Nutrition is the aspect closely connected to physical activity and may affect body composition, sports performance and post-workout regeneration. Using an appropriate diet plan is a proven method to optimize performance improvements in combat sports. In the majority of combat sports athletes are classified according to their body mass in order to minimize differences between competitors. Many athletes induce weight loss in order to gain an advantage over their opponents. The review was undertaken to provide safe, evidence-based protocols helping athletes in weight reduction without negative effects on sports performance. The nutritional requirements for combat sports athletes, sports supplements, gradual and rapid weight reduction strategies are discussed in this review. Nutrition as the aspect closely connected to physical activity may affect body composition, sports performance and post-workout regeneration. The nutritional requirements for athletes are higher than for the average population for many nutrients. In combat sports the recommended nutrition and energy intake may further exceed levels for non-training population 2.The primary component to optimize sports performance in combat sports is ensuring an adequate energy intake. The caloric needs for athletes training with moderate or high intensity may approach 40–70 kcals/kg/day depending on the intensity and frequency of trainings. For elite athletes the recommended energy intake may further exceed these levels The primary source of energy for athletes are carbohydrates. Carbohydrates, as energy substrates, can be oxidized via aerobic metabolism (for instance by glycolytic pathway coupled with Krebs cycle and electron transport chain) or converted into lactate via anaerobic metabolism such as anaerobic glycolysis. As a result, adenosine triphosphate is synthesized and the energy is transferred. The ability to maintain exercise is connected with muscle glycogen content 3.The important macronutrient in combat sports is protein. The recommended intake in an athlete's diet is higher in comparison to non-training people in order to ensure regular muscle protein synthesis. Muscle mass loss can have a negative effect on strength, power and performance 4.The recommended level of fat in athletes' diet is 25–30% of energy intake and the most preferable sources of fat are unsaturated fatty acids, particularly omega-3 polyunsaturated fatty acids 5.Water is a crucial ergogenic aid for athletes and an adequate level of hydration is necessary to maintain exercise capacity 6.The influence on strength, endurance and body composition may also have sports supplements. There are many substances which have a proven impact on the effectiveness of training One of the substances which have a proven ergogenic effect in combat sports is caffeine 2) and hydrogen (H+) cumulate in the blood and muscles. Through the bicarbonate system the body rids itself of the hydrogen and CO2 via their conversion to bicarbonate throughout the renal and respiratory system Another ergogenic supplement with evidence-based influence on performance that may be used in combat sports is sodium bicarbonate. Sodium bicarbonate is an alkalizing agent which is able to augment extracellular buffering capacity The ingestion of beta-alanine could also be beneficial in combat sports. It has been proved that using beta-alanine supplements increase intramuscular carnosine levels. Carnosine is a dipeptide prolifically found in skeletal muscle, composed of histidine and beta-alanine. Carnosine is believed to exert an important acid-base regulation function Probably the most effective dietary supplement to increase high-intensity exercise performance in combat sports is creatine. Injecting creatine supplements can increase intramuscular phosphocreatine (PCr), which is an energy buffer protecting the ATP concentration Using supplements such as antioxidants may be beneficial due to alleviating oxidative stress and inflammation induced by training. Exercise induces enhanced production of reactive species leading to modifications of lipids, proteins and nucleic acid, decreased muscular functionality, histological changes and muscular soreness 7.Using supplements can be connected with adverse effects that can affect the health and sports career of the athlete A special group of food supplements are available as preparation for weight loss. The supposed mechanism of weight loss supplements includes an impact on appetite regulation, carbohydrate absorption, fat metabolism and energy expenditure regulation. According to current scientific knowledge, none of the available, legal dietary supplements are supported by high-quality evidence 8.In most combat sports, athletes are classified in weight divisions according to their body mass. Most athletes tend to reduce body weight in order to qualify for a lighter weight division so that they could fight with lighter, smaller and weaker opponents th day both groups were consuming 15 ml/kg of fluids and were following the same rehydration protocols on 5th and 6th days. Measurements of urea and serum electrolytes didn't exceed reference levels in both groups. There were no differences in physical performance between groups The most popular method of acute weight loss is an increase in exercise and restriction of fluid and food intake. To reduce weight, many athletes use dehydration protocol in order to remove excess water from the body The effects of dehydration can be enhanced by using a low sodium diet. It has been proved that a high intake of sodium leads to an increase of fluid retention. Reducing the intake of sodium several days prior to weigh-in may intensify weight loss 9.The appropriate use of nutrition can enhance sports performance, post-workout regeneration and improve body composition in combat sports athletes. The type, as well as the time of macronutrient intake, may enhance muscle protein synthesis, recovery and tissue repair. The primary source of energy for combat sports athletes should be carbohydrates in order to ensure the proper level of muscle glycogen, which is linked with higher ability to continue moderate and high-intensity trainings.The positive effect of a balanced diet can be enhanced by sports supplements. There are some supplements which have a proven influence on strength, endurance and body composition in combat sports such as sodium bicarbonate, caffeine, creatine, beta-alanine or protein supplements.In most combat sports athletes tend to reduce body mass in order to obtain classification in a lighter weight division. The most preferable way to reduce weight is gradual strategy of fat reduction including low energy deficiency. If necessary, another method of reduction that may be used is rapid weight loss. It has to be noticed that acute weight loss shouldn't exceed 5% of body mass. Otherwise, it could lead to serious health problems, for instance cardiovascular and endocrine system problems or even death. Both strategies of weight loss could decrease sports performance so it's necessary to use safe, evidence-based protocols in order to minimize negative health outcomes. In order to lower the health problems and maximise sport potential, each athlete should be placed under a medical team including doctors and nutritionists. Furthermore, it's crucial to educate athletes about proper nutrition and weight loss in combat sports.


Cross-talk between opioid and adrenergic receptors is well-characterized and involves second messenger systems, the formation of receptor heterodimers, and the presence of extracellular allosteric binding regions for the complementary ligand; however, the evolutionary origins of these interactions have not been investigated. We propose that opioid and adrenergic ligands and receptors co-evolved from a common set of modular precursors so that they share binding functions. We demonstrate the plausibility of this hypothesis through a review of experimental evidence for molecularly complementary modules and report unexpected homologies between the two receptor types. Briefly, opioids form homodimers also bind adrenergic compounds; opioids bind to conserved extracellular regions of adrenergic receptors while adrenergic compounds bind to conserved extracellular regions of opioid receptors; opioid-like modules appear in both sets of receptors within key ligand-binding regions. Transmembrane regions associated with homodimerization of each class of receptors are also highly conserved across receptor types and implicated in heterodimerization. This conservation of multiple functional modules suggests opioid–adrenergic ligand and receptor co-evolution and provides mechanisms for explaining the evolution of their crosstalk. These modules also suggest the structure of a primordial receptor, providing clues for engineering receptor functions. Cross-talk between opioid receptors and adrenergic receptors has been well-characterized both in laboratory and clinical studies. Opioid compounds such as morphine and the enkephalins enhance adrenergic receptor activity ,13,14,15While it is possible, but extremely unlikely, that adrenergic and opioid receptors had separate origins and then underwent convergent evolution that increasingly integrated their functions, it is difficult to conceive of random evolutionary processes and selection mechanisms that would not only permit the receptors themselves to form functionally integrated heterodimers but also lead to each receptor evolving allosteric binding sites for the other’s ligand. We propose that a simpler solution to these puzzles is that the adrenergic and opioid receptors co-evolved from a common evolutionary origin. Structure and function were integrated from the outset, and receptor specificity evolved as a secondary result. However, rather than proposing a typical evolutionary tree-like process in which the receptors evolved from a common ancestor by means of selection on an accumulation of single nucleotide polymorphisms, we propose that the evolution of the opioid and adrenergic receptors occurred through the swapping and rearranging of a shared set of common modules that were selected for their molecular complementarity. Typical evolution through the accumulation of polymorphisms then modified and honed the resulting structures producing the variety of opioid and adrenergic receptors that now characterize vertebrate organisms but retained their shared modular basis.In order to make our argument, we have structured our paper in an unusual form that does not follow the typical introduction, methods, results, and discussion format. Instead, we combine four elements in a synthetic manner, introducing review material and methods where appropriate to the development and testing of our hypothesis. The first element is a general theory of receptor–ligand co-evolution based on modular complementarity and its specific implications for understanding adrenergic and opioid ligand and receptor cross-talk. The second is a review of existing evidence concerning modular complementarity as it relates to adrenergic and opioid ligand co-evolution. The third element consists of novel tests of modular complementarity as it applies to co-evolution and crosstalk in adrenergic and opioid receptors. The final element consists of a series of experimentally testable, logical predictions that follow uniquely from the previous three elements involving the artificial evolution and the engineering of novel receptor–ligand functions. 20 sequences, and a potential receptor protein for that ligand that is 400 amino acids in length could have any of 40020 possible sequences, how could a specific ligand end up “finding” its corresponding receptor within “protein space” and then instantiating the pairing as a viable, genetically encoded set? The problem would appear to become exponentially more difficult when trying to account for the co-evolution of two sets of receptors with two sets of ligands, and still more difficult when the two types of ligands bind to both types of receptors and the receptors are able to bind to each other, altering each other’s functions. The number of permutations that would have to be explored seems to be unreasonably large.One of the most perplexing problems posed by the evolution of any receptor is how it co-evolved with its ligands. This problem, as noted above, is exacerbated when trying to figure out how two different types of receptors co-evolved to be able to form functional heterodimers and becomes even more perplexing when each receptor responds allosterically to the other’s ligands. Given that a peptide ligand that is just ten amino acids in length could have any of 10Some years ago, Dwyer  and this. In thisSubsequently, Root-Bernstein proposed that Dwyer’s theory could be expanded to include hetero-complementary pairs in which two different complementary peptides were involved or in which only one of the two molecules was a peptide. One peptide component of the pair evolved into the receptor, while the other component (the complementary peptide or non-peptide) remained the ligand. This extension permitted Dwyer’s concept to be expanded to explain the evolution of receptors for hetero-complementary peptide pairs and for non-peptide ligands such as the monoamines ,63. The Dwyer has subsequently extended his work to identify shared adhesion binding sites among interleukins and gastrointestinal peptides , while S(1)Since adrenergic compounds modify the effects of opioid compounds and vice versa (see above), adrenergic compounds should be molecularly complementary to opioid compounds, which is to say that adrenergic compounds should bind specifically to opioids.