The experience of craving via exposure to drug-related cues often leads to relapse in drug users. This study consolidated existing empirical evidences of cue reactivity to methamphetamine to provide an overview of current literature and to inform the directions for future research. The best practice methodological framework for conducting scoping review by Arkey and O’Malley was adopted. Studies that have used a cue paradigm or reported on cue reactivity in persons with a history of methamphetamine use were included. Databases such as Medline, EMBASE, PsycINFO and CINAHL were searched using key terms, in addition to citation check and hand search. The search resulted in a total of 32 original research articles published between 2006 to 2020. Three main themes with regard to cue reactivity were identified and synthesized: (1) effects of cue exposure, (2) individual factors associated with cue reactivity, and (3) strategies that modulate craving or reactivity to cues. Exposure to methamphetamine-associated cues elicits significant craving and other autonomic reactivity. Evidence suggests that drug cue reactivity is strongly associated with indices of drug use and other individual-specific factors. Future studies should focus on high quality studies to support evidence-based interventions for reducing cue reactivity and to examine cue reactivity as an outcome measure. Several theoretical models of drug-use behaviors, such as the expectancy model, the dual-affect model, and the cognitive processing models have proposed that external environmental cues can serve as triggers for drug use ,2. Cues Drug cue reactivity is one of the hallmark characteristics in addiction research and numerous attempts have been made to elucidate the underlying learning mechanisms . Drug cuCue reactivity paradigm is a valuable and versatile tool that aids the studying of cue-elicited drug craving. It typically involves the exposure of current or abstinent drug users to visual and/or auditory drug-related cues in order to monitor their reactions to these cues . ExposurMethamphetamine, a highly addictive illicit drug that is also otherwise known as ice, speed, meth and crystal, is an amphetamine-type stimulant that heightens stimulation in the central nervous system . While tDespite amphetamine-type stimulants (mainly methamphetamine) being the second most widely used class of illicit drugs worldwide , no effeThe current review was conducted in line with Arksey and O’Malley’s framework for scoping methodology   . An elecA two-stage review strategy—first at the title/abstract level followed by full-text level—was adopted and each potentially relevant result was examined by two authors (ES and WJ) who worked independently at each stage. Disagreements between the two authors were discussed or consulted with the third author (MS) until a consensus was reached.Studies were screened and included if they had used a cue paradigm or reported on cue reactivity in persons with a history of methamphetamine use . Studies that were conducted in animals or among healthy participants only were excluded. In addition, studies that recruited participants with polysubstance  use and had not reported findings specific to exposure of methamphetamine cues or the use of methamphetamine separately, were also excluded. Upon full-text screening, we decided to further exclude reviews, study protocols, extracts from books or other non-scientific publications, case reports and those that were only available as abstracts , and to include only primary studies.Data was extracted and tabulated to include the Population, Intervention, Comparison and Outcome (PICO) elements by one author (ES) and verified for completeness and accuracy by a second reviewer (WJ). Details of study population and context, cue reactivity paradigm, type of intervention and comparator (if applicable), outcome measures, and findings of interest were extracted and recorded. In order to chart the data, the studies were classified according to the broad areas of application of the cue paradigms and sorted by date of publication. Quality of evidence or formal risk-of-bias assessment for each individual study was not evaluated, as consistent with the scoping review methodology. To summarize the findings, data was synthesized and reported according to key themes. Within each broader theme, data was further sub-categorized to group common effects, factors and intervention types so as to present a more meaningful narrative account of the existing literature. All team members reviewed the themes and consensus was reached for the label of each theme.The combined search identified a total of 163 references, of which 84 were duplicates and were removed. A total of 87 citations  were therefore screened on the basis of title and abstract see  to ensurn = 28), in vivo paraphernalia and/or simulated drug (n = 10) and imagery cues  (n = 4) were the different modalities used in all the studies to elicit responses in a cue