac disorder . Indeed,The results of the study showed that treatment with positive airway pressure (PAP) after two years significantly reduced symptoms of isolated middle insomnia, while other different mechanisms seem to contribute to symptoms of initial and late insomnia in these patients, likely leading to a negative adherence to PAP. This observation suggests the need for targeted treatment for insomnia  that may be beneficial for patients with OSA comorbid with insomnia affecting positively adherence to PAP. Another subsequent study by Castillo et al. supporting the overlap features between the two comorbid disorders showed how sleep apnea patients who underwent diagnostic polysomnography displayed a large spectrum of total sleep time misperception values, with one-third of the cohort underestimating their total sleep time by at least 60 min . FurtherThey postulated that the regional differences might have been related to sex, age and BMI . As a geTo administer proper treatment, it is also crucial to clarify the pathogenetic mechanisms of the disorders in question, as several mechanisms are believed to play a role in the relationship between OSA and insomnia . DiffereRecent studies support the idea that metabolic processes correlate with sleep; the study of metabolite biomarkers offers unique opportunities to provide insights into the pathology of diseases by revealing alterations in specific metabolic pathways . AccordiA comprehensive assessment of both OSA and sleep-related disorders is very important to warrant effective treatments, and an integrated, interdisciplinary approach should be considered. For this reason, efficient diagnostic procedures for a combined evaluation are urgently needed. Consequently, the diagnosis of insomnia needs to be enriched by multiple aspects, including a complete patient medical history, several clinical aspects, the results of the physical examination, the recording of sleep diaries and, in selected patients, other complementary techniques, such as actigraphy . On the The presence of comorbid OSA, according to Lack et al., does not seem to impair the treatment of insomnia. Following a treatment program for insomnia, the substantial improvement in sleep and daytime functioning variables among COMISA patients was in fact comparable to that observed in patients without OSA . In patiThe first-line treatment for moderate to severe obstructive sleep apnea syndrome (OSAS) is continuous positive airway pressure (CPAP) . AlthougOther treatments for OSA include the use of mandibular advancement devices (MADs), positional training and surgery. These treatments are typically recommended based on specific features related to sleep apneas  . A receni) also improved TST and resulted in shorter sleep latency [i in the last years has shown a pivotal role in first-line non-pharmacological treatment for insomnia [i is generally regarded as safe and effective across a variety of comorbid conditions, some aspects remain controversial. For example, it has been shown that sleep restriction exacerbates daytime sleepiness due to OSA [i such as stimulus control, aimed at eliminating the extended periods of wakefulness spent in bed, might improve poor sleeping habits in individuals with comorbid insomnia and OSA [i, or separate treatment alone. Their analysis indicated that almost all the evaluated studies showed a decreased PAP-adherence caused by insomnia. The authors suggested that having comorbid OSA did not impair the treatment of insomnia among COMISA patients. Additionally, insomnia in COMISA patients could be treated with CBTi [i and PAP-therapy are necessary even though distinct treatment strategies may be useful in different COMISA phenotypes.Cognitive and behavioral therapy  [2, improving sleep quality without causing respiratory depression. Consequently, the authors suggested that the statement “hypnotics should not be used in patients with sleep apnea” should be changed to “hypnotics may sometimes be used in patients with sleep apnea” [Even though hypnotics may be used in patients with insomnia, benzodiazepine-based therapies are generally not recommended for patients with OSA . Dolly e = 0.04) . Similar = 0.04) . Other ap apnea” . Two yeap apnea” . Lastly,p apnea” .In recent years, newer non-benzodiazepine sedative hypnotics (NBSH), such as zolpidem, zaleplon and eszopiclone, were developed and studied in patients presenting insomnia with or without concurrent OSA. These compounds refer to non-benzodiazepine (non-BDZ) sedatives acting as BDZ receptor agonists that were shown to have generally a lower harmful impact effect on the airways. According to Luyster et al., non-benzodiazepine