Clinical examinations (assessments) are integral to ensuring that medical students can treat patients safely and effectively. The COVID-19 pandemic disrupted traditional formats of clinical examinations. This prompted Medical Schools to adapt their approaches to conducting these examinations to make them suitable for delivery in the pandemic. This systematic review aims to identify the approaches that Medical Schools, internationally, adopted in adapting their clinical examinations of medical students in response to the COVID-19 pandemic.Three databases and four key medical education journals were systematically searched up to 22 October 2021; a grey literature search was also undertaken. Two reviewers independently screened at title, abstract stage and full text stage against predefined eligibility criteria. Discrepancies were resolved by discussion and involvement of senior authors. Risk of bias assessment was performed using an adapted version of a pre-existing risk of bias assessment tool for medical education developments. Results were summarised in a narrative synthesis.A total of 36 studies were included, which documented the approaches of 48 Medical Schools in 17 countries. Approaches were categorised into in-person clinical examinations (22 studies) or online clinical examinations (14 studies). Authors of studies reporting in-person clinical examinations described deploying enhanced infection control measures along with modified patient participation. Authors of studies reporting online clinical examinations described using online software to create online examination circuits. All authors reported that adapted examinations were feasible, scores were comparable to previous years’ student cohorts, and participant feedback was positive. Risk of bias assessment highlighted heterogeneity in reporting of the clinical examinations.This review identified two broad approaches to adapting clinical examinations in the pandemic: in-person and online. Authors reported it was feasible to conduct clinical examinations in the pandemic where medical educators are given sufficient time and resources to carefully plan and introduce suitable adaptations. However, the risk of bias assessment identified few studies with high reporting quality, which highlights the need for a common framework for reporting of medical education developments to enhance reproducibility across wider contexts. Our review provides medical educators with the opportunity to reflect on past practises and facilitate the design and planning of future examinations. Clinical examinations, or assessments, are integral to ensuring medical students are competent to progress to higher levels of training or a medical qualification , 2. The The COVID-19 pandemic and subsequent implementation of social distancing rules disrupted traditional formats of clinical examinations , 7. TypiHow were clinical examinations adapted in response to the COVID-19 pandemic? What were the successes and challenges of designing and implementing clinical examinations? What are the recommendations for future practices informed by lessons learnt by the study authors? Three systematic reviews have investigated medical education developments due to the pandemic –12 of whOur systematic review was conducted from January to October 2021. Prior to commencing, a study protocol was uploaded onto the Center for Open Science (OSF) registry (10.17605/OSF.IO/R64NZ) . We condnd October 2021 using the same search strategy as described below.Our original search took place in February 2021. However, because of the topical nature of this review and the rate at which new literature is emerging, we conducted a final updating search on 22We searched three electronic databases: MEDLINE, EMBASE and ERIC (Education Resources Information Centre). We piloted our search strategy on MEDLINE. Our final search strategy consisted of three axes which were combined with Boolean operators: (one representing the COVID-19 pandemic) AND  AND . Each axis consisted of keywords with their truncated forms and Medical Subject Headings (MeSH)/subject headings or descriptors specific to the database. We cross-checked our database searches against our searches of indexed journals to confirm the database searches captured papers identified from our journal searches.In addition to our database search, we hand-searched the online publications of four key medical education journals  using the same Boolean combination of axes described above.We also included Google Scholar in our search as it captured international texts and other forms of non-peer reviewed material. Since Google Scholar yields large volumes of irrelevant results, we made the decision prior to the search to stop screening when no results were passing through title and abstract screening after two pages of results (20 hits) .We searched for grey literature on the An International Association for Medical Education (AMEE) website , includiResults were exported onto Endnote reference manager  and subsThe SPIDER model , a searcWe defined a structured clinical examination as an examination in a simulated clinical environment in which candidates perform pre-designed tasks, are examined by appointed examiners, and where multiple candidates are examined in turn. This contrasts with a work-based assessment which we define as individual or small groups of students being assessed on placement in a hospital or clinical environment by a clinician.Inclusion and exclusion criteria were piloted at the title and abstract screening stage (first 100 results) and full text screening stage (first 10 results).Studies describing how one or more Medical Schools adapted their clinical examination of medical students in response to the COVID-19 pandemicStudies describing adaptations to any type of non-work based, structured clinical examination Studies including medical students in any year of studyStudies available as pre-publications and non-peer reviewed material in addition to published articles in peer reviewed scientific journalsStudies from any location and in any languageThe following inclusion criteria were applied:Studies describing any non-clinical, knowledge-based examinations Work based assessmentsStudies including examination candidates with any form of provisional medical registrationOpinion pieces where authors do not include a description of the adaptations to the design and implementation of their own deployed clinical examinationThe following exclusion criteria were applied:Two independent reviewers (SC and ET) screened titles, abstracts and full text articles against the inclusion and exclusion criteria. Rayyan  was usedWe used Excel to create a data extraction form. Initially, one reviewer (SC) piloted the data extraction form on five studies. Two independent reviewers (SC and ET) performed data extraction on a random sample of 15% of the included studies to check for alignment then subsequently SC performed data extraction on the other 85%. Broadly, data were extracted in three categories: study characteristics, what was reportedly done and study author evaluations.Currently, there is no consensus method for assessing study quality in studies included in medical education systematic reviews , 29–31. Our final version of the tool rated underpinning bias, setting bias, resource bias and evaluation bias as high quality, unclear quality, or low quality Table . This toTwo reviewers (SC and ET) performed a risk of bias assessment on a random sample of 15% of the studies to check for alignment and identify major discrepancies followed by SC assessing the remaining 85%. Again, inter-rater reliability was calculated using Cohen’s Kappa, with the same threshold as above.Because of the descriptive nature of the studies, we planned to summarise results in a narrative synthesis using guidance from Popay et al. . This apOur search strategy identified a total of 6,972 hits, which following de-duplication, resulted in 5,628 unique records Fig. . FollowiConsidering the two reviewers, Cohen’s Kappa was 0.79 at title and abstract stage and 0.92 at full text stage, representing substantial agreement.Some studies reported findings from multiple Medical Schools, and some Medical Schools published multiple studies, therefore the 36 included studies reported the approach adopted by 48 Medical Schools. Twenty-two studies were published in 2020 and fourteen in 2021 with an overall date range from March 2020 to September 2021. Studies reported findings from 17 countries in six continents. The largest number of studies came from Asia (39%) and Europe (36%). One study required translation from Spanish into English using an externally sourced translator before inclusion.Twenty-seven studies primarily focused on structured clinical examinations while 9 described clinical examinations as part of a wider focus on medical education developments in the pandemic as a whole.Thirty-four studies described adaptations to OSCEs, one described adaptation to the M3 structured clinical examination , and one described development of the Virtual Clinical Encounter Examination . Of the thirty-four studies describing OSCEs, thirteen described end of year OSCEs; eleven described end of rotation/clerkship OSCEs; five described supplementary OSCEs; and five did not specify the type of OSCE examinations.Out of the 36 studies, only ten reported quantitative outcomes comparing candidate scores from previous years to the present year, and 24 reported either formal  or informal feedback from stakeholders and participants who took part in the clinical examinations. Furthermore, six studies reported the number of COVID-19 cases in participants following in-person clinical examinations.Only three studies scored highly in all four domains in the risk of bias assessment tool Table  35–37].–37.35–37Studies reported either in-person or online clinical examinations amended or newly developed in response to the pandemic; fourteen studies described in-person examinations and 22 described online examinations . Study authors also noted that with sufficient planning and development, online examinations could be effective at examining clinical skills and indicated that in future, online clinical examinations could be very useful for examining students on remote placements and provided an authentic approach to assessing telehealth skills.In both approaches to adapting clinical examinations, candidate scores were reported to be comparable to previous years’ student cohorts and there was generally positive feedback received from participants and stakeholders.Unlike research with outcome measures, most studies included in our review focused on sharing practises, therefore we elected to assess for risk of reporting bias in line with Gordon et al. . It is uThough no prior systematic review has specifically considered the adaptations to clinical examinations required by the pandemic, two systematic reviews examining medical educational developments more broadly have been published; Gordon et al. , 13 and The strengths of our review included the systematicity and methodological rigour we employed throughout. We performed comprehensive literature searches including key bibliographic databases, key journals, grey literature and relevant websites. This meant we encompassed a range of sources and retrieved international studies, including those published in languages other than English. Additionally, we found substantial agreement between the two reviewers at both stages of screening. We effectively piloted eligibility criteria and the data extraction form prior to commencing each stage of our review and adapted a pre-existing medical education development reporting risk of bias tool to make it suitable for use in our review. Furthermore, two reviewers independently assessed a sample of studies for risk of bias, in addition to undertaking extensive discussions with senior authors to determine suitable thresholds for quality ratings in each of the four risk of bias assessment domains. Finally, we updated all searches in October 2021 given the currency of our review.There were some limitations to our study. It was challenging to develop a search strategy that was sufficiently sensitive to capture key papers, yet specific enough to make it feasible to screen the retrieved papers in a timely manner. This may have resulted in missed studies and was compounded by the observation that medical education publications were indexed inconsistently in the databases we searched. The approaches of just 48 Medical Schools were included in our review; however, all Medical