reactivity paradigm. Several studies also designed and employed the use of a methamphetamine virtual reality (methamphetamine-VR) cue model to assess self-reported cravings and physiological changes [n = 4), beach (n = 1), images of artifacts and normal daily actions (n = 2), footage of tropical fish in tank/aquarium (n = 2) or the handling of a glass of water (n = 2) and pine cones, shells, and rocks (n = 1). Few studies also used control cues such as sexual (n = 2), food (n = 1) visual cues, as well as “happy” and “sad” stimuli based on subjective evaluations of the emotional valence of the images (n = 1). Only 8 studies recruited a control group with healthy participants for comparison purpose. Studies (n = 3) that used both control cue(s) and a control group were those that looked at difference in neural reactivity to methamphetamine vs. control vs. neutral cues using functional magnetic resonance imaging, and compared them between drug users and healthy controls. Most studies employed a single item visual analogue scale to assess cue-induced craving (n = 14). Established scales such as the general craving scale , within session rating scale , brief substance craving scale  and methamphetamine urge questionnaire  were also utilized to assess cue-induced drug craving. Depending on the aim of the studies, these measures were administered to participants at different time points . Overall, this scoping review identified several themes with regard to the main applications of drug cue paradigms. As shown in Visual (photographs/images and videos)  and skin conductance while heart rate was significantly decreased only after viewing methamphetamine-related photos or video, but not after exposure to paraphernalia . MethampBesides subjective and physiological responses, the scoping review also found studies examining cue reactivity with functional magnetic resonance imaging (fMRI) and electroencephalogram (EEG). Seven studies that examined fMRI evidence reported functional abnormalities in the brain of those with methamphetamine use disorder, compared to healthy participants. Blood-oxygen-level dependent measures of methamphetamine cue reactivity revealed activation of a broad set of regions, particularly the mesocorticolimbic system which includes the ventral and dorsal striatum, the cingulate cortex, prefrontal cortex (PFC) and insula ,33,34,35The cognitive effects of cue exposure in chronic methamphetamine abusers have been examined in one study. Tolliver and colleagues found the exposure of methamphetamine-related cues to impair participants’ performances (increased rates of both response errors and inhibition errors) on an auditory dual task Go–No Go cognitive test requiring divided attention and inhibition of distracting information .The scoping review identified and characterized major factors that were found to modulate craving upon presentation of methamphetamine cues in users. In this section, we have reviewed available evidence and discussed individual-specific factors that were associated with, or in some cases, predictive of cue-elicited craving see .Three studies explored the relationship between demographics and cue reactivity and reported that differences in cue-induced cravings  were not associated with examined sociodemographic variables such as age, education, employment etc. ,38,39. TFindings from six out of seven studies that examined the relationship between variables relating to drug use and cue reactivity indicated significant effects of substance use-related variables on cue reactivity (except ). EkhtiaThree studies explored the relationship between personality attributes and craving in response to drug cues. Saladin et al. (2012) examined the factor alexithymia—a personality attribute characterized by a difficulty identifying and describing emotions—and predicted that it may contribute to addicted persons’ failure to report cue-elicited cravings. Contrary to their hypothesis, higher scores on the Toronto Alexithymia Scale factor 1 (a measure of difficulty in identifying feelings) were positively associated with cue-elicited craving and authors suggested that their results may need to be replicated . In a stSeveral studies found an association between cue-elicited craving and other measures of cue reactivity. For neural cue reactivity, for example, three studies revealed craving response to be significantly associated with increased activation in different regions of the brain related to the mesolimbic reward pathway during methamphetamine cue processing ,30,33. IWe reviewed eighteen studies that assessed efficacy of measures that targeted methamphetamine cue reactivity and organized them into broad categories of “pharmacological” and “non-pharmacological” methods. Studies using “non-pharmacological” methods were further grouped into non-invasive brain stimulation or behavioral techniques see .Five studies investigated the role of medications in the treatment of participants with methamphetamine abuse