agents have hypnotic and sedative effects similar to those of benzodiazepines, but some—especially those selective for gamma aminobutyric acid (GABA) receptors containing α1 subunits—may have fewer muscle-relaxant effects, thereby offering a more favorable treatment approach to insomnia in OSA . In a piOther authors examined the effects of anti-depressive agents prescribed in OSA patients comorbid with insomnia. Kryger et al. investigated the efficacy and safety of ramelteon in individuals with mild-to-moderate OSA. Ramelteon is a selective melatonin MT(1)/MT(2) receptor agonist indicated for insomnia treatment. The potential effects of ramelteon on apneic and hypopneic events and arterial oxygen saturation in OSA patients was assessed in a randomized, double blind, crossover study, which led to the conclusion that this antidepressant does not worsen sleep apnea when administered to a subject with mild-to-moderate OSA . DecreasSleep hygiene education and pharmacotherapy are the most commonly offered treatments for chronic insomnia disorder. Sleep hygiene education commonly includes information about caffeine, alcohol and nicotine use, exercise, sleep environment, sleep–wake regularity, nap avoidance and stress management . A recenA meta-analysis on the use of melatonin for treating primary sleep disorders showed an approximately 7-min decrease in sleep latency, an 8-min increase in total sleep time and a very small improvement in sleep quality. Adverse events were not discussed . LikewisAccording to Restless legs syndrome (RLS) patients may show insomnia-specific characteristics such as sleep disruptive habits and cognitions , and simThere is increasing evidence that obstructive sleep apnea with insomnia symptoms coexist in epilepsy . Continuous positive airway pressure treatment of OSA in epilepsy improves seizure control, cognitive performance and quality of life . ParasomOSA occurs frequently in narcolepsy and may delay the diagnosis and interfere with narcolepsy proper management. In patients with OSA, cataplexy (a major narcoleptic trait) should be actively looked for to exclude the presence of narcolepsy. OSA and narcolepsy are associated with poor quality of sleep and excessive daytime sleepiness (EDS): treatment with CPAP does not usually improve EDS in narcoleptics with OSA . A new mi with progressive delay of the circadian rhythm with the strategic use of light therapy, natural light exposure and melatonin [Circadian rhythm sleep–wake disorders are a collection of disorders that can occur when patients’ typical sleep times are misaligned. Many people have experienced jet lag, travelling across at least three time zones, or shift work sleep phase disorder, affecting those who have a non-traditional work schedule, resulting in poor sleep and exhaustion during waking hours. It is important to consider whether another sleep disorder might be occurring, such as sleep apnea, that may be exacerbated and associated with new-onset insomnia. Therapeutic options include CBTelatonin . i and hypnotics, it is recommended to evaluate signs and symptoms of OSA [i and CPAP is more effective in improving CPAP use and insomnia symptoms compared to treatment with CPAP alone [OSA is the most common kind of sleep apnea characterized by upper airway collapse during sleep with repeated episodes of apneas and hypopneas leading to oxygen desaturations; it is related to many other medical conditions such as congestive heart failure, hypertension, stroke, arrhythmia and decline in renal function  that mays of OSA . In thiss of OSA . Thus, cs of OSA ,15. In tAP alone . FittingAP alone . For thiAP alone .The evidence summarized in this review indicates that the association between insomnia and OSA is an important, yet unexplored, research area that needs to be studied in depth. Recent research interest has focused on the idea of better management with a multidisciplinary approach and concurrent treatment of the two entities that seems to be promising but needs additional data to determine the effectiveness of combination treatment in this population. Globally, it is advisable to look for comorbidities in OSA patients when their symptoms persist despite adequate treatment. Another important aspect emerging from our study is that the onset of OSA symptoms may be independent from that of other co-occurring sleep disorders. In light of this complex comorbidity profile in OSA patients, it is highly recommended for clinicians to investigate the potential co-occurrence of other sleep disorders, diagnose them as comorbidities and treat them accordingly to preserve sleep integrity and improve daytime quality of life.