(2)Opioid peptides that bind adrenergic compounds (hetero-complementarity) should provide the basis for the molecularly complementary modules upon which evolution has built receptors and transporters for adrenergic compounds so that adrenergic receptors should have opioid-like modules within their ligand binding regions.(3)Self-aggregation (homo-complementarity) of opioid peptides should provide the basis for the molecularly complementary modules which evolution has built receptors and transporters for opioid compounds.(4)Some of these opioid-like regions of opioid receptors should bind adrenergic compounds (heterocomplementarity) acting as allosteric modifiers of opioid receptor function.(5)Similarly, some of the opioid-like regions of adrenergic receptors should bind opioids (homocomplementarity again) and act as allosteric modulators of the receptor.This paper extends these previous results to the opioid and adrenergic receptor classes within a broader theoretical framework concerning the evolution of interactomes as the basis for integrated physiological systems. Complementary pairings between molecules would have been selected during the origins of life because their complexes protect the components from degradative processes so that they survive longer than the individual components; the complexes may have novel properties, and binding provides a basis for building functional interactions ,70,71. IIn other words, Predictions 1 through 5 state that the molecular complementarity of the ligands became embedded in the receptors through both homo- and hetero-complementarity such that the receptors retain some affinity for both ligands.(6)Opioid and adrenergic receptors will therefore be likely to share common, evolutionarily conserved modules.(7)Because opioid and adrenergic receptors can homodimerize as well as heterodimerize with each other, these conserved modules are likely to include some transmembrane regions of the receptors involved in receptor oligomerization.(8)The sum of these shared modules may help to explain how these receptors have integrated functions that result in mutual enhancement of function and how they manage their cross-talk.Each of these propositions is tested below using combinations of previously published results as well as new data from proteonomic homology methods.Some of the predictions made above have already been experimentally investigated. We consider these results in terms of the hypothesis that adrenergic and opioid receptors and their ligands co-evolved by means of conserved, molecularly complementary modules.Prediction 1.Adrenergic compounds should be molecularly complementary to opioid compounds since each molecular species alters the physiological effects of the other.As noted in the Introduction, combining adrenergic and opioid compounds alters the effects of each on its respective receptor. As predicted, adrenergic compounds do bind to opioids and vice versa. Nuclear magnetic resonance spectroscopy, ultraviolet spectroscopy, and capillary electrophoresis have been employed to demonstrate that compounds such as epinephrine, norepinephrine, amphetamine, albuterol, propranolol, etc., bind directly to morphine, methionine- and leucine-enkephalin and endomorphin  17,75,7,77. ThusPrediction 2.Adrenergic receptors should have opioid-like modules within their ligand binding regions.Since adrenergic compounds bind to opioids, primitive versions of opioid peptides such as pro-enkephalin and the endorphins may have provided the basic modules from which adrenergic receptors evolved. Molecular paleontology therefore predicts that remnants of these opioid-like sequences should be conserved in the binding regions of adrenergic receptors. Indeed, an LALIGN comparison of opioid peptides with adrenergic receptors (methods explained more fully in the Prediction 3.Self-aggregation (homo-complementarity) of opioid peptides should provide the basis for the evolution of opioid receptors.Dwyer’s theory proposes that homo-complementary peptides may evolve into ligand–receptor pairs  so that Enkephalins have been found to self-aggregate into homodimers arranged in an anti-parallel beta ribbon conformation ,81,82,83Additionally, beta endorphin self-aggregates to form amyloid fibrils ,87,88,89Homo sapiens for which the ranges of opioid and adrenergic receptor and ligand sequences were available). These homologies occur within the opioid ligand binding site of the opioid receptors, as will also be demonstrated in Prediction 6 below, and additional homologies are found between opioid receptors and pro-opiomelanocortin and beta endorphin in the extracellular loops. These extracellular loop homologies are significant both in terms of the fact that opioids often interact transiently with these extracellular loops prior to being drawn into their high-affinity binding site that exists in a pocket formed by the transmembrane sequences of the receptor )QTY and nfCXCR4QTY receptors that retain activity. Is the dispensability of TM1 also true for ADR and other aminergic receptors? What additional modules can be eliminated without losing all function? Could even simpler constructs such as an endorphin-like extracellular sequence attached to a single conserved transmembrane sequence with an intracellular “tail” comprise another endorphin-like sequence  , and Qinsequence ? Or are Finally, swapping of modular, complementary regions permits us to begin to understand how evolution rapidly and efficiently evolves interactive sets of proteins so that they function as systems rather than randomly exploring the almost infinite possibilities of protein space and the selecting among the permutations. Selecting and re-using complementary modules in novel combinations assures interactivity and communication within protein systems. Understanding the principles of such complementary modular systems will mean that we will be able to harness these effects through molecular engineering to explore biological systems in new ways and more efficiently invent new biomedical applications.


Recent advances in cell based therapies for lung diseases and critical illnesses offer significant promise. Despite encouraging preclinical results, the translation of efficacy to the clinical settings have not been successful. One of the possible reasons for this is the lack of understanding of the complex interaction between mesenchymal stromal cells (MSCs) and the host environment. Other challenges for MSC cell therapies include cell sources, dosing, disease target, donor variability, and cell product manufacturing. Here we provide an overview on advances and current issues with a focus on MSC-based cell therapies for inflammatory acute respiratory distress syndrome varieties and other inflammatory lung diseases. Despite fundamental discoveries regarding the potential benefits of MSCs in pre-clinical models of lung diseases and other critical illnesses, more effort needs to be made to understand the in vivo fate of infused MSCs, particularly in clinical settings. Importantly, in addition to the MSC cell product, conditioned media and bioactive products such as EVs isolated from MSC cultures have been used in pre-clinical settings with promising results, however, these have been reviewed extensively in other publications (Severe respiratory diseases such as the acute respiratory distress syndrome (ARDS) and other inflammatory lung diseases remain a significant cause of mortality worldwide. For many of these end-stage diseases, there is no cure and current treatments aim to treat symptoms and/or delay disease progression. New treatment strategies are therefore critically needed. Mesenchymal stromal cells (MSCs) have been widely studied in the field of regenerative medicine for applications in the treatment of lung diseases and critical illnesses. MSCs are multipotent progenitor cells with the potential to secrete a spectrum of anti-inflammatory mediators, such as cytokines and extracellular vesicles (EVs). However, due to a lack of basic understanding of the ications  and willications . Here, wDespite the enormous interest in using MSCs in clinical settings to treat respiratory lung diseases, the knowledge regarding the exact mechanism of action is limited. Today, the generally accepted hypothesis is that MSCs modulate the immune system through different paracrine effects (such as cytokines and extracellular vesicles), a hypothesis mainly based on pre-clinical studies [reviewed in ].E. coli-induced acute lung injury, where MSCs isolated from TLR4 deficient mice had impaired survival under conditions of inflammatory stress in vitro, and were not therapeutically active in vivo. Mechanistically, it was shown that TLR4 pathway regulates signaling through PAR1 on MSCs and TLR4 stimulation leads to expression and secretion of prothrombin by MSCs . Following lung injury, MSCs were administered and the outcome was evaluated for each different lung injury model with relevant markers including Ashcroft lung injury score, pulmonary elastance, and lung collagen content  exposed to clinical bronchoalveolar lavage fluid (BALF) samples obtained from patients with ARDS were less effective in promoting an anti-inflammatory monocyte phenotype than hMSCs exposed to BALF obtained from patients with other lung diseases including acute respiratory exacerbations of cystic fibrosis (CF) , which might explain why different pathways were activated and thereby different outcomes were obtained. Another important difference is that the control group to which the results were compared to were very different, hMSCs exposed to BALF from other lung diseases vs. hMSCs exposed to healthy control BALF samples.In line with the findings by Islam sis (CF) . Interessis (CF) . This waRDS BALF . In simiex vivo to BALF samples obtained from cystic fibrosis patients who had Aspergillus infection were rapidly killed. This effect on hMSCs was at least partly related to the mycotoxin, gliotoxin, produced by the fungus  treated with MSCs. In these patients, only those who demonstrated high cytotoxic activity against the infused MSCs responded to the treatment. Using a murine GvHD model, it was demonstrated that recipient cytotoxic cells activated MSCs to undergo apoptosis through a perforin-dependent pathway. Perforin-induced MSC apoptosis was then further demonstrated to induce immunosuppression via indoleamine 2,3-diocygenase (IDO) production in recipient phagocytes . This il.ex vivo to inflammatory cytokines such as IFN-γ and TNF-α  each can influence the biological properties of MSCs (reviewed in ) are necdelivery . It is het al. it is also very important to include recognized end-point such as length of ICU, ventilator-free days, and overall mortality. Another important issue to considerate, especially for treatment of COVID-19 induced ARDS, is when to enroll patients and when to initiate MSC treatment, but also which COVID-19 patient population to target (The global coronavirus pandemic has increased the number of initiated MSC clinical trials dramatically, and there are a number of challenges related to the conducting trials such as having a good cell product, as discussed above, enrolling patients, assessing outcomes, and ethical issues. For example, a global pandemic aim to find a rapid and effective treatment however it is important that this occurs in a transparent manner following appropriate ethical guidelines . As disco target . The issThe enthusiasm for using MSC-based therapy to treat severe lung disorders is great and it is currently increasing due to the COVID-19 pandemic. Unfortunately, the very promising pre-clinical studies performed in animal models of lung diseases has not yet translated into improved clinical outcomes in clinical trials. The field is still struggling with the lack of knowledge regarding the fate of administered MSCs, lack of standardized protocols for manufacturing of MSCs, and how to determine which patients are more likely to respond to this type of treatment. Despite these limitations, there are several interesting studies demonstrating that MSCs can be activated and modified in order to enhance their therapeutic efficacy using either genetic modifications or environmental factors including inflammatory cytokines and hypoxia. Moreover, recent studies have used artificial intelligence as a tool to predict which MSCs are better to be used in clinical trials, a strategy that would improve the manufacturing process substantially and hopefully lead to a better and more effective MSC cell product. We can conclude that currently there is no clear path to reconcile the issues discussed in this review, however one potential solution might be that investigators begin to combine all aspects of potential benefits into a unified effort to establish a greater search for where benefit could occur.