Schools internationally would have had to adapt their approaches to clinical examinations in the pandemic. Numerous factors could have influenced whether Medical Schools sought to publish adaptations to their practices, such as local COVID-19 restriction timescales, familiarity with the research and publication process, and staff capacity; however, exploring this is not within the scope of this review. Of the 39 Medical Schools included, a significant proportion of studies originated in Asia, meaning our review may not fully represent the international range of approaches that Medical Schools adopted to facilitate clinical examinations. Finally, our risk of bias assessment was subjective, and occasionally it remained unclear whether studies should be rated low quality, unclear quality or high quality in each of the four domains.Firstly, regardless of the approaches adopted, study authors all concluded that it was feasible to adapt existing clinical examinations and deliver effective assessments despite the restrictions placed on Medical Schools by the pandemic. Commonly stated enabling factors to the success of these adaptations were sufficient workforce and other resources, and the investment in time for planning and carrying out these examinations , and we would urge that medical educators are given adequate time, facilities, and resources in order to plan and deliver the necessary changes.Several study authors describing a move to online clinical examinations concluded that these approaches were unable to assess key domains such as physical examination skills and practical procedural skills. Therefore, adaptations to in-person clinical examinations with stringent infection control measures may be preferred where possible. However, online delivery of clinical examinations may provide a useful tool for examining telehealth skills. The advent of ubiquitous internet usage, which has been expedited by the pandemic, means that telehealth has become a prominent mode of delivery in many settings. Telehealth demands a distinctive skill-set compared to conventional face-to-face consultations, in which medical students will need to gain skills and demonstrate competence in the future , 79. SomIn addition, we would also recommend that Medical Schools and regulatory bodies work together to ensure adequate contingency plans are prepared for future disruptions to critical clinical assessments, for example, having a blueprint for an online clinical examination ready for immediate use. Moreover, we urge medical educators to consider whether their assessment strategy is resilient to future disruptions, for example through reducing reliance on final year high-stakes clinical examinations and more focus on programmatic assessment and work-based assessments. Medical educators could consider a model such as Miller’s pyramid; a framework which emphasises the need for different assessment modalities for different expected outcomes in medical education . This enBecause of the disparities in both the indexing and reporting of clinical examinations described in the studies included in our review, we recommend that medical education research and publication continues to work towards increased systematicity. Examples of highly systematic work that readers might study include BEME guides , 83. IncWe conducted a systematic review to identify the approaches that Medical Schools, internationally, used when conducting clinical examinations of medical students in the COVID-19 pandemic. We identified two broad approaches, adaptation to in-person examinations and a switch to online clinical examinations. Study authors reporting both types of adaptation concluded that conducting clinical examinations was feasible, but it required a significant investment in planning, time, and resource. Of note, a major limitation to online examinations was the inability to examine physical examination skills or practical procedural skills, though other advantages of online approaches are a potential area for further research. We hope our review will be used as a resource by the international medical education community to understand what adaptations have been made in response to the pandemic to help design future clinical examinations in their own institutions that meet local needs; to facilitate reflections on past practises; and to determine what should remain post-pandemic.


The aim of this study was to illustrate recent developments in neural repair utilizing hyaluronan as a carrier of olfactory bulb stem cells and in new bioscaffolds to promote neural repair. Hyaluronan interacts with brain hyalectan proteoglycans in protective structures around neurons in perineuronal nets, which also have roles in the synaptic plasticity and development of neuronal cognitive properties. Specialist stem cell niches termed fractones located in the sub-ventricular and sub-granular regions of the dentate gyrus of the hippocampus migrate to the olfactory bulb, which acts as a reserve of neuroprogenitor cells in the adult brain. The extracellular matrix associated with the fractone stem cell niche contains hyaluronan, perlecan and laminin α5, which regulate the quiescent recycling of stem cells and also provide a means of escaping to undergo the proliferation and differentiation to a pluripotent migratory progenitor cell type that can participate in repair processes in neural tissues. Significant improvement in the repair of spinal cord injury and brain trauma has been reported using this approach. FGF-2 sequestered by perlecan in the neuroprogenitor niche environment aids in these processes. Therapeutic procedures have been developed using olfactory ensheathing stem cells and hyaluronan as a carrier to promote neural repair processes. Now that recombinant perlecan domain I and domain V are available, strategies may also be expected in the near future using these to further promote neural repair strategies. Has3 KO) display frequent seizures and an epileptic phenotype [Hyaluronan (HA) is a major space-filling component of the CNS/PNS providing tissue hydration and a matrix for cell attachment and an environment conducive to cellular survival and cellular migration during CNS/PNS development ,2,3. HA henotype ,7.Schofield originally proposed the term stem cell niche in 1978 to define local hemopoietic tissue environments that had specific molecular and cellular activities that maintained hemapoietic stem cells  had idenEpendymal ciliated neuronal support cells (neuroglia) arising from embryonic neuro-ectodermal tissue in the epithelial lining of the ventricles of the brain and the central canal of the spinal cord have roles in the establishment of neural stem cell niches . ECM andIn the adult mammalian brain, neural stem cells (NSCs) in the ventricular–sub-ventricular zone contain a subpopulation of cells with astroglial properties (B1 cells), which give rise to intermediate transient amplifying progenitors , which transit to the olfactory bulb as a stem cell reservoir. NSCs and IPCs are the primary and secondary progenitor cells of the niche. These cells have long processes that reach into distant regions of the niche, including the adjacent vasculature. NSCs are thus in a unique cellular compartment, and depending on ECM components, they are exposed and may be stimulated to proliferate and undergo differentiation or they may remain as quiescent cells which continue to slowly recycle for self-renewal. Cell–cell, cell–ECM and soluble factor interactions involving HA, perlecan, laminin and FGF-2 thus all have roles in the regulation of NSC behavior. To better understand the complexity of the niche compartment, it has been proposed that it should be considered as three functional regions, proximal (zone I), intermediate (zone II) and distal  . These rThe specialized neural stem cell niche has been termed the fractone in honor of the late mathematician Dr Benoit Mandelbrot due to their fractal-like appearances similar to Dr Mandelbrot’s early computer graphic generated fractal geometric images . The ideFractone ECM structures in the neural stem cell niche  influencA reduction in HS 6-O-sulfation, which is critical for FGF-2 signal transduction, has been observed in the aged sub-ventricular zone, and this reduces cell proliferation through a failure of FGF-2-induced phosphorylation of extracellular signal-regulated kinase (Erk1/2) ,42. IncrHA is the most abundant GAG of neural tissues and, as already discussed in the introduction, has important space filling and hydrating properties and is a cell-friendly ECM component that promotes cell viability and proliferative processes important in tissue development and repair processes. HA is thus a logical candidate to investigate for potential application in neural repair biology. In its native form, HA is a weak scaffolding material, and for it to remain in tissues, it must interact with HA receptors and HA interactive PGs and glycoproteins; the large size of these aggregates physically entraps these HA complexes within collagenous networks in tissues. However, neural tissues lack these collagenous networks, and the immobilization of HA in neural tissues relies on interaction with lectican PGs and neural HA receptors. This is essential to provide HA’s longevity in tissues, since it is rapidly degraded in vivo by hyaluronidase and is highly soluble, with the generated HA oligosaccharides rapidly undergoing dispersal from tissues. For tissue engineering applications HA must therefore be chemically modified and crosslinked or attached to another polymer to form stable, functional composite scaffolds that support cell adhesion and proliferation ,49,50. HA diverse range of HA bioscaffold applications have been developed for tissue repair . HA is aHA can be modified using a number of crosslinking chemistries to alter its viscoelastic properties to prepare scaffolds that mimic the material properties of native brain tissue, which is one of the softest tissues in the human body . DivinylBiomimetic composites of HA with collagen, laminin and CS-PG mimic the native brain structure to promote cell survival and neural differentiation and neural outgrowth in brain repair strategies ,73. HA–lOlfactory ensheathing cells (OECs) support axonal regeneration and remyelination with the appropriate formation of axonal nodes of Ranvier and improvement of nerve conduction velocity. OECs derived from nasal mucosa are of clinical interest, since these cells are amenable for harvest for such autotransplantation procedures . ResearcPerlecan is also a major functional component of the stem cell niche and has many attributes with regard to tissue development and repair processes ,103,104.The fine structure (sulfation position and density) of the HS side chains of perlecan is an important regulatory determinant in the differentiation of pluripotent stem cells in the niche environment in neural tissues . InteracCollagen–HS porous scaffolds containing NSCs have been used to treat a rat model of traumatic brain injury, established using a controlled cortical impact . Brain eSignificant improvements in bioscaffold microfabrication methodology has permitted the miniaturization of these platforms. Lithography and direct laser printing have been applied to prepare 2D patterns and 3D scaffolds to shape hydrogels and synthetic polymers to create niche-like structures for single neural cell culture . ArtificWith a greater understanding of the roles of key functional components in the stem cell niche environment that promote neurogenesis, repair and regeneration following trauma, it is logical that these components should be developed for use in neural repair strategies. The use of neural stem cells from the olfactory bulb and HA either as a stem cell delivery vehicle or as a component of composite new generation bioscaffolds is a powerful synergistic combination that is enabling the more effective repair of neural tissues. By understanding the roles of HA in the fractone stem cell niches of the sub-ventricular zone and sub-granular region of the dentate gyrus of the hippocampus, it has been possible to formulate tissue repair procedures using HA either as a delivery vehicle or as a component of composite bioscaffolds to improve neural repair. Further components of the niche environment such as perlecan and laminin also regulate stem cell proliferation and differentiation, and these are promising agents that may also find application in novel neural repair strategies in the future. Perlecan has already shown promise in the repair of the blood–brain barrier following ischemic stroke.