Exercise has been used to define mechanisms related to improved vascular health, which may involve the microbiome. Motivated by the clinical significance that both exercise and the gut microbiome have; the objective of our work is to assist in defining the gut-vascular axis while identifying biomarkers of gut microbial health linked to vascular function. In this  In the context of vascular health, products of microbial metabolism have been shown to promote processes that increase cardiovascular disease (CVD) risk , 2. Emers [LSS])  to map sin vitro exercise mimetic (LSS) in cultured EC to define the molecular mechanisms which protect and/or improve endothelial health stimulated by both exercise and biomarkers of gut microbial metabolism  (The scope of our work is to fill the gap in knowledge concerning the role of exercise in CVD risk and the interactions within the gut-vascular axis by (i) comparing and contrasting human gut microbial profiles between exercise-trained  and sedentary individuals; (ii) influencing beneficial gut microbiome adaptations, via supervised exercise training, in individuals with and without HTN to identify functional shifts in metabolic capacity and specific microbial taxa associated with exercise-induced improvements in BP; and (iii) utilizing an A.The rising trend of specific risk factors that can make one prone to CVD, including obesity, high BP, and lack of exercise, obliges the quest for defining their relationship with the gut microbiome and identifying more efficient approaches to preventing and altering the trajectory of these disorders . Studiesin vitro data from our lab informed us that butyrate has anti-inflammatory properties on EC and can attenuate EC dysfunction. Currently, we are working to define SCFA signaling pathways related to inflammatory processes in EC function. Additionally, we are utilizing our in vitro exercise mimetic in cultured EC to determine distinct signaling pathways associated with SCFA and our exercise stimulation on EC inflammatory profiles. In the near future, we expect to define how SCFAs, the microbes that produce them, and exercise may superimpose their effects to improve endothelial function to mitigate HTN.Unpublished data in a previous study informed us that there may also be a significant vascular component to gut inflammation  and initB.Concerning exercise, our work assesses how exercise improves vascular health . Again, Defining the impact of exercise-induced microbial alterations on vascular health versus canonical exercise-induced vascular adaptations is a significant challenge in this field. Our lab’s approach is to work to isolate mechanistic avenues that link exercise-induced gut-derived SCFAs to endothelial function and the gut-vascular axis. Future studies must include germfree mouse cecal/fecal transplant studies in HTN pathology and isolated vessel studies. We are realizing that our work, utilizing exercise as a novel tool to elicit shifts in microbial metabolic profiles in mild to moderate HTN, has significant potential in revealing functional shifts of the gut microbiome linked to improved vascular health and potentially other chronic diseases. Our work in identifying gut metabolic profiles (specific taxa and circulating biomarkers) of gut microbial health is essential to developing strategies in predicting and reducing CVD risk while advancing therapies to improve gut microbial health.


The recent tragic death of three children in a clinical trial to treat X-Linked Myotubular Myopathy by delivering myotubularin with an AAV8 vector notwithstanding, AAV remains a highly promising therapeutic gene delivery platform. But the successful use of AAV vectors to treat an increasing number of diseases also makes establishing protocols to determine therapeutically relevant titers of pre-existing anti-AAV antibodies and approaches to deplete those antibodies more urgent than ever. In this mini review, I will briefly discuss (i) our knowledge regarding the prevalence of anti-AAV antibodies, (ii) the challenges to measure those antibodies by methods that are most predictive of their influence on therapeutic efficacy of AAV gene transfer, and (iii) approaches to overcome the formidable hurdle that anti-AAV antibodies pose to the successful clinical use of AAV gene therapy.Adeno-associated virus (AAV) vector-based gene therapy is currently the only  Thirty years ago, the Chicago Tribune published a front-page article entitled “Gene therapy poised to reinvent medicine.” While, after three humbling decades of intense research, we have not achieved this lofty goal, it is safe to say that we made tremendous progress toward establishing gene therapy as an important tool to treat both inherited as well as acquired diseases. Among the currently available gene therapy platforms, vectors based on adeno-associated virus (AAV) have clearly emerged as one of the most promising gene delivery vehicles.in vivo gene therapy treatments have been approved by regulatory agencies for clinical use  Even wild-type AAV has not been shown to cause any disease, (ii) In contrast to other currently available gene delivery methods, in non-dividing or very slowly dividing cells AAV results in the long-term expression of the therapeutic payload, even though the AAV vector DNA persists mostly as extrachromosomal episomes , (iii) AUntil recently, AAV gene therapy had an unblemished safety profile . Sadly, One of the drawbacks of AAV is its limited