Identifying the cause of non-traumatic coma in the emergency department is challenging. The clinical neurological examination is the most readily available tool to detect focal neurological deficits as indicators for cerebral causes of coma. Previously proposed clinical pathways have granted the interpretation of clinical findings a pivotal role in the diagnostic work-up. We aimed to identify the actual diagnostic reliability of the neurological examination with regard to identifying acute brain damage.Eight hundred and fifty-three patients with coma of unknown etiology (CUE) were examined neurologically in the emergency department following a predefined routine. Coma-explaining pathologies were identified retrospectively and grouped into primary brain pathology with proof of acute brain damage and other causes without proof of acute structural pathology. Sensitivity, specificity and percentage of correct predictions of different examination protocols were calculated using contingency tables and binary logistic regression models.The full neurological examination was 74% sensitive and 60% specific to detect acute structural brain damage underlying CUE. Sensitivity and specificity were higher in non-sedated patients (87/61%) compared to sedated patients (64%/59%). A shortened four-item examination protocol focusing on pupils, gaze and pyramidal tract signs was only slightly less sensitive (67%) and more specific (65%).Due to limited diagnostic reliability of the physical examination, the absence of focal neurological signs in acutely comatose patients should not defer from a complete work-up including brain imaging. In an emergency, a concise neurological examination should thus serve as one part of a multimodal diagnostic approach to CUE. Coma of unknown etiology (CUE) is a challenging emergency. CUE defines an impairment of consciousness for reasons other than traumatic brain injury or cerebral hypoperfusion in cardiac arrest. Causes of CUE include neurological, neurosurgical and medical conditions many of which are time-sensitive and life-threatening emergencies , 2. EarlThe purpose of this study was to determine and quantify the predictive value of the neurological assessment with regard to detecting acute structural supra-tentorial or infra-tentorial brain damage as the cause of CUE in emergency patients. We aimed to identify those neurological examination findings that serve this purpose with high reliability. The significance of the clinical examination in the initial emergency management of CUE should be evaluated by empirical evidence.Charité Campus Virchow-Klinikum is a tertiary care university hospital located in Berlin, Germany, caring for approximately 70,000 emergency patients per year.n = 1027 prospectively recruited adult emergency patients presenting with CUE between May 2013 and January 2017 [We previously reported on Data were collected by neurologists in the emergency department who consulted the patients immediately, following a predefined management procedure . PatientNeurological examinations were performed according to a predefined protocol . ResultsEmergency management of patients presenting with CUE followed standardized operating procedures . After cSummarized, two board-certified neurologists  reviewed all available clinical, laboratory, radiology and (if available) autopsy findings. Main diagnoses were from one of three categories: (I) primary brain pathology with proof of acute structural or inflammatory brain damage , (II) primary brain pathology without acute structural damage  or (III) acute systemic disorders affecting the brain secondarily . Whenever acute structural brain damage was present and could explain the disorder of consciousness directly (by its location and/or size) or indirectly , patients were given a main diagnosis from category I. This holds true even in the presence of coexisting further potential coma explaining pathologies from category III, e.g., in a patient with subdural hematoma and severe alcohol intoxication. If categorization of the underlying coma explaining pathology was divergent between the two investigators, a detailed re-evaluation and discussion of patient data were performed until consensus was reached. Table or bilaterally. Pathological flexion or extension responses were always flagged as indicators of CNS (namely midbrain or brainstem) pathology. Meningism  [Digitalized clinical findings, diagnoses and adjacent variables were extracted from the patients’ files and anonymized. For one out of 854 patients, no examination was documented. This dataset was thus excluded from analysis. All asymmetrical findings  were considered suggestive of primary structural brain pathology (“red flag”). Exceptions from this rule were extreme bilateral dilatation of pupils with loss of contour which was interpreted as a sign of elevated intracranial pressure until proven otherwise and positive pyramidal tract signs being flagged as suspicious when present unilaterally eizures) .R2 and Hosmer–Lemeshow-Tests (HLT). p values below 0.05 were considered significant. We used IBM SPSS Statistics 26  for analysis.We retrospectively calculated the neurological examination’s sensitivity and specificity to detect acute structural brain pathology causing CUE. We applied various filters to the data and performed different calculations for (A) the whole examination including all items , (B) single items of the examination and (C) combinations of a limited number of items. Whenever a red flag appeared in one clinical item, the examination  was marked suggestive of structural brain damage. We contrasted this information with the patient’s main diagnosis that was established after diagnostic work-up . Sensitivity and specificity of clinical tests were calculated using contingency tables. Sensitivity was defined as the ability of a clinical test  to detect patients with acute primary structural brain pathologies (proportion of positive tests in affected patients). Specificity was defined as the ability of a clinical test  to only detect such patients (proportion of negative tests in patients with other causes of coma). The degree of association between a positive neurological examination and underlying acute structural brain damage was assessed by calculating binary logistic regressions. Model quality and goodness of fit were evaluated by Nagelkerke’s (pseudo-) We report on 853 emergency patients who were unconscious for unknown reasons at the time of examination in the ED and at least until all emergency diagnostics were completed .R2 = 0.144, HLT not significant; Table R2 = 0.285, HLT not significant), sensitivity of the full neurological examination increased to 87% while specificity remained at 61%. In sedated patents , sensitivity was at 64% while specificity was at 58%  and SPs (61%).When comparing the three different examination protocols Table  across aThis analysis of structured clinical examination data from patients presenting with CUE points out several important limitations of the neurological examination as a tool to detect the correct etiology in an emergency.First, the full neurological examination had a low sensitivity and specificity to detect acute structural brain damage in unconscious patients. In our cohort, the percentage of overall correct predictions by clinical findings was only 65%, which is clearly insufficient given the severity of the clinical problem at hand. In statistical terms, predictions did not fit observations sufficiently which is reflected in an insignificant goodness of fit in binary logistic regression models. Second, the administration of sedatives, which happened in the emergency for various reasons and mostly outside the responsibility of the emergency physician in charge, further decreases the diagnostic value of the clinical examination. While this is hardly surprising, it is important to point out, that even in a sub-group analysis of non-sedated patients, the predictive value of the examination is below optimal standards. Third, the full neurological examination of unconscious patients was not associated with a higher sensitivity, specificity, or percentage of overall correct predictions compared to a concise examination protocol that only consists of four items and can be completed with little effort and almost regardless of specialist medical training in a minimal amount of time.While several studies evaluated the prognostic value of results of parts of the clinical examination as integrated in the FOUR Score , data onThere are several reasons that may explain the shortcomings of the neurological examination as a diagnostic tool in unconscious patients. Ideally, primary structural brain pathologies underlying disorders of consciousness should be clinically detectable by abnormal neurological findings. However, even in patients with unimpaired consciousness, the sensitivity of many clinical tests such as testing for meningism is well below 100%. A recent meta-analysis reported a sensitivity of 46.1% for nuchal rigidity in patients with suspected meningitis . A CochrFurther, significant abnormal findings in unconscious patients are also found in patients without acute brain damage and systemic disorders. Obviously, constricted pupils are caused by opioid intoxication more often than by isolated pontine damage. But maybe less obviously, the whole range of possible metabolic disorders (depending on their severity) may also cause asymmetrical neurological abnormalities that may be misleading , 24–29. Another problem is that many patients presenting with CUE have, in fact, more than just one underlying pathology that could explain the disorder. As reported earlier, one third of patients who were managed according to the same standard operating procedure were found to have multiple coma-explaining conditions . For exaFurthermore, there is a significant interobserver variability associated with neurological observations in general and specifically so in unconscious patients . Subtle And not least, iatrogenic sedation  may have masked neurological signs that should be present following a causal brain lesion. In our cohort, patients who eventually turned out to have acute structural brain damage were slightly more likely to be sedated at the time of examination  and thus even more difficult to discriminate by clinical findings. However, in a time-critical emergency, clinicians need to focus and rely on robust decision-supporting tools that can be applied regardless of unswayable factors such as pre-hospital application of sedatives.Calculating sensitivity and specificity for single items of a neurological examination is a somewhat artificial approach to determine their diagnostic value since these items usually only test for a specific dysfunction and not for any kind of brain damage. However, the approach is not too far from clinical reality, where examiners are faced with a variety of testing methods on the one hand and an even wider variety of conditions to be detected on the other hand. There is no single test that is sufficient and there are tests that are statistically insignificant on their own. However, there appears to be a selection of tests better suited to the problem than others as is reflected by their relatively higher sensitivity and, in fewer cases, statistical significance in regression models. This seems to be true for pupil size even regardless of sedation. Predefined answers recorded in the examination protocol applied in this study Fig.  did not In this large