Vascular spasm is well known and studied in the arterial system. There are only a few cases reported related to central venous spasms. We present the case of a 63-year-old male with an extensive medical history, including deep vein thrombosis (DVT), who underwent peripheral insertion of a central catheter in his left upper extremity with subsequent development of left upper extremity edema. The central catheter was removed before the patient underwent a contrast-enhanced computed tomography of the chest which revealed severe narrowing of the left brachiocephalic vein, consistent with venospasm in the clinical setting. Nitroglycerin might be useful to prevent vasospasm, or it might also be used for treatment. In our case, the catheter was removed, and no subsequent treatment was necessary. Although large-caliber venous spasms have been reported, they are rare and less studied compared to arterial spasms. The few similar cases of large venous spasm are associated with the percutaneous intervention, including inferior vena cava spasm, left axillary vein spasm, and left subclavian vein spasm -3. To ouThere is evidence, however, with a small sample size, that pre-procedural intravenous administration of 200 μg nitroglycerin can reduce the severity of axillary venospasm during pacemaker placement . There aWe present the case of a 63-year-old male with an extensive medical history, including deep vein thrombosis (DVT). A peripherally inserted central catheter (PICC) line was inserted in the left brachial vein and advanced centrally without immediate adverse events. A few hours later, the patient developed left upper extremity pitting edema. At this point, the PICC line was removed, and a contrasted chest computed tomography (CT) was performed for suspicion of venous thrombosis, considering the patient's previous vein thrombosis history. The chest CT revealed severe narrowing of the left brachiocephalic vein just posterior to the sternum without evidence of a filling defect. The narrowing of the brachiocephalic vein can be seen in Figure The physiology of vascular vasospasm is not fully understood. The most established and accepted mechanisms are endothelial dysfunction, nitric oxide release, and enhanced vascular smooth muscle cell contractility as disruptions of vascular tone ,7. HowevIn this study, we report that large vessel venospasm occurred after instrumentation of the brachiocephalic vein, adding to the body of evidence suggesting endothelial dysfunction due to physical is likely to be a risk factor for vasospasm. Clinicians should be aware of the risk for central venospasm after PICC line insertion, a condition that may mimic DVT, another common adverse event associated with PICC placement. The difference is considerable as anticoagulation would not be indicated for a patient with vasospasm. The key CT finding of acute vein thrombosis is normal or slightly enlarged vessel diameter with a luminal filling defect. However, severe narrowing of the vessel lumen with the unimpeded flow in a patient with relevant history is a sign of venospasm.Central venous spasm can occur after PICC line insertion, a condition that may mimic acute DVT. This case report adds to a small but growing body of literature that instrumentation of large vessel veins can cause venospasm. It is important to differentiate venous spasm from vein thrombosis as anticoagulation would not be indicated for a patient with vasospasm. Radiologists should recognize the severe narrowing of the vasculature with the unimpeded flow in a patient with a central line as a sign of venospasm.


Birds have extremely elevated metabolic rates during migratory endurance flight and consequently can become physiologically exhausted. One feature of exhaustion is oxidative damage, which occurs when the antioxidant defense system is overwhelmed by the production of damaging reactive oxygen species (ROS). Migrating birds have been shown to decrease the amount of oxidative lipid damage during stopovers, relatively stationary periods in between migratory flights. It has therefore been argued that, in addition to accumulating fuel, one of the functions of stopover is to restore the oxidative balance. If this is so, we would expect that migrating birds are unlikely to resume migration from stopover when they still have high amounts of lipid damage.Turdus philomelos) and northern wheatears (Oenanthe oenanthe), a medium- and long-distance songbird migrant, respectively. We measured malondialdehyde (MDA) concentration, a biomarker for oxidative lipid damage, and total non-enzymatic antioxidant capacity (AOX), an overall biomarker of protection against ROS. Stopover departure decisions were determined using a fully automated telemetry system set-up on our small island study site.To test this hypothesis, we measured parameters of the oxidative balance and related these to stopover departure decisions of song thrushes (The decision to resume migration was not related with MDA concentration in either study species, also not when this was corrected for circulating fatty acid concentrations. Similarly, AOX did not affect this decision, also not when corrected for uric-acid concentration. The time within the night when birds departed also was not affected by MDA concentration or AOX. However, confirming earlier observations, we found that in both species, fat individuals were more likely to depart than lean individuals, and fat northern wheatears departed earlier within the night than lean conspecifics. Northern wheatears additionally departed earlier in spring with more southerly winds.We found no support for the idea that stopovers departure decisions are influenced by parameters of the oxidative balance. We discuss possible reasons for this unexpected finding. In most avian migrants, the temporal schedule and associated speed of migration is strongly dependent on the number and duration of stopovers , 49. StoAnalogous to fuel accumulation, one could argue that recovery of the oxidative balance is a function of stopover; where fuel stores decrease while flying and increase during stopover, oxidative damage increases while flying and decreases during stopover. What is missing to complete the argument are studies showing that, comparable to migrants’ fuel stores, also the amount of oxidative damage affects migrants’ stopover departure decisions. A similar argument could be constructed for migrants’ antioxidant defences, however, the dynamics of antioxidant capacity during the flight-stopover cycle are not as clear as for oxidative damage; it is unknown whether antioxidant capacity changes during flight and ambiguous if or how it changes during stopover (see above).Oenanthe oenanthe, wheatears hereafter), and a nocturnal medium-distance migrant, the song thrush (Turdus philomelos). Departures from stopover were determined with a fully automated radio-telemetry system constructed at our small island study site. MDA is a frequently used biomarker of current and rapid oxidative damage to lipids. Therefore, in the context of short stopovers it is more informative and reliable as an indicator of ROS exposure and antioxidant efficiency than, for example, DNA damages which require long-term exposure to ROS before accumulation can be detected. Also, MDA is a secondary product of peroxidation of polyunsaturated fatty acids ) Stopovers allow migrating birds to accumulate fuel in the form of lipids (fat), and are thought to also allow birds to recover physiologically. One specific type of recovery at stopover is the reduction of oxidative lipid damage, which has been shown to occur in multiple bird species. Perhaps, therefore, recovery of the oxidative state is a function of stopover. In two species of migrating birds, we found that stopover departure decisions were associated with the birds’ lipid stores, but not with the amount of oxidative lipid damage. The latter result provides no further support for the idea that migrating birds make stopovers to improve their oxidative state. That we generally failed to observe an association between stopover departure decision making and migrants’ oxidative state may be explained by an over-ruling effect of the birds’ fuel stores on stopover departure decisions.