cargo capacity of ~5 kb. Even though the median size of a human protein is only 375 amino acids  30–60% of all individuals harbor antibodies that neutralize AAV transduction . Interesin vitro assay) is/was one of the exclusion criteria. While this makes inherent sense, the absence or presence of NAbs is ill defined. The in vitro assays employ an AAV reporter vector  of the serotype used in the respective clinical trial. Pre-treatment patient serum/plasma samples are then serially diluted and incubated with an AAV reporter virus with a pre-determined number of vector genomes. After addition of the virus to cells (most often HEK293 cells) and incubation for a defined amount of time, the expression of the reporter protein is measured for each serum dilution as well as samples that did not receive any patient serum. Expression levels of each serum dilution sample are then normalized to the no-patient serum control. In general, the neutralizing titer is defined as the highest dilution that reduces transduction by ≥50%, although other levels of reduction have been used as well [e.g., 29% .Unfortunately, the AAV dose/MOI is not the only parameter that can influence NAb titers. Two other critically important parameters are the purity of the vector preparation, especially as it relates to the absence or presence of monomeric or oligomeric capsid proteins. Moreover, depending on the vector production method and purification scheme, the final vector preparations can also contain empty viral particles, particles with truncated vector genomes or particles with plasmid or genomic DNA. All of those particles are essentially inactive virus, and their presence in an NAb assay will artificially lower the NAb titer . It is ain vivo assay to determine neutralization by human or non-human primate (NHP) sera [e.g., . In my view, going forward, it will be essential that regulatory agencies such as the FDA and the EMA will create standardized in vitro assays for each AAV serotype/variant. These assays should be sensitive and rely on laboratory equipment that is commonly available in academic laboratories. Ideally, these assays will be validated in vivo in mice and NHPs. Importantly, the assays should include commercially available reagents, such as a neutralizing, monoclonal antibody, to allow the validation of the assay in each laboratory using it. Only standardized assays will allow a valid comparison of NAb levels reported by different groups.Currently, AAV gene therapy is rapidly expanding with more than 200 ongoing or completer clinical trials  levels , 22. HowIn theory, the most appealing approach to overcome the challenge of pre-existing NAbs is to introduce mutations into the AAV capsid that prevent the binding of NAbs. However, the significant cross-reactivity among AAV serotypes  shows thin vitro by incubating IVIG or human sera with beads that have AAV particles covalently coupled to them. Moreover, my colleagues and I were able to demonstrate that performing hemapheresis with such beads can fully restore liver transduction in animals with NAb titers that without hemapheresis show none to negligible transduction  , a streptococcal cystein protease, that can cleave IgG into F(ab')s and Fc , 29. Tres and Fc . StrikinAV-LK03) .Using IdeZ, a homolog of IdeS produced by a different streptococcal strain, Asokan and colleagues  also couPre-existing anti-AAV antibodies are a vexing problem for AAV gene therapy because they can severely limit the patient population that could benefit from AAV gene therapy. This becomes all the more frustrating as the field rapidly advances and many more AAV therapeutics will become available to treat an increasing number of genetic diseases. Nonetheless, over the last couple of years, significant progress has been made in overcoming this formidable obstacle and to extend therapy to patients with pre-existing neutralizing antibodies, either as a result of infection by wild-type AAV or prior treatment with an AAV vector. Because of the ease of use, the IgG cleaving proteases IdeS and IdeZ are particularly attractive to use in AAV gene therapy patients with pre-existing anti-AAV antibodies , 30. HowBecause of significant differences in assay conditions, a comparison of neutralizing titers obtained by different laboratories is currently impossible. As the use of AAV gene therapy rapidly expands it will, in my view, be absolutely essential that validated assays that are approved by regulatory agencies for any given AAV serotype or variant will be used to determine the amount of neutralizing factors in sera. Until such assays are validated and approved, I urge my colleagues to report very detailed descriptions of their assays used and to report the neutralizing activity of sera/plasma samples as AAV particles neutralized per μl serum/plasma. While imperfect, this should allow at least a “reasonable” comparison of results obtained by different laboratories.Clearly, a significant amount of future research will be required to “solve” the problem that neutralizing antibodies and factors pose to the expansion of the eligible patient population. Nonetheless, recent developments offer a great deal of optimism that we are on the way to being able to include patients into clinical trials and treatment regimens that owing to the presence of neutralizing antibodies and factors are currently excluded from treatment with AAV gene therapy.The 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.