cohort of acutely unconscious patients presenting with the full range of possible underlying pathologies, the clinical neurological assessment proved not to be sufficiently reliable in finding the correct diagnosis. In an acute emergency, our data support a quick and simple neurological assessment instead of a lengthy examination Fig. . It may In our analysis, the evaluation of motor function of the limbs (including assessment of muscle tone and tendon reflexes) does not relevantly improve sensitivity or specificity despite being considerably more work. Likewise, we left out assessment of meningism, vestibulo-ocular and corneal reflexes because they appeared with very low values for sensitivity. They add little to the diagnostic value of the examination protocol, most likely because they are either very likely to disappear in deeper stages of coma or particularly unreliable to assess. In an emergency that needs urgent ABC management, there is often little tolerance for the examiner, especially in the head and neck area of the patient. In addition, by being simpler and easier to reproduce, the proposed four-item protocol may help minimize interobserver variability that can result from varying qualifications of the personnel involved , 32.To the best of our knowledge, this is the first study that prospectively assessed the value of a structured neurological examination in terms of identifying an acute structural brain damage in a large real-life cohort of patients with CUE. Nevertheless, the value of studies on routinely acquired clinical data and especially on clinical observations is associated with intrinsic limitations, the most substantial of which are interobserver variability and documentation bias. Further, we cannot exclude an examiner bias, since we did not analyze the effects of clinical experience of the neurologists performing the examination.not in favor of omitting the neurological examination. Next to its initial purpose of accurately assessing the level of consciousness, a full and thorough neurological examination is required in any unconscious patient as soon as circumstances allow. It is the most important monitoring tool for the individual patient’s recovery or decline and may trigger further diagnostics and guide treatment decisions in conjunction with all available information. In an acute emergency, however, less may be the same in terms of predictive value and maybe even more in terms of critical time being saved to save lives. From our data, we conclude that clinical neurological information on unconscious emergency patients should not solely determine clinical decisions but rather be seen as one of several complementary sources of information within a multimodal diagnostic approach to CUE. All CUE patients should receive a complete and thoroughly executed set of diagnostics as quickly as possible, embedded in a structured management routine including early brain imaging [Although it is not a sufficient diagnostic tool, we are certainly  imaging . Further


Traumatic Brain Injury (TBI) is a leading cause of morbidity and moribundity in the United States  plays a major role in the development of human gender including transgender identity by directly or indirectly (epigenetics) altering gene expression and behavior  effects from gonadal influence along with the XYSteroid hormone levels and receptors are markedly different in natal males and females throughout most of their lifespan and clearly play a role following TBIs Arnold, . In termTBI has historically been viewed as a problem that predominantly affects natal males, as they are both more likely to receive TBIs and have less of the circulating neuroprotective steroids: estrogen and progesterone  via gender confirmation surgery (Coleman et al., If natal females are indeed better protected from TBI, then individuals undergoing trans-feminine transition may see better outcomes following hormonal transition as they typically take exogenous estrogen and progesterone, as well as anti-androgens. Typically, younger-aged cisgender women appear to be protected against neuronal damage, compared with cisgender men, but lose this advantage in their post-menopausal years (Niemeier et al., Individuals undergoing trans-feminine gender affirming surgeries sometimes remove the testes, a significant source of testosterone (Coleman et al., The bulk of research into hormone use following TBI has been done in adult or possibly aged populations. However, a significant number of individuals begin this sort of transition in early adulthood or adolescence (Smith et al., Although we have placed a great emphasis on the lack of research in TGNC individuals, sex/gender as a biological variable is underrepresented in TBI research and requires further analysis. The increase in the number of individuals undergoing hormonal or surgical treatment to aid in gender transition calls for a substantial increase in TBI research in these underserved and marginalized populations. There is a need for evidence-based guidelines, common hormone plans, and clinically translatable diagnostic and prognostic models. Furthermore, expanding our knowledge of how exogenous hormone use affects TBI could have profound effects not only on TGNC populations, but also cisgender males and females. Such work and studies will not only help to develop better treatment options for those identifying as TGNC, but will also create a conceptual framework which can be used to extrapolate to others undergoing hormone replacement or depletion in the future.KD and SG-G contributed equally the development and authoring of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over-the-counter (OTC) use of antibiotics contributes to the burgeoning rise in antimicrobial resistance (AMR). Drawing on qualitative research methods, this article explores the characteristics of OTC sales of antibiotic in Nepal, its drivers and implications for policy.Data were collected in and around three tertiary hospitals in eastern, western and central Nepal. Using pre-defined guides, a mix of semi-structured interviews and focus group discussions were conducted with dispensers at drug stores, patients attending a hospital and clinicians. Interviews were audio-recorded, translated and transcribed into English and coded using a combination of an inductive and deductive approach.Drug shops were the primary location where patients engaged with health services. Interactions were brief and transactional: symptoms were described or explicit requests for specific medicine made, and money was exchanged. There were economic incentives for clients and drug stores: patients were able to save money by bypassing the formal healthcare services. Clinicians described antibiotics as easily available OTC at drug shops. Dispensing included the empirical use of broad-spectrum antibiotics, often combining multiple antibiotics, without laboratory diagnostic and drug susceptibility testing. Inappropriately short regimens (2–3 days) were also offered without follow-up. Respondents viewed OTC antibiotic as a convenient alternative to formal healthcare, the access to which was influenced by distance, time and money. Respondents also described the complexities of navigating various departments in hospitals and little confidence in the quality of formal healthcare. Clinicians and a few dispensers expressed concerns about AMR and referred to evadable policies around antibiotics use and poor enforcement of regulation.The findings point to the need for clear policy guidance and rigorous implementation of prescription-only antibiotics. Previous research in Nepal has shown that around 30% to 60% of the population self-medicates.More than two-thirds of community pharmacies dispense antibiotics without prescription and more than one in three patients attending private pharmacies receive at least one antibiotic.Qualitative research can provide essential insights to improve the implementation of existing regulations and develop new strategies to address the development and spread of antimicrobial resistance.In light of their ubiquity and low costs, drug stores were the main source of antibiotics.Interactions between drug store staff and patients were transactional in nature: akin to visiting grocery stores, where signs and symptoms were traded for a quick remedy.This quick fix circumvented perceived barriers to obtaining a prescription from clinicians and boosted the commercial incentives of drug dispensers.High over-the-counter (OTC) use of antibiotics was also attributed to ambiguous and evadable policy.Control and management of OTC use of antibiotics in Nepal, as in other low-income and-middle-income countries, needs a multi-pronged and comprehensive approach.Reinforcing formal healthcare, increasing awareness of antibiotic and AMR among patients and drug dispensers, and reforming existing policy with local and regional AMR surveillance to regulate the OTC use of antibiotics is crucial.Antimicrobial resistance (AMR) is a burgeoning health threat that claims an estimated 700 000 lives every year.3Among antimicrobials, antibiotics are commonly prescribed in low-income and middle-income countries (LMICs), where around 50% of patients receive at least one antibiotic,3 10 11The private sector, mainly OTC drug shops or pharmacies, dominates Nepal’s health service: out-of-pocket payments make up nearly 70% of all health expenditure.3 4 15Researchers have examined various dimensions of antibiotic use and AMR in Nepal, for instance, antibiotic use in primary health care,Nepal is divided into seven provinces and its health system comprises federal, provincial and local level healthcare facilities.This qualitative study was conducted with patients, dispensers and clinicians and follows a standard consolidated criteria for reporting qualitative studies (COREQ) guideline .23 Membe24 2510.1136/bmjgh-2021-005829.supp1Supplementary dataDispensers refer to anyone who sells antibiotics at a pharmacy/drug shop, including drug sellers  and qualified pharmacists. Dispensers work in 'drug shops' , a term used in this article for private or public pharmacies or drug stores where antibiotics are available for purchase. Focus group discussions (FGDs) were conducted with patients and clinicians; semi-structured interviews (SSIs) were undertaken with patients, clinicians and dispensers.The study was co-ordinated by at least one clinician working at each site, where major tertiary hospitals were purposively selected. Patients were recruited from various departments who attended each of the tertiary hospitals. Clinicians were approached from various levels and departments. Dispensers were recruited from drug shops from around these tertiary hospitals. All participants were selected purposively for this study. Patients with experience of visiting drug shops in the past were interviewed regarding their reasons for seeking healthcare at tertiary health centres, and why and under what circumstances they visit drug shops. This allowed us to compare why people attend hospital versus drug shops. Recalling past experience has been an established method to explore the health-seeking behaviour.26A total of 90 respondents from three different study sites participated in the study . Respond10.1136/bmjgh-2021-005829.supp2Supplementary dataThe recruitment of participants for SSIs and FGDs continued until theoretical saturation was reached, whereby no new findings emerged from subsequent interviews.27The lead interviewers  received training in