This manuscript is the product of the authors’ discussions, literature overview, and consultation with experts in the field, and identifies important gaps in the evidence base for substance use disorder (SUD) treatment effectiveness within criminal justice (CJ) settings. Lacking from the extant literature are longitudinal investigations of treatment related outcomes during and after incarceration. Such studies could provide rich contextual data about treatment delivery and effectiveness across the CJ continuum, and would provide important insight into individual characteristics  as well as institutional and environmental factors . We also identified the importance of reproducibility within CJ research, and the unfortunate reality of too many single studies conducted in single (or relatively few) correctional facilities. Some of this has been because the studies designed to produce that evidence are not prioritized for funding, which has continually placed researchers in a position where we cannot make firm conclusions or recommendations based on available evidence. The importance of replicating the foundational studies in this field cannot be overstated. We hope this article spurs other researchers to join in the healthy process of questioning the existing state of the CJ-based SUD treatment research, what should be re-examined, and how we can lay a stronger foundation for the future. Individuals involved in the criminal justice (CJ) system are disproportionately impacted by substance use, with estimates of substance use dependence or abuse among correctional populations ranging from 58 to 63% for individuals incarcerated in prisons and jails, respectively . ImproviDespite the known substantial need for SUD treatment among individuals who are involved in the justice system, there is a dearth of scientific evidence regarding effective treatments for this population. While researchers have outlined the key components of corrections-based treatment  than those in jail. Individuals in county jails are often incarcerated for relatively short periods of time, often weeks to months but sometimes only hours or days , which makes SUD treatment assessment, planning, and delivery challenging. Understanding which specific treatment modalities are most appropriate for specific correctional contexts is a critical gap in our collective knowledge about SUD treatment effectiveness among CJ populations. Furthermore, organizational climate can facilitate or inhibit adoption and implementation of evidence-based SUD treatment services in correctional settings . This haIn this paper, we outline key areas in which sizable knowledge gaps exist with respect to SUD treatment effectiveness in correctional settings, including: how much treatment (dosage) is sufficient to achieve positive gains (enough); to what extent co-occurring disorders should be addressed; what are the appropriate goals of treatment; whether or not mandating treatment to those who don’t want it is worthwhile; and how evidence-based practice can be better represented through executive/legislative policy or action. We recognize the inherent challenges in conducting health services research within CJ settings . HoweverThis manuscript was an outgrowth of an ongoing effort by ASAM to revise current SUD treatment guidelines for CJ settings. The first author (NZ) is a co-Chair of the committee convened by ASAM to revise these guidelines. The other coauthors are affiliated with the NIDA funded Justice Community Opioid Innovation Network (JCOIN), a national network of researchers and CJ and SUD practitioners working on implementation of medications to treat opioid use disorder (MOUD) in CJ settings. The ASAM committee identified a dearth of literature regarding evidence-based SUD treatment outcomes in CJ settings as a primary challenge for revising current guidelines for SUD treatment in CJ settings. We worked independently of the ASAM committee to highlight important areas where further research is needed. For each area discussed below, we conducted targeted literature searches in each area. We completed a review of EBSCOhost , and Pub Med in our targeted searches to identify examples in the extant literature associated with each area. We then met as a group to review the state of knowledge and to reach consensus regarding the evidence-base, or lack thereof, associated with each area.The prominent tools used to assess service needs among those in correctional settings—Level of Service Inventory-Revised (LSI-R), Correctional Offender Management Profiling for Alternative Sanctions (COMPAS), and Ohio Risk Assessment System (ORAS) which are typically criminal legal system risk and need assessment tools—can be costly  and time consuming. Costly, burdensome assessments may actually pose a barrier to treatment. A study examining service-need determinations based on scores from the LSI-R and COMPAS compared to results of four yes/no questions regarding need for education, vocational assistance, substance use treatment, and housing showed that the single-item indicators identified 70–90% of those flagged as having high need for these services on the LSI-R and COMPAS instruments . The latThe literature has long relied on the positive correlation between treatment retention and reductions in substance use, dating to the cohort studies of Drug Abuse Treatment Outcomes (DATOS), which found that those individuals who were in treatment 90-days or more tended to have reduced drug use and re-arrest relative to those with shorter treatment durations , 74. DATprescribed treatment duration. That study, conducted by Rawson et al. [We are only aware of one randomized controlled trial assessing n et al. , was conn et al. . The abon et al.  are moreThe requirement (for methadone) or recommendation  of counseling seems to be an intuitive complement to MOUD alone. It is worth noting that there is significant variation in counseling services for SUD and the legal requirements do not specify the nature, type, or length of services. As with the prior section describing treatment dose–response relationships, it is unclear the extent to which the nature and duration of counseling would provide additional benefit, especially among incarcerated populations. It is therefore unsurprising that the evidence that counseling improves outcomes over and beyond MOUD is mixed, with the strongest support derived from post-hoc analyses. In an experimental comparison (of a non-CJ sample), Ling and colleagues  found noThe majority of people who are incarcerated have had lifetime exposures to trauma, including the experience of being incarcerated itself , with trIn particular, while there is increasing understanding of the importance of integrating trauma-informed care (TIC) into carceral treatment and settings , we knowIt may be common sense that any clinical intervention or policy initiative should have clearly stated goals, but goals of SUD treatment vary across—and even within—criminal justice agencies. In this section, we pose several unanswered questions that merit further debate and study.Health Affairs Forefront that “the magnitude of the drug overdose crisis demands out-of-the-box thinking and willingness to jettison old, unhelpful, and unsupported assumptions about what treatment and recovery need to look like. Among them is the traditional view that abstinence is the sole aim and only valid outcome of addiction treatment.” suggesting that we should look at reduced drug use, use of alcohol, or other indicators of changes in use patterns.In 2002, addiction researcher Thomas McLellan  publisheby half relative to probation as usual . A receual , the exams  the benefits of coercion. These presumptions are unlikely to be challenged when a facility struggles to find enough participants to fill its designated SUD treatment beds (or when cost of screening/assessment is a prohibitive barrier). Consider, in contrast, a facility with limited SUD treatment slots. Can we justify filling beds with those deemed to have a substance use problem (regardless of their desire to participate) even if it means excluding others who specifically requested it? Evidence would suggest not. Parhar and colleagues  reviewedWhat we do know about the effectiveness of SUD treatment for coerced participants poses an important ethical question: Does the existing evidence for psychosocial treatment justify overriding individual choice in whether to participate? After conducting a systematic review of compulsory treatment evaluations in several countries, Werb and colleagues  found this evidence-based into formal policies and practices implemented through executive/legislative policy or action. Policymaking and policy implementation often are slow and incremental, with significant gaps between initial formulation and actual implementation. Advancement of MOUDs in correctional settings is one example, though the gap between what national leaders in the field recommend and policies actually put into place is significant, though improving somewhat in the past 1–2 years. For example, a recent study surveyed state prisons in states with disproportionate opioid overdose mortality and found that while all prison systems within the states surveyed reported offering at least one medication to treat OUD, only 7% of the 538 individual prisons within the states offered all three medication types ; 61% of the 538 prisons did not offer any type of MOUD [While this paper speaks to additional research and knowledge gaps relative to SUD treatment in correctional systems, another shortcoming of the current legal-health systems rests in the lack of integration of what  of MOUD . A recen of MOUD . Other mOrganizations as diverse as the National Institute on Drug Abuse, American Society of Addiction Medicine, American Medical Association, National Governors Association, and National Commission on Correctional Health Care (NCCHC) have endorsed MOUD treatment as a best practice. The Legislative Analysis and Public Policy Association even developed model legislation for states and localities to use as a foundation for introduction of legislation to provide MOUD in correctional settings  through Some programs that do offer MOUD suffer operationally due to lack of trained providers, misunderstanding or lack of information within correctional institutions, and other factors , 77. EveAnother step toward expanding best practice-based OUD treatment in correctional facilities is being pursued on the legal front. The U.S. Department of Justice has challenged existing MOUD policy in correctional facilities, citing potential violations of the Americans