or dependence by targeting cue reactivity. These studies assessed oral naltrexone, aripiprazole and bupropion using slightly different randomized trial designs  in clinical samples. Three studies assessed the potential effects of 50 mg naltrexone (NTX) in attenuating cue reactivity and generally found NTX to be a promising pharmacotherapy through reducing cue-induced craving or other neural and physiological cue reactivity and altering functional connectivity ,44,45. Fn = 4) or transcranial direct current stimulation (tDCS) (n = 2) of the dorsolateral prefrontal cortex (DLPFC) to evaluate changes in cue-induced craving [Six studies assessed either repetitive transcranial magnetic stimulation (rTMS)  program to reduce attention bias towards drug-related stimuli was tested in one study which indicated that the ABM training did not lead to reductions in craving for methamphetamine or in attentional bias to methamphetamine-related stimuli . The autThis review seeks to identify and describe the type of available literature related to cue reactivity and the use of cue paradigms in methamphetamine research. A scoping review was chosen as it helps to provide a framework to (1) map the key concepts and insights, (2) summarize and share existing research findings and (3) determine gaps in the current literature. To the authors’ knowledge, only one article published in 2010 had provided a preliminary review on studies that apply the different cues as main methods of craving induction in laboratory settings among human methamphetamine dependents . Six stun = 16) followed by China (n = 11), with an under-representation of studies from other parts of the world. Importantly, three overarching themes were identified in this review and they provided us with further knowledge on (1) the effect of methamphetamine cue exposure, (2) possible factors that correlated with cue reactivity, and (3) strategies that could modulate cue reactivity.The current scoping review identified 32 relevant articles that either involved the use of a cue reactivity paradigm in their studies or allowed us to understand more about cue reactivity in methamphetamine abuse or dependence. Our results revealed high variability in the terms of reporting and conduct of the included studies and sources of heterogeneity include differences in sample groups, methodologies, type of cue reactivity paradigms, interventions and comparators, outcome measures and even research purposes across the studies. The included articles were predominantly from two countries—USA  for use in methamphetamine dependence . CravingOur review suggested that oral naltrexone (NTX), an opioid receptor antagonist, could be a potential pharmacotherapy for preventing relapse to methamphetamine. Three studies examined the efficacy of NTX in modulating or reducing cue-induced craving and all revealed positive results in favor of the drug ,44,45. FFor behavioral intervention, the use of either cue extinction or counterconditioning strategies appeared to be promising in preventing relapse due to cue reactivity based on findings from our review. Studies among healthy humans have consistently shown that conditioning, the process by which contextual cues becomes associated with methamphetamine through repeated pairing, is the key to understanding addiction and problematic drug use ,61. LiteAlthough it is not the main tenet of a scoping review, it must be acknowledged that we did not formally assess the methodological quality of the studies, particularly those that evaluated interventions. Among which, we also included one qualitative study and few of these studies did not include a control group with only a pre-post study design. The majority of studies had small sample sizes except one, which had over 1000 study participants. The current review also did not include grey literature , which may have led us to miss out some relevant studies or possible interventions. The restriction of searches to well-established academic databases and exclusion of grey literature may also lead to a potential publication bias as studies with null findings are less likely to be published in peer-reviewed journals.Cue reactivity studies have been shown to be useful for understanding how craving would lead to continued drug-seeking behaviors and relapse among abusers in a real-life environment. Exposure to methamphetamine-associated cues can significantly induce measurable craving or other autonomic reactivity in laboratory settings. Our scoping review provides insights into the type of cue reactivity, as well as in identifying and characterizing specific factors that modulate this reactivity in methamphetamine research. The use of cue reactivity paradigms also has important implications for the development of new pharmacological and psychosocial interventions for methamphetamine relapse prevention. Further studies on cue-induced craving are necessary to explore the effects that this notion could bring to treatment approaches.