interview techniques from SP  and BA , who have over 10 years’ experience of qualitative social science research. The lead interviewers conducted face-to-face SSIs and FGDs at a convenient (and quiet) location for the respondents, and were working as clinicians at these institutions during the study. For instance, most of the FGDs and SSIs with patients were conducted in the departmental meeting rooms. FGDs and SSIs with clinicians were conducted in the duty rooms within the departments. SSIs with dispensers were conducted in quiet locations behind the busy drug stores. Each of the FGD/SSI lasted between 30 and 60 min.All FGDs and SSIs were conducted with the help of a thematic guide prepared and reviewed by BA, SP and CP . Themes 10.1136/bmjgh-2021-005829.supp3Supplementary data10.1136/bmjgh-2021-005829.supp4Supplementary data10.1136/bmjgh-2021-005829.supp5Supplementary dataOral informed consent was obtained from each participant before the FGDs and SSIs, with consent audio-recorded. All respondents were assured about the confidentiality of their responses. Brief informal discussions on their occupation were conducted to facilitate their candid expression and participation. No incentives were provided to any of these participants.Interviewers conducted SSIs and FGDs in Nepali and audio recorded the discussion using mobile phones, in addition to writing down the field notes. No repeat interviews were conducted with any of the participants. All recorded interviews/discussions were later transcribed and translated into English by SS, RY, SP and JA. Transcripts were cross-checked by SP and lead interviewers  against the audio files for missing utterances and subtle nuances.Thematic analysis of the transcripts was conducted in parallel with data collection by two investigators (BA and SP). None of these transcripts were shared with participants for their opinion. All transcripts were collated in NVivo for line-by-line coding. First, using a deductive approach, data were categorised based on the themes for the FGD/SSI guides; additional categories and themes were added, inductively, to incorporate the emerging themes from the transcripts.The practice of buying medicine and antibiotics OTC depended on multiple factors, which were described by dispensers, patients and clinicians . DispensIn the next section, the characteristics of OTC microbial sales, their drivers, consequences and relevant policies are described.Almost all patients described their experience of visiting drug shops for their own, family members’ or friends’ illnesses. Often the interactions were focused and brief: they resembled retail transactions, whereby medicines were purchased for specific illnesses, rather than patient consultations. Selling antibiotics for infectious diseases was one strand of everyday business in the drug shops: all dispensers explained such a business-as-usual approach and described how sometimes antibiotics were dispensed empirically when patients presented with signs and symptoms of infectious diseases. Taking no responsibility for the outcome, dispensers did not generally follow-up after selling antibiotics.We don’t call them for follow ups. As we are not doctors, we ask them to take those medicines for certain days and ask them to visit a doctor if the medicine doesn’t work within the period .Dispensers often described how patients hesitated to visit hospitals because of the costs, time and complexity involved:Usually doctors ask to carry out different tests. So, I think they feel burdened to spend money on such tests and thus, they try medicines first .Also, patients who travel a long distance from rural regions are poor, constrained by time and opportunity costs, and are unfamiliar with the ‘complex’ systems at the hospitals.Clinicians echoed dispensers’ comments regarding how patients from rural regions face challenges related to visiting hospitals and communicating with doctors, particularly referring to the gaps in doctor–patient relationship, and their unfamiliarity with the hospital system.For people from remote regions… [hospital is] a different world for them. They are scared to visit hospitals. They fear how to speak to doctors, they believe doctors are high up . There are gaps in doctor–patients’ relationships; and they do not know what to speak, how to express the problem and where to go in a hospital… all kinds of hesitations, you know All patients described visiting drug shops for mild to moderate illnesses. Direct and indirect expenses incurred in travelling to the hospitals and the cost of laboratory tests at hospitals were identified as the main barriers to seeking care at hospitals:If you visit big hospitals they ask you to do blood tests even if you have simple fever; they don't give you medicine without test. But if you go to a pharmacy and ask for medicine worth Rs.10, it works, and it actually works. So, if Rs.10 medicine works, why go for blood test and other tests at hospital? .Patients were even more fearful of the excessive costs of visiting private hospitals or clinics. Although patients did weigh other options and discuss with family members where to seek care, a nearby drug shop was often described as the most appealing, despite their confidence in the clinicians working in hospitals.Particularly in rural areas, the poor accessibility of the formal healthcare sector was viewed as a prominent driver of patients seeking care at dispensaries. A few patients, who came from rural regions to attend the hospital, also mentioned specific conditions  for which they sought treatment from traditional healers whilst also visiting drug shops in parallel. Patients also expressed strong opinions about the quality of services at the hospitals. Complaints centred on waiting times, a lack of access to essential medicines and commercial interests embedded in clinicians’ practice at private clinics.‘patients’ demand’ for particular antibiotics. These demands were reportedly rooted in patients’ past experiences of cure from specific antibiotics or those of friends and family. Clinicians also reported that patients visit drug shops and make demands for specific medication using as prompts, the empty blisters/bottles, and old prescription that cured them or a family members’ illnesses.Dispensers explained the OTC sales of antibiotics in terms of Although antibiotics were easily purchasable, to the extent that patients could ask for specific medications, dispensers described only providing antibiotics to close social contacts. This contradiction potentially stemmed from an awareness that this practice contravened prescribing guidelines.We give antibiotics to people who have close relation with us when they have fever Although a few clients may demand specific antibiotics from drug shops, most of the interviewed patients did not seem to know what the term ‘antibiotic’ meant. They reported describing their symptoms to dispensers who would make decisions. Hence, patients placed their trust in dispensers to make a presumptive diagnosis. Such interactions led to repeat visits and strengthened the social relationships perpetuating the cycle of antibiotic dispensing.Patients also described OTC as a social practice that was inherited through the generations in urban, peri-urban and rural settings. Younger generations seem to follow their parents in seeking OTC medications as a way of circumventing the treatment-seeking at hospitals or from clinicians.I think it’s because of the ongoing practice in our society. The young generations follow their parents. They have seen their parents going to pharmacies before going to clinics/hospitals, thus they are doing the same. This practice has become a trend in our society. They are doing what they are seeing for years .Clinicians were vocal about the excessive pressure from pharmaceutical companies and their representatives who visited clinics and drug shops to encourage them to prescribe particular brands of antibiotics. These pharmaceutical representatives often provide incentives (both monetary and non-monetary) and have undue influence on the prescription.Dispensers had commercial interests in serving patients and building goodwill for future visits; they also sold the antibiotic that the pharmaceutical representatives incentivised. Patients did not explicitly acknowledge that drug shops might be motivated by profit rather than providing the most appropriate care. Although clinicians recognised that there were many drivers of OTC sales of antibiotics, including patients’ demands, few were critical about the business motives of dispensers with regard to the sale of antibiotics.According to dispensers, there was varied expertise among the other drug shops around them. They described how, in other drug shops, sometimes an owner would assess patients and dispense antibiotics based on experience alone. The license certificates that hang on the wall of drug stores are often bought from a license holder and hold no relation to the personnel selling medicines OTC.‘double-edged sword’ because it could either cure a broad range of diseases, and if not cured, patients were asked to visit doctors. Patients reported that, in general, drug shops provide higher-dose medicines that were likely to cure their illnesses without having to undergo various tests at hospital, which ultimately would save their time and money. In contrast to dispensers’ claim that they were hesitant to dispense antibiotics, patients described how drug shops usually provided antibiotics easily, and in high doses.Dispensers were asked specifically about the antibiotics sold for infectious diseases: in most cases, they described antibiotics provided for 2 to 3 days and sometimes in sub-standard doses. Most dispensers provided broad-spectrum antibiotics including injectables to cover wide range of diseases. ‘Providing broad-spectrum antibiotics for only few days’ was considered a A clinician shared his experience of how a relative received a few doses of broad-spectrum antibiotics from the drug shop, developed respiratory complications and required intensive care treatment for 19 days.There was a patient (my relative) with 1–2 days fever who took multiple medicine from a drug shop at first and came to the hospital after 3–4 days and… suddenly had Pneumonia… and then ARDS [Acute Respiratory Distress Syndrome] and was to be admitted for 19 days .a double loss’ if treatment did not work and the patient had to visit a formal health centre. To prevent this, dispensers were inclined to sell high-dose and newer-generation antibiotics. The dispensers also mentioned patients’ lack of compliance in completing the dose once relieved from the symptoms.Dispensers were aware of the sensitive nature of these antibiotics in terms of their judicious use based on a clinician’s prescription or confirmatory diagnosis. Some were quite vocal about the adverse implications of their potentially inappropriate presumptive treatment. However, they did not spontaneously mention the development of AMR. They described this leading to ‘The broader adverse effects of sub-standard doses and lack of compliance were not easily translated to antibiotic resistance. Many dispensers seemed complacent about the eventual ineffectiveness of the antibiotics due to OTC use; only a few made the connection between the effectiveness of the antibiotic and resistant bacterial strains.It means the medicine will stop working with same effectiveness later as it should. Gradually, it will stop being effective .When