with Disabilities Act (ADA) . BecauseWhile the pace of adoption has long been lagging, coordinated efforts advancing evidence-based practices, at least around MOUD treatment in correctional facilities, are producing results. Since the beginning of 2020, legislation in at least a dozen states has established or expanded access to MOUD, and most states now have at least one correctional facility that provides some form of MOUD . AdditioA few states, Massachusetts and Maryland notably, have recently passed legislation that incorporates evidence-based best practices for OUD treatment , correctional facilities. A benchmark of science is the extent to which research findings are reproducible and transportable. Most of us are familiar with publication bias, the phenomenon that studies with null findings are less likely to be published in the scientific literature . Howeveren lower .It will be important to take the lessons learned around adoption and implementation of MOUD in correctional settings and apply these to other treatment modalities. However, policy makers have another important role in this space, namely to promote SUD related research within correctional institutions. Often correctional facilities put into place significant barriers to conducting research in their facilities, including banning researchers or withholding important data around current policies, practices and procedures . PolicymWe conclude with a set of study topics that, if rigorously conducted, would substantially advance our understanding of how best to deliver SUD treatment to patients in prison and jail settings. We are aware that some of these topics have been studied before, but they are revisited here because the existing body of literature still lacks the necessary rigor, consistency in findings, and sufficient studies to provide a solid empirical foundation to guide clinical practice or policy decisions in this critical public health domain.As mentioned above, more research is needed regarding SUD treatment dose response. This should be assessed in correctional settings using community-based standards for appropriate treatment duration through randomized or prospective studies rather than correlational observation studies. For treatment modalities that include pharmacotherapy, such as MOUD, limited evidence exists with respect to ancillary psycho-education or counseling components that can accompany pharmacotherapy to optimize treatment effectiveness. Furthermore, we have highlighted in this article that most individuals involved in the criminal justice system have complex co-occurring disorders, so more research is needed in order to understand which community-based evidence-based practices for COD are feasible and effective within carceral settings. Relatedly, research on how treatment modalities can be adapted or modified to integrate a more trauma-informed approach are needed in order to address the significant burden of trauma that many CJ-involved individuals endorse. Finally, given that 60% of incarcerated adults are people of color  and thisWe also strongly recommend a rethinking of currently accepted goals for treatment outcomes for correctional populations. For example, the primary outcome many correctional institutions prioritize for criminal justice involved individuals with SUD is recidivism. To date, findings on the impact of SUD treatment on recidivism are equivocal. This begs the question as to whether or not recidivism is the most appropriate SUD treatment outcome among CJ-involved populations. As the National Academy of Sciences recent report on recidivism and success measures, other measures of functionality and well-being are worthy outcomes . Future There has been considerable debate on whether or not to mandate SUD treatment for CJ populations. However, “coerced” status is often inferred from one’s CJ status rather than directly measured through individual interviews. More research is needed that: (1) more accurately measures perceived coercion among individuals with SUD in CJ settings; and (2) whether the outcomes of SUD treatment justify overriding personal choice and, in some cases, displacing individuals with SUD who seek treatment voluntarily. Increasingly, CJ-based services, including SUD treatment, are informed by algorithms that incorporate specific individual attributes, including demographics, criminal and substance use history, to predict needs and likely treatment outcomes . HoweverIn conclusion, our discussions, literature overview, and consultation with experts in the field have identified clear and important gaps in the current evidence base with respect to treatment effectiveness within CJ settings. While there are important clinical considerations during treatment delivery within prisons and jails , it is difficult to make firm clinical recommendations without sufficient effectiveness research. One fundamental aspect that is lacking from the extant literature is a natural history of treatment related outcomes during incarceration. Such a longitudinal investigation could enroll participants prior to incarceration and conduct follow-up both during and upon release from incarceration, to provide rich contextual data about treatment delivery and effectiveness across the CJ continuum. This kind of research would provide important insight into individual characteristics  as well as institutional and environmental factors .As we stated at the beginning of this paper, the issues raised (and accompanying research priorities proposed) are undoubtedly incomplete. But is it our hope that others will join the healthy process of questioning the existing state of the CJ-based SUD treatment research, what should be re-examined, and how we can lay a stronger foundation for the future.


Sepsis is infection sufficient to cause illness in the infected host, and more severe forms of sepsis can result in organ malfunction or death. Severe forms of Coronavirus disease-2019 (COVID-19), or disease following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are examples of sepsis. Following infection, sepsis is thought to result from excessive inflammation generated in the infected host, also referred to as a cytokine storm. Sepsis can result in organ malfunction or death. Since COVID-19 is an example of sepsis, the hyperinflammation concept has influenced scientific investigation and treatment approaches to COVID-19. However, decades of laboratory study and more than 100 clinical trials designed to quell inflammation have failed to reduce sepsis mortality. We examine theoretical support underlying widespread belief that hyperinflammation or cytokine storm causes sepsis. Our analysis shows substantial weakness of the hyperinflammation approach to sepsis that includes conceptual confusion and failure to establish a cause-and-effect relationship between hyperinflammation and sepsis. We conclude that anti-inflammation approaches to sepsis therapy have little chance of future success. Therefore, anti-inflammation approaches to treat COVID-19 are likewise at high risk for failure. We find persistence of the cytokine storm concept in sepsis perplexing. Although treatment approaches based on the hyperinflammation concept of pathogenesis have failed, the concept has shown remarkable resilience and appears to be unfalsifiable. An approach to understanding this resilience is to consider the hyperinflammation or cytokine storm concept an example of a scientific paradigm. Thomas Kuhn developed the idea that paradigms generate rules of investigation that both shape and restrict scientific progress. Intrinsic features of scientific paradigms include resistance to falsification in the face of contradictory data and inability of experimentation to generate alternatives to a failing paradigm. We call for rejection of the concept that hyperinflammation or cytokine storm causes sepsis. Using the hyperinflammation or cytokine storm paradigm to guide COVID-19 treatments is likewise unlikely to provide progress. Resources should be redirected to more promising avenues of investigation and treatment. Sepsis refers to disease caused by infection that can manifest a spectrum of illness ranging from influenza-like discomfort to organ malfunction or death. The standard of care to treat sepsis includes controlling the infectious source, rapid administration of antimicrobial drugs, and supportive measures like volume infusion, oxygen and ventilatory support and use of vasopressors to augment blood pressure . HoweverThis theoretical report brings to light underappreciated problems with the hyperinflammation concept. According to this concept, pro-inflammatory cytokines are produced by host cells after recognizing microbial components and pro-inflammatory cytokines enter the circulation and spread inflammation systemically. Unregulated excessive production of these cytokines is assumed to cause sepsis as an undesired consequence of inflammation run amok. On close inspection, however, we uncover defects with this concept. Two defects we call attention to are conceptual confusion and failure to establish a cause-and-effect relationship between cytokine storm and sepsis. Conceptual confusion refers to a lack of precise understanding of concepts including inflammation, cytokine storm, and sepsis. These ideas are too vague to be used as optimal guides for research or treatment. Lack of clear understanding what these concepts refer to creates challenges in linking investigation and clinical study to sepsis. For example, no consensus defines which cytokines are pro-inflammatory and this complicates interpretation of studies associating specific cytokines with sepsis. These associations may represent true causes of sepsis, non-causal effects of sepsis, or unrelated chance associations with sepsis. A specific understanding of which cytokines are in fact pro-inflammatory would assist interpretation of studies associating specific cytokines with sepsis. Similarly, lack of adequate concepts of inflammation and cytokine storm complicate understanding of sepsis since sepsis pathogenesis depends on these background concepts. An overview of the concepts inflammation, cytokine storm, and sepsis indicates confusion that we believe weakens the relevance of research results to sepsis. We also assessed evidence supporting a cause-effect relationship between cytokine storm and sepsis. Not only is rationale supporting existence of this relationship weak, but available data suggest such a relationship cannot exist.Finally, we are struck by resistance of the cytokine storm concept of sepsis to yield to falsifying clinical observation. There has been