One class of enhancers, termed super-enhancers, is highly active and cell-type specific, implying their critical role in modulating cell-type specific gene expression. Deletion or mutations within these CREs adversely affect gene expression and development and can cause disease. In this mini-review we discuss recent studies describing the potential roles of two CREs, enhancers and binding sites for CTCF, in early mammalian development.Cell fate decisions are the backbone of many developmental and disease processes. In early mammalian development, precise gene expression changes underly the rapid division of a single cell that leads to the embryo and are critically dependent on autonomous cell changes in gene expression. To understand how these lineage specifications events are mediated, scientists have had to look past protein coding genes to the  In this mini-review we will focus on both classes of CREs specifically, as well as their mechanisms and roles in driving early cell fate decisions during early mammalian development. The examples discussed below do not encompass all the literature available by any measure but reflect selected key studies in the field focusing on embryonic stem cells (ESCs). For further information on enhancer and CTCF functions, we suggest these reviews  in driving cell fate decisions by regulating gene expression. Nuclear architecture is the dynamic, three-dimensional organization of chromatin and its interaction with itself and regulatory proteins within the nucleus. The development of high-resolution imaging and chromosome conformation capture technologies, which permit a direct interrogation of long-range DNA:DNA interactions, has facilitated these important aspects, which regulate gene expression. Two types of  reviews .CDX2, which turns off the pluripotency gene Pou5f1, which encodes the pluripotency critical transcription factor Oct3/4  distal to the gene promoter, which are located immediately adjacent to the transcriptional start site (TSS). Binding of these TFs drives enhancers to promote or repress gene expression through multiple mechanisms that are beyond the scope of this review . Next-geNanog is near three SEs  of the three SEs at the Nanog locus demonstrates that they physically interact with the Nanog promoter, but CRISPR-based deletion of each SE differentially alters expression  dynamics upon deletion of the −5SE shows a complete loss of RNAPII at the Nanog promoter, indicating that the −5SE modulates Nanog expression by recruiting RNAPII or by promoting its conversion to its initiating form through phosphorylation of Ser 5 of the C-terminal domain  from the −5SE  disrupt maintenance of pluripotency in ESCs when deleted , which is classically characterized by mandibular underdevelopment along with other characteristics . PRS is Sox9 . Sox9 plelopment  and enhaelopment . PRS asspression . These tShh and Sox2 enhancers has shown that physical proximity may not be necessary, further expanding the question of the mechanisms that enhancers use to modulate gene expression.Although all the nuances of how enhancers determine cell fate are still being investigated, we do understand that their role is critical to a range of developmental processes. Two mechanistic insights not discussed here but also under investigation are whether and how enhancers modulate gene expression by regulating transcriptional bursting  and/or pNanog, Pouf51) is sufficient to alter gene expression, indicating these INs facilitate proper gene expression regulation, perhaps by creating and/or preventing enhancer-promoter interactions. A key point to consider is that INs and TADs were identified using different techniques and therefore may be identifying the same phenomenon but at different scales due to methodological differences.DNA is organized within the nucleus on multiple levels . Double-The insulator protein CTCF contains 11 highly-conserved zinc-finger DNA binding domains . InsulatHoxA locus, deletion of CTCF sites within the locus alters the enrichment of the repressive H3K27me3 mark, permitting aberrant HoxA gene expression during the differentiation of ESCs to motor neurons (in vivo (Many studies have shown