probed, dispensers alluded to the implications of antibiotic resistance, such as a reduction in effectiveness of the antibiotics. Even if they were not aware of the mechanisms of resistance, many dispensers attributed declining effectiveness of antibiotics to patients’ incomplete adherence, and r dispensers’ or clinicians’ practice of providing high doses of antibiotics including their own practice of providing high dosage with multiple antibiotics.Clinicians and dispensers explained the adverse consequences of prior empiric use of antibiotics, such as complexities of disease presentation  and outcome (not responsive to previously effective antibiotics). Reasons and potential measures for the mitigation of the high empirical treatment with antibiotics, mostly in the peripheral region of Nepal, were fiercely debated by clinicians; the OTC practices were particularly seen as rooted in health system constraints such as lack of laboratory facilities.Some dispensers linked rising AMR to widespread use of antibiotics in animal foods, particularly chicken. These concerns were echoed by clinicians, who further explained the misuse of antibiotics in agriculture and animal food, mostly referring these practices to how farmers themselves over-used these antibiotics.Awareness of antibiotic prescribing policy was fairly consistent among the respondents. Dispensers explained that all medicines, except for narcotics and the medicines for mental health, could be sold at drug shops without a clinician’s prescription. A few were aware that some of the medicines that they sell need a prescription from health workers:…[antibiotics] come under Group C so it can be sold and also it needs prescription .To meet the legal requirements to dispense antibiotics, many of drug shops offer an appointment with healthcare providers, such as physician, health assistant, community medicine assistant (CMA) or auxiliary nurse midwife (ANM), who are certified for clinical decision-making to varying degrees. These clinicians generally work within the local clinics/hospitals. However, dispensers  often dispensed all available antibiotics on their behalf from drug shops without their prescription, and without taking a full history, physical examination or laboratory investigation:If we look in most of the pharmacies, only a single person dispensing medicines is a pharmacist, whereas he is helped by CMAs or ANMs on paper. CMAs and ANMs have mostly covered the pharmacies. What shall I say; they are allowed to prescribe medicines but are not allowed to sell it [and dispensers are allowed to sell only with the prescription]? The policy that antibiotics must be prescribed by healthcare workers, who could be a CMA, but that CMAs can not not dispense the medicines promotes confusion. This ambiguous policy also obliges drug dispensers to look for some kind of legitimate approvals to display on the walls of the drug shop which equally helped them to cover up the informal drug dispensing practice. Most dispensers were quite relaxed in terms of dispensing antibiotics, whereas some were unaware of policies regulating antibiotics except for few drugs that strictly required prescriptions.In addition to the gaps in policies around dispensing antibiotics, most clinicians targeted their frustrations at the lack of enforcement of the existing regulations, and patients’ practice of buying medications from the drug shops without a prescription.In Nepal, with limited healthcare coverage and a high burden of infectious diseases,Drug shops are a recognised part of health system infrastructure in LMICs, including Nepal.Patients’ visits and interactions at drug shops resemble retail transactions.34Dispensers’ lack of competence was a major concern among clinicians and (other) dispensers. Most drug shops, particularly in rural Nepal, are run by unqualified personnel (non-pharmacists) who learn during their work and operate using someone else’s license  or have no license at all.33Many studies have explored access to formal healthcare services, identifying barriers such as distance, cost and perceived quality of services in Nepal and other LMIC settings.41Patients from rural regions often perceive doctors to be from a higher social class. They seem less approachable and patients find it difficult to express their problems to them. This (perceived) social distance could lead to poor doctor–patient relationships, unmet expectations and resentment.35In Nepal, drug shops began to open shortly after the establishment of the first allopathic health centre, Bir Hospital, in 1890. The number of drug shops increased greatly in the late 2000s: from around 400 in 1979 to 7000 in 1992 and ~29058 in 2021.45The practice of providing multiple and broad-spectrum antibiotics for a wide group of illnesses echoes findings from previous studies in Nepal: antibiotics were dispensed for viral infections and self-limiting gastro-enteritis.49Previous studies have highlighted antibiotic dispensing practices in drug stores,43 54The empiric use of broad-spectrum antibiotics often in high doses was driven by dispensers’ intention to avert the ‘double burden’ . The OTC use of broad-spectrum antibiotics is likely to drive AMRPatients are not aware of the potential pitfalls of purchasing medicines OTC from dispensers. They were often unaware of purchasing antibiotics. They hence almost never realised that buying these medicines OTC—without supervision—can promote AMR, reducing their future effectiveness. These findings resonate with those of previous surveys in Nepal, which reported very limited knowledge of antibiotics among non-science studentsOnly a few dispensers and clinicians had specific knowledge of policies regarding antibiotics. Policies addressing the widespread use/misuse of antibiotics OTC in Nepal were regarded ambiguous and poorly enforced. Specifically, lack of stringent policies regulating dispensing of antibiotics, and ambiguity in prescription versus dispensing require urgent attention. Clinicians also reflected on the lack of adequate health infrastructure and the essential nature of antibiotics in terms of preventing deaths due to infectious diseases. This resonates with the broader recognition that antibiotics are often crutches for fractured health system infrastructure, poverty, poor water and sanitation, and high burden of infectious diseases.61The lack of knowledge about antibiotics among patients, and inadequate knowledge about the adverse consequences of OTC antibiotics and AMR among dispensers has critical implications for national policies and strategies. Community and stakeholder engagement strategiesThis is the first study to explore the perspectives from three most relevant stakeholders  on the OTC use of antibiotics from tertiary hospitals located in different provinces of Nepal. OTC practices were compared across these regions: respondents from urban and rural regions described high OTC use of medications combined with particularly poor quality of care in the rural regions. Because of limited health services, including the drug shops in rural regions, patients reported a reliance on local health services/drug shops before deciding to travel in search of quality healthcare and reflects other evidence that indicates the urban–rural divide in health services across Nepal.Approaching dispensers for interview was particularly difficult because of their hesitance around revealing their practice of selling antibiotics. During SSIs, they often began cautiously and the interviewer was forced to ask about dispensing practices indirectly to probe further their perception and practices. Although interviews were conducted in a way to build rapport, using non-leading questions and responding in a non-judgemental manner, power difference between clinicians and patients may have engendered social desirability bias.This study focused on human health alone and did not address the widespread use of antimicrobials in livestock and agricultural products. Future research embracing ‘One health approach’ would be useful to examine attitudes to antibiotic use and the development of resistant infections among livestocks.Purchasing antibiotics OTC is an established practice in Nepal with many drivers. Faced with constraints of money, time and distance, the complexities of navigating services in hospitals, and intimidating social hierarchies, patients often visit drug stores. When in drug stores, decisions to dispense antibiotics—often in the absence of laboratory investigations—were influenced by patient demand (driven by a preference for rapid symptom relief) and dispensers’ intention to gain goodwill. Antibiotic doses were often inappropriate and there was a perceived lack of compliance and follow-up. Current national policy on OTC purchasing of antibiotics was recognised as ambiguous and poorly enforced. Policies to address inappropriate antibiotic use and AMR should be embedded in a broader drive to improve health infrastructure. Specific AMR-related initiatives must be multipronged: re-allocating the resources in rural regions to scale up the quality health services, strengthening antimicrobial resistance surveillance, and stakeholder and community engagement, and awareness.


We find that paying attention to redox states illuminates which reactions are endergonic or exergonic. Our results highlight the role of acetate in proto-metabolic cycles, and its connection to other prebiotic molecules such as glyoxalate, glycolaldehyde, and glycolic acid.How did metabolism arise and evolve? What chemical compounds might be suitable to support and sustain a proto-metabolism before the advent of more complex co-factors? We explore these questions by using first-principles quantum chemistry to calculate the free energies of CHO compounds in aqueous solution, allowing us to probe the thermodynamics of core extant cycles and their closely related chemical cousins. By framing our analysis in terms of the simplest feasible cycle and its permutations, we analyze potentially favorable thermodynamic cycles for CO However, at its core , represeAs pointed out by Pross  in this 2 as its carbon source to build biomass. While proto-life on the early Earth may have had a variety of carbon sources, the ability to incorporate C1 molecules into larger structures remains fundamental for a self-sustaining system to emerge  term) in the reaction quotient Q would then result in different ΔG values. These only address equilibrium thermodynamics in a perturbative way. Temperature, pH, and solute activities are also expected to impact kinetics. Our group is working to calculate baseline activation barriers for reaction types in these cycles, although this requires the lengthy process of optimizing transition states.This baseline provides a preliminary step to exploring the effect of temperature, pH, and different effective concentrations (activities) of the various solutes including the reference species. One could recalculate the relative free energy changes using ΔOnce baseline kinetics is established and we have made better estimates of the rate constants, we plan to incorporate both thermodynamic and kinetic parameters into a dynamic network model that tracks the fluxes of chemical species as they cycle through their myriad reactions. Our hope is that this will provide a preliminary model (with its attendant limitations) that represents the establishment of proto-metabolic cycles under non-equilibrium conditions, and we hope to provide a richer story in the future.