repeated and near exceptionless failure of studies designed to suppress hyperinflammation to treat sepsis. This record has generated little introspection about the viability of the hyperinflammation concept. Cytokine storm appears to be unfalsifiable. Substantial efforts to understand and treat COVID-19 as a manifestation of cytokine storm underscores continued dominance of this concept and establishes it is alive and flourishing in 2022. We believe the remarkable resilience of cytokine storm needs explanation, and we attempted to understand reasons for persistence. The strategy we chose was to consider the hyperinflammation concept of sepsis as a scientific paradigm as described by We believe the concept that hyperinflammation is the cause of sepsis is the dominant account of this disease. We therefore assessed pivotal notions associated with this account.th century Virchow added loss of function or functio laesa to this list. While much factual knowledge was accrued regarding clinical (bedside), histological, and molecular components of the inflammatory response, there remains no established unifying concept relating these components. No concise definition of inflammation has been established , tumor (swelling), calor (heat), and dolor (pain). This description is attributed to Celsus in the first century AD. In the 19ablished . In addiablished . Sepsis Confusion about inflammation has been noted and reviewed by This is a nebulous term with an uncertain definition. Use of “cytokine storm” dates to at least 1993 in a description of graft vs host disease . A recenSepsis describes significant clinical disease following infection of any kind. The global significance of this disease is underscored by a report revealing 48.8 million sepsis cases and 11.0 million sepsis deaths that accounted for 18.7% of global deaths in 2017 . In 2017This is an analogy invoked to describe the role of inflammation in host-pathogen interaction . ProperlThe concept host inflammation causes organ damage or death following infection appears to date to two discoveries . One wasTNF does not directly damage tissues, but TNF initiates biological activities that can culminate in tissue damage. Proposed mechanisms invoked to link TNF excess to toxicity in sepsis include polymorphonuclear neutrophil (PMN) chemotaxis directed into tissues . Tumor nTo evaluate the claim hyperinflammation or cytokine storm causes sepsis, we focus on the strength of evidence for TNF as cause for five reasons. First, we believe sound theoretical reasons support TNF as a pivotal cause of inflammation (manuscript in preparation). Tumor necrosis factor has been labeled the “master regulator” of inflammatory cytokine production , and refhttps://www.medrxiv.org/content/10.1101/2021.12.13.21267720v1.full.pdf. We found healthy persons have approximately 5.5 pg/ml TNF in the circulation, and in sepsis patients TNF concentration increases to approximately 60 pg/ml. Therefore, the case for a necessary increase in cytokines in sepsis is reasonable with levels approximately 10-fold elevated compared to healthy persons. This criterion of causality establishes a consistent relationship between sepsis and increased pro-inflammatory cytokines.Evidence that TNF is necessary for sepsis amounts to showing TNF is elevated in sepsis patients compared to healthy controls. Remarkably, no report comprehensively quantifies circulating concentrations of TNF in healthy persons and in patients with sepsis. Therefore, we conducted a systematic review and meta-analysis that addressees this lacuna in the literature and submitted our manuscript that is currently being subjected to peer review. This manuscript is based on a prospectively published research plan , and theTo show TNF is sufficient for sepsis, TNF should be capable of producing sepsis. The best available data derive from studies reporting injecting humans with TNF and observing for sepsis manifestations. Recombinant TNF has been injected intravenously into volunteers, usually as attempts to treat cancer . In geneet al emphasized and expanded on this observation in 2017: “Over the last 3 decades, more than 100 phase 2 and phase 3 clinical trials have been performed testing various novel pharmacologic agents and therapeutic interventions in an attempt to improve the outcome of patients with severe sepsis and septic shock; all of these efforts ultimately failed to produce a novel pharmacologic agent that improved the outcome of sepsis” . The CTAP dashboard lists types of treatments under consideration with “safe to proceed investigational new drugs”. As of 10 March 2022, CTAP listed (excluding vaccines) more than 120 immunomodulator (mostly anti-inflammation) therapies, followed by “other” treatments with more than 110, neutralizing antibodies or cell and gene therapies with over 60 each, and antiviral approaches with more than 50 entries. This snapshot reflects the degree to which inflammation suppressing approaches influence research priorities in developing COVID-19 therapies. Anti-inflammation drugs to treat COVID-19 were investigated early during the pandemic. Following rapid conduct of clinical studies to treat COVID-19, accepted cytokine-suppressing treatments that reportedly lower mortality include drugs that block IL-6 biological activity and the immunosuppressive corticosteroid dexamethasone  as a treatment for patients with severe sepsis or septic shock  refers to the tendency for history to repeat itself. Since COVID-19 is an example of sepsis , hyperinflammation as the cause of disease was therefore implied . The Ranic shock . This stic shock . It is aet al invoke Kuhn’s thoughts on scientific progress to describe the course of anti-inflammation research in sepsis  all costs. A quote from Structure of Scientific revolutions is telling in this regard where Kuhn writes “it is a poor workman who blames his tools for a bad outcome” . Kuhn reMechanisms that explain maintenance of the sepsis cytokine storm paradigm likely include two characteristics of paradigms. First, Kuhn’s emphasis on the role of a social structure that accompany paradigms is of special importance. Institutions enable and promote modern bioscience and they are heavily biased for care and maintenance of the current paradigm. Most scientists and physicians are educated to understand sepsis as a manifestation of cytokine storm and textbooks actively maintain the cytokine storm concept by depicting exemplars that encapsulate the contemporary approach to solving problems in infectious diseases. Journals favor manuscripts that offer experimental results that favor the hyperinflammation concept, and even lay press publications largely support the concept. Most importantly, funding agencies are populated with proponents of cytokine storm since adherence to this concept defines expertise in this field. A successful career in the modern era of bioscience requires large sums of funding to conduct research. Millions of US dollars are required to support even modest careers. Given these considerations, outlier scientists skeptical of cytokine storm face substantial difficulties obtaining resources with which to pursue such a controversial line of investigation. In fact, it is doubtful adherents to the current paradigm would view the work of anyone challenging that paradigm as conducting legitimate research. Funding agencies are unlikely to expend limited resources to support “speculative” experiments with a perceived small possibility of scientific return. Committing one’s career to being an outlier or “lone wolf” presents high risk for failure and such individuals are likely to receive little institutional, emotional, or financial support during career development. Second, conceptual confusion in the sepsis field is characterized by imprecision and weak definitions . This caSummarizing the points above, at least two characteristics of the cytokine storm paradigm foster persistence of this concept despite inability to deliver clinical treatment. First, social institutions formed around the cytokine storm paradigm direct the flow of many elements of science into this paradigm. A sepsis investigator encounters an educational history, funding agencies, and publishing organizations that channels the conduct of science to support the existing paradigm. Second, conceptual confusions in the sepsis field provide escape mechanisms that stymie falsification. Contradictory results are explained by failure of experimental design or conduct instead of paradigm failure. According to Kuhn, during normal science one possibility not to be considered is that investigation is conducted under direction of a faulty paradigm. Kuhn’s model of scientific progress appears to provide an understanding of how the current sepsis paradigm has survived despite apparent pressures to be discarded.Elements of theory within a paradigm establishes the menu of tools available for experimentation. This is a component of the theory-laden or theory-infection quality of experimentation . Since wIf further experimentation is unlikely to provide meaningful advancement in sepsis therapeutics, we need to entertain alternative approaches. When applied to Kuhn’ model, the cytokine storm sepsis paradigm is in a state of normal science crisis. According to Kuhn, a scientific crisis is characterized by accumulation of anomalies that cannot be accommodated by an existing paradigm. Over time, accumulation of contradicting data can reach a point where the paradigm fails to function as a driver of scientific advancement and fails to solve puzzles. Normal science anomalies include contradictory observations  and an iIf we accept the verdict that the hyperinflammation sepsis paradigm is in crisis, resolution of the crisis will likely require a “paradigm shift.” Two impediments pose challenges for altering the current paradigm. The first impediment is Kuhn’s observation that the presence of crisis in science does not entail a search for paradigm alteration. In fact, shedding an existing paradigm is often the solution of last resort if it is considered at all. History shows anomalies or contradicting observations can be tolerated for prolonged periods of time and mechanisms are generated within the existing paradigm to explain anomalies .As pointed out above, examination of commentaries acknowledging the presence of anomalies or failure of sepsis research to produce clinical benefit confirms this. The response to anomalies has been to invoke numerous suggestions for tweaking or rehabilitating the existing hyperinflammation sepsis paradigm. However, there has been no call to abandon the hyperinflammation