In one case study, Faiss and Rapillard also reported the benefits of 150 repeated sprints at a simulated altitude of 3,300 m over 10 days in a professional cyclist. Advancing our understanding of RSH-induced adaptations also requires relevant research in sport-specific ecological test settings to be conducted. In this context, the effects of a 4-wks in-water swimming specific RSH program on freestyle swimming performance have been evaluated by Camacho-Cardenosa et al., but failed to induce further benefits than similar training in normoxia, possibly due to the selection of a sub-optimal hypoxic dose.The repeated-sprint training in hypoxia (RSH) paradigm requires the completion of maximal, short duration  efforts interspersed with incomplete recovery periods (≤60 s) in hypoxic environment  involves the repetition of long (~30 s) “all-out” efforts and is an effective training strategy for upregulating mitochondrial biogenesis and exercise capacity  induce greater adaptations in cycling endurance performance that may be related to muscle perfusion and metabolic changes .Other systemic or local LLTH methods based on the repetition of “all-out” efforts can be used to induce a potent physiological stimulus, up-regulate signaling pathways and eventually maximize performance outcomes. For instance, when hypoxia is induced by voluntary hypoventilation at low lung volume (VHL) , national sport institutes , professional clubs  or national federations , and private gyms  are now equipped with altitude simulation facilities. However, innovative LLTH methods such as resistance training in hypoxia can also be implemented in high performance centers located at moderate natural altitude ranging 1,800–2,400 m . In testing the viability of using mean propulsive velocity to adjust the load in the countermovement jump at a terrestrial altitude of 2,320 m, Girard et al.) and their physiological and performance consequences remain unresolved. The effectiveness of pre-acclimatization strategies for high altitude exposure should be explored (Fulco et al., Although an extensive number of LLTH studies have been published so far, several issues regarding the implementation of many innovative methods described in the updated panorama that we proposed (All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Research led by Katrin Andreasson suggests that fixing age-induced metabolic defects in myeloid cells would suffice to reverse cognitive impairment and to restore synaptic plasticity to the level of young subjects, at least in mice. This opens up the possibility to develop rejuvenating strategies by targeting immune dysfunction. Ageing is accompanied by many molecular and cellular alterations, which lead to a gradual deterioration of physiological functions. While lifestyle and genetics have a big impact on the speed of these ageing processes, ageing per se is usually seen as not easy to revert . NeurocoThe immune system is drastically affected with ageing . While tCellular metabolism has emerged as a key player in the regulation of immune function –9, start2 (PGE2) signaling in the ageing myeloid compartment, mediated by the age-dependent upregulation of EP2, one of the four PGE2 receptors. They show that this signaling cascade is also altered in human monocyte-derived macrophages (MDMs). PGE2, also known as dinoprostone, is a lipid messenger which acts as a key mediator of inflammation, downstream of the cyclooxygenase 2 (COX-2) pathway. Among other responses, this increase leads to the activation of glycogen synthase (GYS1), the key enzyme converting glucose into glycogen. This channeling of glucose away from glycolysis might thus be at the origin of the metabolic dysfunction of macrophages.In a recent publication, Minhas and colleagues have elucidated this cascade using an impressive set of in vitro and in vivo experiments in mice and in human myeloid cells . They fo2, specifically in the myeloid cells  of aged mice proves to be indeed an effective strategy at multiple levels. First, it rescues the expression of some of the immune factors upregulated with age, both in the plasma and in the hippocampus. Second, the loss of EP2 also reduces glycogen levels, normalizing the metabolic state and the associated mitochondrial defects observed in old macrophages. A similar effect is also mimicked by directly inhibiting GYS1 via shRNAs in human MDMs. Third, strikingly, aged EP2 cKO mice appear to be completely protected from a decline in hippocampal-related memory functions with ageing.Conditional knockout of EP2 cKO microglia were able not only to promote Aβ clearance, but also to prevent synaptic injury and memory deficits [2 ablation remained somewhat unclear. With their recent study, Minhas et al. tried to disentangle this further [2 antagonist in aged mice, they could show a significant reduction in the levels of age-associated immune factors and a global restoration of the metabolic profile. This treatment is sufficient to rescue the age-related long term potentiation defects in the hippocampus, along with cognitive function.Interestingly, the same group showed that these mice displayed beneficial effects when crossed to a mouse model of Alzheimer’s