Multiple implementation scholars have highlighted the importance of designing implementation strategies to fit the existing culture, infrastructure, and practice of a healthcare system. Worldwide, healthcare systems are adopting improvement models as their primary approach to improving healthcare delivery and outcomes. The prevalence of improvement models raises the question of how implementation scientists might best align their efforts with healthcare systems’ existing improvement infrastructure and practice.In several recent articles, authors have called for aligning the fields of implementation and improvement science. In this paper, we call for implementation science to also align with improvement practiceWe describe three challenges and five benefits to aligning implementation science and improvement practice. Challenges include (1) use of different models, terminology, and methods, (2) a focus on generalizable versus local knowledge, and (3) limited evidence in support of the effectiveness of improvement tools and methods. We contend that implementation science needs to move beyond these challenges and work toward greater alignment with improvement practice. Aligning with improvement practice would benefit implementation science by (1) strengthening research/practice partnerships, (2) fostering local ownership of implementation, (3) generating practice-based evidence, (4) developing context-specific implementation strategies, and (5) building practice-level capacity to implement interventions and improve care. Each of these potential benefits is illustrated in a case study from the Centers for Disease Control and Prevention’s Cancer Prevention and Control Research Network.To effectively integrate evidence-based interventions into routine practice, implementation scientists need to align their efforts with the improvement culture and practice that is driving change within healthcare systems worldwide. This paper provides concrete examples of how researchers have aligned implementation science with improvement practice across five implementation projects.The online version contains supplementary material available at 10.1186/s43058-021-00201-1. Contributions to the literatureTension continues to exist between the fields of implementation science and improvement practice. This paper presents both the challenges and benefits to aligning the two fields.This paper argues that greater alignment between implementation science and improvement practice has the potential to strengthen the implementation of evidence-based interventions.Five case studies provide concrete examples of how Cancer Prevention and Control Research Network members are achieving the benefits of aligning implementation science with improvement practice.In several recent articles, authors have called for aligning the fields of implementation and improvement science with the goal of strengthening both fields’ potential to improve care –3. TheseImplementation science and improvement science both aim to produce generalizable knowledge to guide the improvement of healthcare services and health outcomes but differ in their approach. Implementation science focuses on increasing the uptake of evidence-based interventions (EBIs) whereas improvement science focuses on iteratively measuring and improving care processes and systems 2). Improvement science has its foundation in the field of improvement practice, which also focuses on improving care processes and systems 1). Improvement practice is distinct from improvement science in that its goal is to produce the local  knowledge needed to improve care in a specific setting [. Improve. ImproveAcross the USA and worldwide, healthcare systems are adopting Lean Six Sigma, quality improvement, and other improvement models as their primary approach to improving practice –10. TablThe widespread adoption of improvement models is driven in part by requirements that health systems develop improvement programs to qualify for health insurance reimbursement and other sources of funding –15. ConcA central tenet of implementation science is that implementation strategies will be most successful when they align with healthcare systems’ existing culture, infrastructure, and practices . ConsistAs detailed below, three primary challenges have impeded the alignment of implementation science with improvement practice.Implementation science and improvement practice both promote specific approaches, referred to as “strategies” in implementation science and “interventions” or “tools” in improvement practice , 2. WhilAs noted above, implementation science and improvement practice have different objectives. Implementation science aims to provide generalizable knowledge to improve healthcare services and outcomes. In contrast, improvement practice seeks to generate local knowledge to improve healthcare services and outcomes in a specific setting . The empSystematic reviews of the literature have identified wide variation in how healthcare systems are using improvement models and tools –32. For In this paper, we advocate for the benefits of implementation science and improvement practice aligning with and building on each other’s strengths. We do not argue for the value of generalizing the findings from improvement projects or for the effectiveness of improvement practice at improving health services and outcomes. Rather, we highlight five potential benefits of aligning with improvement practice as a means of strengthening research/practice partnerships, fostering local ownership of EBI implementation, generating in-depth knowledge of the practice context, and developing context-specific implementation strategies.Implementation science typically requires that researchers engage with healthcare providers, staff, and multiple levels of system leadership in the healthcare systems where they plan to implement EBIs. Engaging these stakeholders is challenging due to competing demands for their time and attention. Furthermore, these stakeholders may resist the input of researchers outside their organizations, as reflected in a recent article where authors refer to implementation science as tending toward “scientific imperialism” and an “overall approach that disrespects and undervalues primary care as a co-producer of knowledge” . This obIn improvement teams, providers and staff work together to plan and test improvements in their unique healthcare setting, thereby promoting local ownership of the change in practice . PartnerEliciting local stakeholders’ knowledge of their local context is key to ensuring that an EBI and its implementation align with a practice setting’s needs, assets, and priorities. Recent years have seen a growing emphasis on the importance of this type of “practice-based” evidence, which Leeman and Sandelowski defined as “evidence concerning the contexts, experiences, and practices of healthcare providers working in real-world practice settings” . ImproveAnother primary component of many improvement models is the PDSA cycle. In conducting PDSA cycles, improvement teams design changes that they then rapidly test in multiple, small experiments with the goal of developing “fit-for-purpose solutions” to improve care . In contStrengthening healthcare systems’ use of improvement models and tools is important for both the improvement teams and implementation scientists to achieve their maximum potential. The literature includes examples of implementation scientists partnering with healthcare systems to bolster application of their improvement models and tools, usually in support of a specific EBI . EffortsAs summarized in Fig. The CPCRN is a national network funded by the Centers for Disease Control and Prevention (CDC) and National Cancer Institute (NCI). Since 2002, CPCRN centers have conducted practice-engaged research to accelerate the adoption and implementation of cancer prevention and control interventions , 48. In Researchers at the University of North Carolina (UNC) Chapel Hill are partnering with a large, academic primary care practice that serves 13,000 adult patients and provides over 39,000 visits per year. The practice has extensive experience with quality improvement (QI) and participates in a system-wide improvement collaborative.Recently, UNC researchers partnered on a clinic-initiated project to address a sudden drop in colorectal cancer (CRC) screening rates. In October 2018, the practice’s QI committee noted a steep decrease in CRC screening rates while reviewing standard clinical quality measures. A subgroup of the QI committee was formed to study the problem and implement potential solutions. The team conducted a root-cause analysis and found that prior interventions, in which CRC screening home test kits were distributed during annual clinic visits, increased CRC screening rates. However, the interventions were not sustained over time, resulting in a drop in screening rates. To identify a more sustainable solution, the team engaged UNC researchers with expertise in mailed CRC test kit outreach, a CRC screening intervention that does not require a clinic visit and has limited impact on clinic workflows. Researchers partnered with the team on multiple PDSA cycles to develop a protocol for mailing CRC test kits, targeting patients who had previously completed a screening kit but were now overdue . With funding from state-level tobacco tax revenue, the CCSP has built extensive statewide partnerships, including partnerships with nearly 150 clinic systems and other organizations. The cornerstone of CCSP over the last decade has been the use of patient navigation to deliver multi-level EBIs to increase CRC screening. Patient navigation is an evidence-based intervention that employs patient navigators to guide patients through the health care system and reduce barriers to screening and treatment . In 2018CCSP provided technical assistance to aid clinics in adapting the process flow template and creating a process flow diagram that was tailored to each clinic’s existing staffing, workflows, and referral arrangement with CT lung cancer centers/hospitals. For example, the more urban of the two pilot clinics shares an electronic health record (EHR) with its CT provider. This shared system allows their primary care team to have a streamlined process flow diagram for referring patients and tracking processes and outcomes Fig. . In contUNC researchers are incorporating process flow diagramming throughout the design, implementation, and evaluation of their National Institutes of Health (NIH)-funded Scaling Colorectal Cancer Screening through Outreach, Referral, and Engagement (SCORE) study. In collaboration with two community health centers, SCORE is testing the effectiveness of an intervention that combines mailed CRC screening home test kits, patient reminders, and navigation to follow-up colonoscopy for patients with a positive test result. Process flow diagrams are being used to highlight key differences in the process steps and to delineate roles and resources needed based on feedback from clinic staff about their local context and standards of care. The research team iteratively developed the process maps through multiple stakeholder-engaged sessions. The maps reflect input from program directors and staff at the community health centers where SCORE is being implemented. Each diagram documents all steps in the process and identifies which individuals perform each step. The team created a separate diagram for each of the core components of the SCORE intervention, which include developing a registry of eligible patients, conducting a mailed screening program, and navigating patients with a positive screening test to follow-up colonoscopy.Early iterations of the process flow diagrams were used to plan implementation of the intervention. Visualization of the planned steps helped identify outstanding questions about both the intervention and implementation, as well as prioritize decision-making. These decisions included (a) determining the total number of patient contacts ; (b) optimizing the order of intervention activities and the time intervals between activities; and (c) identifying the appropriate individual or organization to perform specific steps. The process flow diagrams were then used to develop a comprehensive set of implementation outcomes to be measured. For example, fidelity and cost measures were mapped onto each process step, noting which tool(s) would be used for measurement, the frequency of measurement, and where these data would be tracked. The ongoing assessment of these measures during implementation will provide insight into the time and resources required for implementation, potential challenges to implementing the intervention as intended, and opportunities to create efficiencies. The evidence gathered from the SCORE process flow diagrams will be used to inform the adaptation and sustainment of the intervention in community health centers across the state.Researchers at the NYU Grossman School of Medicine are partnering with six community health centers (three pediatric clinics and three school-based clinics) in Southwest Brooklyn to evaluate and support the implementation of their HPV vaccination programs for low-income adolescent populations. As a first step, semi-structured key informant interviews are being conducted with multi-level stakeholders, including medical directors and other organizational leaders, nurse practitioners, frontline staff, and administrators across all six clinics. Guided by the RE-AIM framework , these iInformed by findings from these key informant interviews, the NYU team will convene a virtual learning collaborative and provide technical assistance to support each community health center as they conduct iterative PDSA cycles to refine existing implementation strategies and develop new ones as needed to address barriers to implementation. In addition to PDSA cycles, clinics will learn to use additional improvement tools that guide root cause and strength/weakness/opportunity/threat (SWOT) analyses  for use in identifying and addressing potential threats as community health centers seek to build capacity for and sustain high-quality HPV vaccination programs in the future.Researchers at the University of Washington (UW) provide implementation and evaluation technical assistance to community health centers across Washington State that participate in the CDC’s Colorectal Cancer Control Program. In this role, UW researchers partnered with three community health centers to evaluate a mailed CRC screening home test kit intervention to increase CRC screening. The three community health centers operate 45 medical clinics that mail out more than 2000 CRC test kits annually. UW researchers met individually with community health centers to learn about their CRC screening workflows and identify data capture points for monitoring and improvement. During these visits, community health center partners reported that their current quality monitoring systems lacked the capacity and flexibility to comprehensively monitor and evaluate implementation of their mailed screening kit programs.Researchers developed a simple Microsoft Excel tracking tool for partners to use; tool contents were tailored based on one-on-one consultations with each community health center to align with their needs and resources. The tool included patient-level implementation variables to track such as demographic data, key dates , date screening kit was returned), test result, referral to colonoscopy (if needed), and colonoscopy outcome. Researchers pre-programmed reports in the tool so that community health centers could generate reports to monitor implementation in real time. Feedback from community health center staff on the tool and its adaptations has been predominantly positive. One community health center was able to work with their EHR vendor to add some of the suggested implementation measures directly into their EHR. A second community health center made enhancements to the tool when it became clear that their system had significant gaps in closing the colonoscopy referral loop and recording follow-up data. The third community health center reported that the automated reports enhanced their ability to provide real-time reporting to leadership, whereas previous reports were less timely due to data lags caused by processing internal data requests. In turn, community health center partners have supported UW’s research efforts over the past 5 years by providing letters of support for research applications and participating in emergent research opportunities.The number of EBIs available to prevent and control cancer continues to increase , 57, andInvestments are needed to build the capacity within both implementation science and improvement practice. Ideally, implementation scientists would include orientation to improvement practice in the multiple trainings being offered to build capacity in the field of implementation science  and woulAdditional file1:.