or cytokine storm concept . This coStandard of care for patients with sepsis includes rapid administration of antimicrobial drugs and supportive measures. However, despite advances in these areas, persistent substantial mortality is observed in sepsis. For this reason, attention has focused on adjunctive therapies targeting the pathogen-triggered host inflammatory response to infection. This approach follows a line of reasoning that posits hyperinflammation as the final common cause of organ malfunction or death. The COVID-19 pandemic has thrust hyperinflammation or cytokine storm to the forefront of discussion by scientific investigators, clinical trialists, and the lay public. Associating COVID-19 with cytokine storm was expected since severe COVID-19 is an example of sepsis, or infection sufficient to cause organ malfunction or death. Since sepsis is thought to be caused by hyperinflammation, a link between COVID-19 and hyperinflammation was forged. A substantial research tradition identifies pro-inflammatory cytokines as pivotal mediators of inflammation and are thus implicated as potentially reversible causes of host inflammation in response to infection. This accounts for use of the term “cytokine storm” to characterize sepsis hyperinflammation. Unfortunately, despite expenditure of ample resources to materially affect sepsis mortality by blocking inflammation, we have been unable to affect sepsis mortality using this concept. There is no indication we are on the verge of reversing this trend.We believe the hyperinflammation or cytokine storm approach to sepsis is an imperiled concept. In this report we identify three elements that we believe are at the heart of difficulties with this concept. The three elements include 1) confusion and imprecision in descriptions of the hyperinflammation concept, 2) weak evidence supporting the idea hyperinflammation is the cause of sepsis, and 3) persistence of the hyperinflammation concept despite inability of therapies based on this concept to treat sepsis. First, we analyzed concepts that underly the hyperinflammation approach of sepsis. We found a surprising degree of conceptual confusion in pivotal ideas. The terms “inflammation” and “cytokine storm” are vague and imprecise. Considering inflammation, no explanation or definition precisely characterizes the systemic inflammation that presumedly causes sepsis. In fact, contemporary understanding of inflammation continues to refer to ideas present since antiquity. Prior sepsis definitions emphasized fever or hypothermia, tachypnea, tachycardia, and leukocytosis or leukopenia. How these patient data relate to inflammation is unclear. Inflammation manifests differently in separate individuals, and it can present in different ways in the same individual. An adequate account of inflammation must derive from an understanding of regularities present in all manifestations of inflammation. We believe such an account of inflammation must originate from a theory-driven characterization that directs selection of clinical or laboratory indicators that can identify and quantify inflammation. Relatedly, the term “cytokine storm” is vague and imprecise. We assume cytokine storm refers to excessive production of molecules that cause overexuberant inflammation and therefore sepsis. However, there is no consensus on which cytokines are relevant for production of inflammation. Moreover, there is no concept of what cytokine concentrations constitutes an excessive amount. Conceptual confusion has adverse scientific and clinical consequences. Given suppression of inflammation as a goal of sepsis therapy, we need to identify the right molecular targets to suppress. We believe progress in sepsis requires re-thinking concepts of inflammation and cytokine storm. Therefore, we have developed a novel conception of inflammation that attempts to demystify these concepts (manuscript in preparation).Second, we analyzed the status of evidence supporting a cause-effect relationship between hyperinflammation and sepsis. Using criteria of necessity, sufficiency, and a pivotal interventionist criterion for causality, hyperinflammation or cytokine storm fails to satisfy criteria as a cause of sepsis.Third, we have been struck by remarkable resilience of the hyperinflammation concept to contradicting clinical assessment. The concept persists with apparent indifference to a remarkable record of repeated inability to deliver clinical success . It appeKuhn refers to normal science as science conducted under the direction of a monpolistic paradigm. The paradigm is not questioned, and anomalous or contradictory observations are explained by errors of experimental design or experimental conduct. The underlying paradigm remains immune to disconfirmation and is insulated from experimental assessment. The Kuhn account of scientific progress anticipates problems that can originate from a faulty paradigm. The lack of precision or conceptual vagueness in the cytokine storm paradigm enables contradicting observations to be interpreted as consistent with hyperinflammation and cannot be used as falsifying examples. Failed clinical sepsis trails are nearly always viewed as failures of experimental design or conduct and are not used as reasons to challenge the underlying paradigm . This ac


To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through Although usually recognized as a kind of ‘fish’ like actinopterygian fishes, cartilaginous fishes (chondrichthyans) form a distinct class of vertebrates with more than 1,200 species, known mostly as sharks and rays . Mainly Callorhinchus milii .The sequencing strategy in the Squalomix Consortium is designed to accommodate genomic characteristics of cartilaginous fishes, mostly with large, repetitive genomes. In the standard protocol formulated in January 2021 , we star akajei  for whicThe Squalomix Consortium aims not only to sequence and analyze the genomes but also to tightly interact with other research groups whose target species list contains cartilaginous fishes including other EBP-affiliated projects (see below). To maximize mutual benefit among those projects, some animal samples from our collection could be provided for genome sequencing at other sites. The Squalomix Consortium offers laboratory experiments for genome size quantification or karyotype analysis for species listed by other consortia, provided that fresh cells are available. The sample transfer will be processed in accordance with the Nagoya Protocol and other relevant regulations. Inclusive cooperation respecting complementary expertise is expected to overcome the long-standing difficulty in studying elasmobranchs sustainably and contribute to disentangling the marine ecosystems for effective conservation.https://transcriptome.riken.jp/squalomix/). This archive also has a link to the up-to-date listing of the species for which genome sequences are available, filed by the GenomeSync database (http://genomesync.org/). The archive website also hosts a gateway to genome browsers powered by JBrowse2 that allow users to visualize specific genomic regions and load additional tracks including base composition, gene models, repetitive elements, and aligned RNA-seq reads . This includes the Vertebrate Genomes Project (VGP), whose data production format employs a suite of modern promising solutions including optical mapping and Hi-C scaffolding as well as long-read and short-read sequencing, to cover all vertebrate species . The Darwin Tree of Life (DToL) Project partly links with VGP and aims to sequence all eukaryotic species in Britain and Ireland. DToL’s first chondrichthyan genome is that of the small-spotted catsharkScyliorhinus canicula, the egg-laying species most widely studied in developmental biology and endocrinology . The recently launched European Reference Genome Atlas (ERGA) also plans to produce reference chromosome anchored genomes of multiple species from this geography including cartilaginous fish aiming to empower conservation efforts .Products from this consortium are deposited in NCBI under the BioProject ID PRJNA707598 and are available at our Squalomix sequence archive . However, this is due to the imprecise use of the term "class" in vertebrate taxonomy where both Chondrichthyes and Mammalia are designated as classes. Thus, classes are not of equal temporal rank. Furthermore, the authors' statement is not quite true, because even Agnatha has the taxonomic rank as a vertebrate class and would thereby be even earlier than Chondrichthyes. I would recommend the authors to describe Chondrichthyes instead as one of the two lineages resulting from the first bifurcation or divergence in (the infraphylum of) Gnathostomata (jawed vertebrates). This, by the way, means that Osteichthyes is as old as Chondrichthyes! Please correct the grammar of the expression "Despite these outstanding evolutionary and biological importance…". It probably needs to be rephrased, perhaps like this: "Despite the outstanding evolutionary and biological importance of chondrichthyans…" (or elasmobranchs if you prefer to focus on these). Shouldn't "giga basepairs" be one word as one would surely write megabasepairs and kilobasepairs. Figure 1: The expression "Other osteichthyans" is imprecies. I assume the authors want to avoid the term "Sarcopterygians" for this group pterygi means fins, and tetrapods of course don't have them. Maybe it's better to just delete "Other osteichthyans" and let this branch be called "Tetrapods, lungfishes and coelacanths) .Are sufficient details of methods and materials provided to allow replication by others?YesIs the rationale for creating the dataset(s) clearly described?YesAre the datasets clearly presented in a useable and accessible format?YesAre the protocols appropriate and is the work technically sound?YesReviewer Expertise:Gene family evolution, pharmacology of G protein-coupled receptors, mechanism of long-term memoryWe confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this Data note, the authors describe and wrap-up all available material generated through their consortium named Squalomix, in which a set of biological material and sequence data obtained in elasmobranch organisms are made available to the research community. The rationale, protocol, material and data availability are clearly described in this Note.Are sufficient details of methods and materials provided to allow replication by others?YesIs the rationale for creating the dataset(s) clearly described?YesAre the datasets clearly presented in a useable and accessible format?YesAre the protocols appropriate and is the work technically