disease. EPdeficits . However further . By injeOverall, these data support an upstream role of peripheral myeloid cells in orchestrating the process of brain ageing, underscoring the important cross-talk between the immune and the central nervous systems. This study nicely illustrates the importance of the cellular metabolic state of myeloid cells: it highlights that not only the availability of glucose, but also its channeling into different pathways (glycolysis versus glycogen synthesis) contributes to maintaining proper myeloid function .2 signaling in myeloid cells to a youthful state is enough to prevent age-dependent cognitive decline However, it should be noted that, although GYS1 knockdown recovered aging-related metabolic and inflammatory changes in human macrophages, all interventions reversing molecular and cognitive changes in aged mice were performed by inhibiting the whole signaling downstream to E2 receptors. Thus, the improved cognitive outcome could also be the result of the general attenuation of PGE2 signaling, not only due to restored metabolic function. Furthermore, it was recently shown that that aging-related sarcopenia is mediated by decreased PGE2 signaling in both myofibers and muscle-resident macrophages [2 signaling prevented sarcopenia in old mice, while its disruption was sufficient to induce sarcopenia in young mice [2-mediated effects during aging might be tissue-specific and these differences should be considered in future therapeutic approaches.Surprisingly, restoring the PGErophages . Strikinung mice . These d


The emerging evidence on the interconnectedness between the gut microbiome and host metabolism has led to a paradigm shift in the study of metabolic diseases such as obesity and type 2 diabetes with implications on both underlying pathophysiology and potential treatment. Mounting preclinical and clinical evidence of gut microbiota shifts, increased intestinal permeability in metabolic disease, and the critical positioning of the intestinal barrier at the interface between environment and internal milieu have led to the rekindling of the “leaky gut” concept. Although increased circulation of surrogate markers and directly measurable intestinal permeability have been linked to increased systemic inflammation in metabolic disease, mechanistic models behind this phenomenon are underdeveloped. Given repeated observations of microorganisms in several tissues with congruent phylogenetic findings, we review current evidence on these unanticipated niches, focusing specifically on the interaction between gut permeability and intestinal as well as extra-intestinal bacteria and their joint contributions to systemic inflammation and metabolism. We further address limitations of current studies and suggest strategies drawing on standard techniques for permeability measurement, recent advancements in microbial culture independent techniques and computational methodologies to robustly develop these concepts, which may be of considerable value for the development of prevention and treatment strategies. The past few years have witnessed significant headway in the exploration of mechanisms underlying cardiometabolic diseases such as type 2 diabetes (T2D), obesity, and related cardiovascular comorbidities. Specifically, microbiome research has gained significant traction, placing it at the center stage of a plethora of noncommunicable diseases, ranging from autoimmune disorders of the gut such as inflammatory bowel disease (IBD)  to a widWhile the crosstalk between the gut microbiome and peripheral organs such as the brain, liver, adipose tissue, muscle, and pancreas has emerged as a crucial factor for homeostatic systems, the search for channels of communication has led to the revival of the concept of “leaky gut”. This concept is based on the notion of translocation of whole bacteria, bacterial products such as metabolites, and bacterial wall components into the circulation and distant tissues, contributing to remote organ injury in metabolic disease. Although bacterial translocation is a physiological process crucial for host immunity , “patholAs subclinical inflammation has been evidenced in obesity leading to local and systemic insulin resistance and the development of cardiovascular disease, it is important to track similar mechanisms of action as seen in SBP in metabolically active organs and to understand the role of remote organ–bacteria interaction in the pathogenesis of noncommunicable diseases, which in turn could open novel avenues for prevention and therapy.Although growing beyond the scope of infectious disease for several decades, microbiome research has seen a remarkable boost in the last two decades thanks to the development of sequence-based techniques for the detection of microbes which escape traditional culture-based approaches. More importantly, there have been efforts to establish systematic approaches to decipher structure and function of the gut microbiome in health and disease with the Human Microbiome Project (HMP)  as well Early indications on the connection between gut microbiome and obesity stem from studies in both mouse models and humans ,22,23,24Examination of