Searching the CrePortal by recombinase activity or specific recombinase gene driver provides users with a recombinase alleles and transgenes activity tissue summary and matrix comparison of gene expression and recombinase activity with links to generation details, a recombinase activity grid, and associated phenotype annotations. Future improvements will add cell type-based activity annotations. The CrePortal provides a comprehensive presentation of recombinase allele and transgene data to assist researchers in selection of the recombinase allele or transgene based on where and when recombination is desired.Recombinase alleles and transgenes can be used to facilitate spatio-temporal specificity of gene disruption or transgene expression. However, the versatility of this in vivo recombination system relies on having detailed and accurate characterization of recombinase expression and activity to enable selection of the appropriate allele or transgene. The CrePortal ( Along with individual research laboratories, large-scale mouse gene targeting projects, such as KOMP, EUCOMM, NorCOMM, and TIGM , have delivered a vast number of conditional-ready loxP-flanked alleles to the scientific community  has data pertaining to 3299 recombinase alleles and transgenes driven by 1230 unique promoters assayed in over 3718 tissues. Of the 3299 recombinase alleles and transgenes, 1166 are inducible. Beyond the basic cre/lox system, temporally and spatially restricted recombination can be achieved using different recombinases . Refinement of recombinase expression and activity can be achieved using recombinases fused with inducible elements   is a freely accessible public database that houses comprehensive information about existing recombinase driver strains and their functional activity. This includes integrated data curated from compiled literature about these lines and via direct submissions of characterization data from resource development programs, providing the community with a comprehensive source of recombinase driver tool strains and information about them.Characterization data that describe the timing and distribution of recombination activity in mouse lines are often fragmented in multiple publications, making it challenging for users to find critical information for good experimental design or to compare the relative utility of different driver strains for a given application. The catalog of these available mouse strains has grown in recent years. However, there are still significant gaps that limit our ability to dissect gene function in certain tissue types and developmental stages. Moreover, despite continued advances and improvements in approaches to engineer Cre driver lines, the fidelity of recombinase activity is not always ideal. Limitations reported in the function of various recombinase lines include expression in non-target tissues (“off-target” activity), inconsistent activity, incomplete or mosaic deletion in a target tissue/cell type, and/or insertional mutagenesis  to help identify optimal anatomical terms for searching. As well, the CrePortal search field contains an autofill feature to assist in identifying the desired structure term. The EMAPA-directed acyclic graph structure improves the likelihood of search success by returning all results that are subclasses of the search term. A toggle on the CrePortal search allows users to search by anatomical structures in which the recombinase activity has been detected or assayed, allowing users, if they so choose, to review the structures in which the absence of recombinase activity has been demonstrated. The “and nowhere else” check box allows users to narrow their search to reporters whose activity has been detected in the specified structure (or its substructures) but nowhere else.Cre reporter data represent allele patterns created through the tissue-specific activity of the Cre recombinase rather than gene expression patterns. However, from a strictly technical point of view, Cre reporter data are similar to knock-in reporter in situ data, a data type that GXD has long captured  against activity patterns for recombinases driven by that gene is now available Fig. . This alWhether accessed through CrePortal search results or MGI’s quick search field, specific, detailed information about individual recombinase alleles and transgenes is presented in conjunction with an expandable grid of systems and structures versus time points derived from annotated recombinase activity Fig. . The recTissue specificity information can be accessed by choosing an anatomical system in the expression summary grid Fig. . The rechttp://www.informatics.jax.org/home/recombinase as described above. CrePortal data access is also available through MouseMine (http://www.mousemine.org) .The CrePortal provides free public web access to data from http://www.informatics.jax.org/mgihome/submissions/recombinase_submission.cgi) or by bulk submission initially via MGI user support. Workflows via programmatic submissions are handled on a case-by-case basis and include the submission of Cre characterization data from the JAX Cre Repository.Finally, the CrePortal supports direct submission from individual investigators and from projects of all images and annotations, ensuring seamless access to all data alongside curated results from the literature. A “Your Observations Welcome” button is available at the bottom of the recombinase activity section of allele detail pages and can be used to report additional activity or problems using the line Fig. . SubmissA significant part of the data in the CrePortal comes from projects that perform a systematic characterization of recombinase-expressing mouse lines, including our work as part of the JAX Cre Repository. Despite the power and versatility of cre driver strains, full awareness and consideration of the caveats and limitations to their use are critical for good experimental design  activity, but frequently this does not include a full assessment of activity in non-target tissues. Existing Cre lines are often used for entirely new research purposes, and depending on the experiment, off-target activity might confound interpretation of results. Therefore, the widespread adoption of individual Cre lines as generalized tools further necessitates systematic evaluation of all potential recombinase actively. Because JAX is the single largest distributor of Cre driver strains, with several hundred unique lines available, we have built an efficient and high-throughput workflow to systematically assess Cre driver functionality using a sensitive reporter strain and characterizing activity in a broad range of tissues at multiple time points (Heffner et al. The development of conditional-ready alleles for spatio-temporal or inducible gene knock-out or transgene expression has expanded our knowledge of gene function in the context of both anatomical structure and developmental stage. The ever-increasing availability of recombinase-expressing alleles and transgenes are limited in their usefulness by the inability of researchers to select the optimal recombinase allele or transgene based on their activity patterns and potential phenotypic impact absent complete characterization of the recombination activity. The CrePortal provides an invaluable resource to identify and evaluate the potential location and time of recombination for suitability in a given experiment depending on the researcher’s hypothesis. The availability of both summarized and detailed recombinase activity data with comparison to driver gene expression patterns gives researchers a complete and searchable context for annotated and expected recombinase activity. Furthermore, the CrePortal supports direct submission of data reporting recombinase activity, simplifying centralized access to large-scale Cre driver generation and/or characterization programs.Future directions for the CrePortal include the retrieval of data associated with specific cell types and improved search and visualization tools. For example, the current anatomical tissue search will not support a search for “Purkinje cell,” an output cell of the cerebellar cortex. The Grid2 and Pcp2 gene drivers express recombinase in Purkinje cells but are annotated to “Purkinje cell layer” in the CrePortal; however, additional cell types exist in this tissue. Furthermore, the striatonigral and striatopallidal projection neurons in the striatum of the brain selectively express the Drd1 and Drd2 dopamine receptors, respectively (Gong et al. As the number of Cre lines and data continue to increase, it is imperative that there are effective visualization tools to assist with effectively sorting through available information. We will develop additional search and display tools to provide data as aggregate comparisons, with the ability to drill down to specific characterization data. For example, a differential search for a Cre line with expression in tissue X at mid-gestational age and NOT in adult or vice versa may be useful to observe changes in a temporal manner or to study changes in adult tissues when expression in embryo tissues may result in lethality. This will require the addition of developmental stage-specific search capabilities for Cre activity at the portal, similar to the GXD Differential Expression Search interface. We will continue to develop new search and visualization tools as new technologies arise.