sound?YesReviewer Expertise:Developmental genetics, EvoDevoI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The most common non-pharmacological intervention for both peripheral and cerebral vascular health is regular physical activity , which improves function across a range of exercise intensities and modalities. Numerous non-exercising approaches have also been suggested to improved vascular function, including repeated ischemic preconditioning (IPC); heat therapy such as hot water bathing and sauna; and pneumatic compression. Chronic adaptive responses have been observed across a number of these approaches, yet the precise mechanisms that underlie these effects in humans are not fully understood. Acute increases in blood flow and circulating signalling factors that induce responses in endothelial function are likely to be key moderators driving these adaptations. While the impact on circulating factors and environmental mechanisms for adaptation may vary between approaches, in essence, they all centre around acutely elevating blood flow throughout the circulation and stimulating improved endothelium-dependent vascular function and ultimately vascular health. Here, we review our current understanding of the mechanisms driving endothelial adaptation to repeated exposure to elevated blood flow, and the interplay between this response and changes in circulating factors. In addition, we will consider the limitations in our current knowledge base and how these may be best addressed through the selection of more physiologically relevant experimental models and research. Ultimately, improving our understanding of the unique impact that non-pharmacological interventions have on the vasculature will allow us to develop superior strategies to tackle declining vascular function across the lifespan, prevent avoidable vascular-related disease, and alleviate dependency on drug-based interventions. As the highway connecting every organ within the human body, the vascular system plays a unique and central role in maintaining health, while simultaneously holding the potential to disrupt multiple regions when not optimally functioning. As a result, vascular dysfunction plays a central role in disease progressions throughout the body, including in cardiovascular disease, stroke, and dementia  within a given vessel and the impact that flow-independent changes in the circulating environment can have on the endothelium. This review will explore the isolated and combined effects of these two adaptive pathways, both within and between intervention approaches.As physical activity plays such a central role in vascular health, it is of little surprise that exercise-based interventions are one of the most commonly utilised non-pharmacological approaches for improving vascular function  facilitates longer term adaptations in vascular function, including improvements in basal brachial artery function as measured by FMD ; characterised by short periods (4–5 min) of peripheral ischemia through inflation of a cuff to supra-systolic pressure, has also been linked to benefits in vascular function  ; via immersion in water; or by a combination of these manipulations. The most common of these approaches are heat-based interventions that aim to induce a significant elevation in core temperature, which have subsequently shown the potential to drive adaptive improvements in endothelial function  intervention periods . However, there is growing interest in examining vascular responses outside of these regions, including within the microvascular of the coronary and cutaneous circulation. While acute flow responses are difficult to determine in humans, increases in the size and vasodilatory capacity of the coronary arteries are seen following exercise training  but not at high intensities (75% of VO2max), while high-intensity exercise alone drove significant increases in markers of oxidative stress. The authors hypothesised that this elevation in oxidative stress may have resulted in an inhibition of vasodilatory mechanisms, which attenuated any adaptive benefits that could have been derived from the elevations in systemic perfusion seen at high intensities. Interestingly, the same effects are not seen in interval-based high-intensity exercise, which have been shown to drive greater vascular adaptive responses than traditional steady-state moderate-intensity exercise  within the occluded limb, leading to the hypothesis that vascular adaptations to interventions may be dependent on transient changes in flow/perfusion   interactions. The most well studied of these pathways is the VE-cadherin–PECAM-1–VEGFR2/3 mechanosensory complex comprising of VE-cadherin, platelet endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial growth factor receptor 2 and 3 (VEGFR2/3), which was first identified by Tzima et al. . In respEvidence also suggests that shear stress can be detected through conformational changes in the extracellular matrix and ECM–cell interactions. However, this is less well understood, in large part due to the high level of complexity and regional specificity  both in terms of acute signalling responses and subsequent adaptive changes. While the mechanosensory pathways are activated under different flow profiles and the nature of the relationship between different sensors is not well understood, clear differences are seen in subsequent signal pathway responses between flow patterns Fig.  Nakajim. In vitr3), of either a constant or pulsatile nature  and NF-κΒ  and extreme (2–8% inspired O2) hypoxic “doses”; including beneficial vs. detrimental impact on blood pressure and systemic inflammation  can have on blood flow (see “Ischemic preconditioning”), environment-based interventions also induce significant changes in the internal environment within blood vessels and the endothelium . In the case of ischemic preconditioning, much of our understanding regarding the impact that changes in temperature or oxygen availability have on the endothelium comes from research focused on pathological conditions. Interestingly, in vitro endothelial cells respond to hypoxia by elevating signalling via NF-κB and HIF-1α  induced locally by the endothelium itself in response to changes in flow; particularly in relation to the release of vasoactive substances and inflammatory cytokines; (2) produced by other tissues and released into the circulation, or (3) induced in an endocrine manner within the endothelium as a positive feedback loop response to circulating signalling factors released from other tissues.In vivo the endothelium is rarely impacted in isolation and more commonly changes in flow are driven alongside changes in both proximal and distal tissues, making the role of circulating signalling factors central to understanding the holistic impact of non-pharmacological interventions. The importance of changes in circulating factors is seen across a range of interventions and is best evidenced in exercise and passive heating, which despite their differences in aetiology , both produce marked elevations in core temperature and blood flow  plays in angiogenesis and vascular function. In vivo human research has demonstrated that both acute and chronic exposure to heat or hypoxia results in similar elevations in extracellular HIF-1α  is drastically altered when cells are preconditioned under arterial levels of shear stress .To improve on our current understanding, interventions should look to mimic the flow profiles seen within a given intervention, utilising the wealth of in vivo data already available on the nature of these responses. As already highlighted (see “Flow-independent adaptation in vivo”), both the magnitude and frequency of pulsatile flow can have an impact on endothelial responses (Zhang and Friedman 2 and 5% CO2, which provides oxygenation levels well in excess of most physiological settings, and “hypoxic” conditions often at the other extreme, reducing oxygen levels closer to anoxic conditions, rather than those which are physiologically relevant to non-pathological conditions (Al-Ani et al. Physiologically relevant models are also essential in the wider context of environment-based interventions, as both baseline and treatment conditions are often not representative of the conditions seen in vivo, particularly within a healthy population. This is demonstrated in models of hypoxia, with normal/ “normoxic” culture conditions generally set at 21% OThe incorporation of samples taken from in vivo experiments when simulating intervention conditions could also improve the physiological relevance of in vitro experiments. Alongside omics-based approaches to better understand the composition of these samples, this approach will also improve our understanding of how changes in the circulating microenvironment may interact with the endothelium, as well as how chronic interventions alter the nature of this microenvironment. This approach was recently taken by Brunt et al. , who demRecent advances in cell culture techniques alongside increased affordability of cutting-edge approaches, such as lab-on-chip systems, are likely to play an essential role in addressing the shortcomings within the current literature, particularly as in vivo research expands beyond large arterial vessels and incorporation of smooth muscle cells. Future research should look to incorporate these advanced culture methods and integrated approaches between in vivo and in vitro research, particularly focussing on the selection of optimal culture conditions and accurate simulation of the magnitude, frequency and duration of intervention responses. This will help to build a more complete picture of the manner in which non-pharmacological interventions interact with the vascular endothelium and ultimately pave the way for the rapid analysis of in vitro endothelial responses in parallel with in vivo research.Non-pharmacological interventions have the capacity to drive significant changes in blood flow and, as a result, induce beneficial adaptations within the endothelium. Our understanding of how the cellular mechanosensory mechanisms of the endothelium respond to flow are relatively well developed and, coupled with research into the impact that the circulating microenvironment has on the endothelium, we can begin to interpret how these mechanisms may be altered during interventions. However, research to date has largely focused on resting and pathological scenarios. Targeting future research to address the lack of studies into intervention responses will help to shed light on the potential benefits that non-pharmacological approaches can have on vascular health.