that CTCF plays a role during development (reviewed in  neurons . In the  neurons . As a di(in vivo . These aIn an auxin-inducible depletion system in mESCs, loss of CTCF reduces proliferation, indicating that CTCF is integral to the maintenance of pluripotency . The acuWap, but also the non-mammary-specific Ramp3. Ramp3 lies outside of the chromatin loop formed by two CTCF sites flanking Wap and the associated SE. Deletion of the separating CTCF sites greatly increases the interaction between the SE and Ramp3 and Ramp3 expression, thus showing that the CTCF sites insulate against this interaction to maintain proper expression (MYC to up-regulate MYC and drive oncogenic transformation. These SEs lay between two CTCF sites that form an IN neighborhood and modulate expression (Several studies have focused on the interaction between CTCF and enhancers, specifically SEs. CTCF has been found to be bound near most SEs and within some SEs . One stupression . In a vapression . These spression , supportNanog locus, what is CTCF’s role? Is the IN truly the two sites just 5′ and 3′ of the gene, as implied in Dowen et al., and can other nearby CTCF sites compensate? And are these CTCF sites encouraging and/or preventing enhancer:promoter interactions? Beyond Nanog, the question still remains whether there are different classes of CTCF binding sites that can be identified through a biological signature. Given that loss of CTCF can cause an up-regulation and down-regulation of gene expression, how can we identify which role it plays for any given gene? Similarly, there are still questions regarding enhancers and their role in cell fate decisions: specifically are all super-enhancers and the same? It is clear from the data regarding the Nanog +60E that this cannot be true, because although it has all the defining features of a super-enhancer, in ESCs, at least, it does not play a significant role. Perhaps it is a redundant enhancer, becoming fully functional if the −5SE and −45SE are out of commission or is utilized in other cell types such as primordial germ cells (The full potential of CTCF sites and enhancers in regulating gene expression is still being investigated and a number of questions remain. As an example, in view of the evidence that CTCF sites modulate SE function, at the rm cells . Can we PA wrote the first draft of the manuscript. SR and PA edited the manuscript together. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We present the case of a 74-year-old female patient who presented to the emergency department with lower extremity weakness found to have a fixed frequency square wave artifact in all leads of her electrocardiogram (ECG). After troubleshooting, faulty external cardiac monitor leads were identified as the cause of this unique artifact.The ECG is an important diagnostic tool for medical providers. Electrocardiogram artifacts are extremely common, and knowledge of artifacts is necessary to prevent inappropriate interpretation, diagnostic error, and unnecessary workup. Medical providers should have a low threshold for suspicion when ECG findings do not correlate with the patient’s chief complaint or history of present illness. They must also be familiar with the most frequent ECG artifact variants and be prepared to follow a stepwise approach to troubleshoot less frequent variants. A 74-year-old female presented to the emergency department for progressive left leg weakness that had resulted in multiple falls. An electrocardiogram (ECG) was obtained per Electrocardiogram artifact is common and can be generated by physiologic and non-physiologic causes as summarized in the What do we already know about this clinical entity?Electrocardiogram (ECG) artifact as a general topic has been studied extensively.What is the major impact of the image(s)?This image represents a new, not previously documented, case of ECG artifact, and the accompanying discussion presents a more holistic list of other potential causes of artifact.How might this improve emergency medicine practice?Recognition of ECG artifact is necessary to prevent inappropriate interpretation, diagnostic error, and unnecessary workup in the emergency department.


Post-traumatic stress disorder (PTSD) is triggered by an individual experiencing or witnessing a traumatic event, often precipitating persistent flashbacks and severe anxiety that are associated with a fearful and hypervigilant presentation. Approximately 14–30% of traumatized individuals present with the dissociative subtype of PTSD, which is often associated with repeated or childhood trauma. This presentation includes symptoms of depersonalization and derealization, where individuals may feel as if the world or self is “dream-like” and not real and/or describe “out-of-body” experiences. Here, we review putative neural alterations that may underlie how sensations are experienced among traumatized individuals with PTSD and its dissociative subtype, including those from the outside world  and the internal world of the body . We postulate that alterations in the neural pathways important for the processing of sensations originating in the outer and inner worlds may have cascading effects on the performance of higher-order cognitive functions, including emotion regulation, social cognition, and goal-oriented action, thereby shaping the perception of and engagement with the world. Finally, we introduce a theoretical neurobiological framework to account for altered sensory processing among traumatized individuals with and without the dissociative subtype of PTSD. The ability to interpret sensations from the external world and from within the internal body influences one’s perception, which informs how we navigate and communicate with our surroundings. Sensory processing refers to the ability to register, modulate, and organize incoming sensory information from one’s internal and external worlds, and in turn, guides adaptive and goal-oriented behavioral responses to sensory stimuli . A singlhypersensitivity. This can subsequently lead to decreased attunement with other sources of sensory information required for guiding informed decision-making  may experience emotion dysregulation, where heightened bodily sensations due to extreme fluctuations in arousal may promote dysregulated affect and impulsivity . Here, an-making . For exaAmong individuals with PTSD, it is thought that prefrontal cortex activation is decreased, causing disruption to top-down cognitive neural networks responsible for executive functioning, including multisensory integration, informed decision-making and emotion regulation . ConsequIn the first objective of this review, we describe the concept of sensory processing. Sensory processing provides a contextual framework where an individual can organize incoming salient information from the external world to shape a cohesive depiction of his/her inner world, which then guides behavioral responses to meet situational demands . On a neexteroceptive sensations, which inform the perception of our environmental surroundings  – This brainstem structure is critical for autonomic regulation and generates raw affective sensations in response to threatening stimuli.Superior Colliculus (SC) – This brainstem structure is responsible for coordinating an orienting response to stimuli from the external environment and works in tandem with the periaqueductal gray to coordinate innate reflexive behaviors when threat seems imminent.Vestibular Nuclei (VN) – This brainstem structure subconsciously acquires sensory information about self-location and aids in relaying sensory information to key structures in the cortex for multisensory integration, including the insula and the prefrontal cortex.Locus Coeruleus (LC) – This brainstem structure is critical for initiating sensory signaling processes in the brain that help facilitate decision-making and motor responses. It also controls norepinephrine release in the body during stress.Vagus Nerve (VG) – This brainstem structure is involved in carrying out bodily responses to the internal viscera in response to external sensory information.Thalamus – This is an important relay structure that acts a sensory gateway between the brainstem and the cortex. Specifically, the mediodorsal region aids in translating incoming sensory information involved in cognitive functioning and the pulvinar region helps prioritize attention to visually threatening sensory stimuli.Insula (Ins) – The posterior aspect of the insular cortex receives interoceptive and exteroceptive sensory information from subcortical structures, including the brainstem and the thalamus. The anterior insula aids in emotion processing and directs salient sensory information to the prefrontal cortex for multisensory integration, as well as other specialized sensory regions in the cortex.Primary Sensory Cortex (Prim Sens) – This brain region is more formally known as the postcentral gyrus and is key for somatosensory processing, including detecting touch and helps govern proprioceptive sensory information associated with the agency of self-movement.Posterior Temporal Lobe – The posterior temporal lobe spans the temporoparietal junction, which is important for embodiment, where an individual can feel present in his/her own body. It also plays a key role in auditory processing.Posterior Parietal Lobe – This area of the brain works closely with the dorsal prefrontal cortex to help guide attentional processing in response to salient sensory information. It is also involved in spatial reasoning and in coordinating planned movements.Visual Cortex (VC) – This area of the brain is critical for receiving and processing incoming visual sensory input.Dorsolateral Prefrontal Cortex (dlPFC) – This area of the prefrontal cortex is key for emotion regulation and multisensory integration, where it aids in understanding and interpreting incoming sensory information from multiple modalities  and ultimately provides contextual meaning underlying a sensory experience.The third objective of this review to is to identify neural aberrations in PTSD that may overlap with neural networks involved in sensory processing. Post-traumatic stress disorder is characterized by extreme arousal states, emotion dysregulation, and persistent negative alterations in cognition and mood . Among iIndividuals that present with the classic form of PTSD may experience intrusive memories of past traumatic experiences and may show persistent hypervigilance concerning their surroundings, even in the absence of threat . In addiFirst, individuals with PTSD have previously shown consistent hyperactivation of subcortical structures as compared to healthy individuals in previous neuroimaging studies, particularly at the level of the midbrain in the brainstem . SpecifiSecondly, incoming interoceptive and exteroceptive sensory information from the brainstem is typically relayed to the insula, which is a critical hub for receiving sensory information and directing it to other cortical areas in the brain for higher-order processing . CriticaFinally, individuals with PTSD have shown impairments in executive dysfunction that can be traced to the prefrontal cortex . The preadditional depersonalization and derealization symptoms associated with emotional detachment (Notably, approximately 14–30% of traumatized individuals present with the dissociative subtype of PTSD characterized by tachment . Criticatachment . In additachment . Hypoacttachment . OverallThe fourth and final objective of this review is to outline an integrative model that identifies sensory processing as an important theoretical framework for investigating post-traumatic stress disorder . We discAt the base of the hierarchy , continuNotably, persistent alterations in arousal may predispose traumatized individuals to be hypervigilant of their surroundings for fear of encountering trauma-related reminders. This state of defensive posturing may compromise one’s ability to utilize typical sensory processing avenues to interpret external sensory information continuously received from their surroundings . If trauExteroceptive sensory information is continuously acquired from the environment to inform the relationship between one’s self and surroundings . The vesdisembodiment, where body ownership is compromised and one’s own mental agency to exert movement is limited (Notably, resting-state vestibular nuclei connectivity patterns in the dissociative subtype of PTSD provide critical insight into how depersonalization and derealization symptoms may negatively impact one’s capacity for exteroceptive sensory processing and multisensory integration.  limited . In addiOn balance, the current literature suggests individuals with PTSD and its dissociative subtype experience significantly altered subcortical resting-state connectivity patterns with cortical structures that together may make them more susceptible to aberrations in sensory processing. In addition, these findings emphasize the importance of classifying individuals with PTSD separately based on the presence of the dissociative subtype, where, at the cortical level, individuals with and without the dissociative subtype show distinct alterations in the multisensory integration of interoceptive and exteroceptive information.As described above, one of the hallmark symptoms of PTSD involves alterations in cognition and mood, where individuals with PTSD frequently experience persistent negative trauma-related emotions and associated changes in perception of the self and the world . Here, cAs mentioned above, the insula is a key node for creating an interoceptive inference based on the convergence of exteroceptive and interoceptive sensory information from the environment  and is tSpecifically, parallel to propagate trauma-related symptoms. These contrasting patterns are discussed in Similar to the classic PTSD symptom profile, It is of note that previous resting-state data from our group show differing functional connectivity patterns involving the prefrontal cortex. Taken together, the current findings overwhelmingly suggest that the insula plays a pivotal role in translating sensory information to higher-order frontal structures involved in the central executive network underlying higher-order cognitive functions, including emotion regulation . UltimatThe next phase of the hierarchy see  is basedCritically, the eventual reintegration of traumatic memories may facilitate one’s attainment of the embodied self, forming the apex of the theoretical hierarchy proposed see . ThroughThe proposed theoretical model identifies potential compensatory neurobiological underpinnings that can provide further insight into how sensory-based psychotherapeutic interventions can be effective for trauma-informed clinical treatments . Here, iOverall, incorporating sensory processing into the lens through which we study post-traumatic stress disorder and other trauma-related disorders is critical for informing clinical treatment approaches that integrate mind and body. The theoretical hierarchy outlined in this paper addresses how studying neural networks in post-traumatic stress disorder through a bottom-up perspective that investigates sensory transmission from the brainstem to the cortex may, in turn, be critical for understanding how top-down cortical processes shape individuals’ engagement with their environment and others in the aftermath of trauma. We suggest that investigating how sensations stemming from traumatized individuals’ internal and external worlds are translated in the nervous system is paramount for understanding the neural pathways underlying embodiment, the agency to interact with others and emotion regulation processes. Accordingly, delineating further the neural underpinnings of sensory processing at the subcortical and cortical levels in individuals with post-traumatic stress disorder appears necessary to enhance our understanding of the neurobiological mechanisms underlying this often debilitating disorder.All authors listed have made a substantial, direct and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.