identical twins, who were either concordant or discordant for obesity, showed that the development of obesity is related to microbiome shifts deviating from a “healthy core microbiome”, reflected by a decreased microbial diversity and altered representation of metabolic pathways . Gut micBifidobacteria and Verrucomicrobiae [Akkermansia muciniphila, which has been associated with reduced weight and improved insulin sensitivity in mouse models [Similarly, a bacterial signature is noted in T2D: In the largest study to date, reduced microbiome diversity as well as significant reduction in butyrate producers were evidenced in 345 Chinese patients with T2D, while the number of opportunistic pathogens increased . Comparaicrobiae . A repree models  and humae models  as well e models .Verrucomicrobiae after fecal microbiome transfer (FMT) was associated with an antidiabetogenic effect, while increased Proteobacteria was associated with insulin resistance. Consistent with weight loss intervention, the improvement of insulin sensitivity was largely driven by baseline intestinal microbiota composition [Evidence from cross-sectional studies was corroborated in intervention studies: Gut microbiome transfer from healthy subjects into patients with T2D resulted in a significant improvement in insulin sensitivity over a period of six weeks ,33. To tLactobacilli and that replenishing lost strains was associated with a reduced induction of Th17 Cells and reduction in hypertension [Similar observations were made on the relation of gut microbial diversity with nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD) ,35,36, artension .Prevotella and Bacteroides is an artificial product of data compositionality. The authors further associated the occurrence of low-cell-count enterotypes with Crohn’s disease, further underlining a relationship between intestinal bacterial load, microbiome composition, and inflammation [Quantitative changes of the microbiome have also been reported in the literature for several metabolic diseases. Sabaté et al. reported a small intestine bacterial overgrowth (SIBO) prevalence of 17.1% in subjects with severe and morbid obesity . In thatammation .Eggerthella lenta-induced Th17 activation and subsequent colitis [Quantitative and qualitative microbiome changes do not occur in pure isolation, and the interaction between diet and the gut microbiome on the one hand and impact of diet on intestinal permeability on the other have been the subjects of several recent extensive reviews and original work ,51,52,53 colitis  and that colitis . However colitis , mediati colitis . Similar colitis  and regu colitis .In conjunction with evidence that environmental factors such as diet or other industrial food additives , as E. coli and other Enterobacteriaceae and Enterococci have been more often shown to translocate than other gut commensals [Beyond the interplay between overall microbiome composition, inflammatory intestinal tone, and permeability, it is worth noting that some bacteria are likely to translocate more readily than others, this being related to their ability to forgo or debilitate host defense mechanisms. Gram-negative bacteria such as mmensals ,69. Notwmmensals .Although many associative studies have shown links between quantitative and qualitative changes in gut microbiome and metabolic disease, a closer look at the three main protective instances is warranted to discern mechanisms involved in connecting these events with metabolic pathologies.The gut-associated lymphoid tissue (GALT) is the largest immunological organ of the human body. The innate immune system as a first line of defense entitles highly conserved recognition of microbe-associated molecular patterns (MAMPs), active on epithelial surfaces as well as within endosomes via TLRs or cytoplasmic NLR . Although perpetrating commensals are rapidly killed by macrophages, intestinal dendritic cells (iDCs) carry small amounts of live bacteria, which can then induce selective Immunoglobulin A (IgA) production in mesenteric lymph nodes (MLNs). These have been suggested to be at the center of bacterial translocation, as they restrict iDCs to the mucosal immune compartment. When MLNs are surgically removed, specific commensal IgA response is abrogated and bacterial systemic penetration is possible, as commensals can be retrieved from the spleens of animals without MLNs .Activated B-cells and T-cells are recruited after bacterial translocation. B-cells produce commensal specific IgA which abrogates the translocation beyond the gut mucosa . Mice deAkkermansia muciniphila, which has been shown to reverse high-fat diet-induced metabolic disorders, including insulin resistance, and which has been shown to increase endocannabinoids tone controlling inflammation [Bacteria close to the intestinal surface of the intestine are monitored and kept at a distance by several secretory elements encompassing antimicrobial proteins (AMPs) secreted mostly by Paneth cells after sensing of whole bacteria or lipopolysaccharides (LPS) via TLR-dependent MYD88 activation . These iammation . Moreoveammation  as well ammation . In factammation  or surgiammation . Anotherammation . HFD leaThe mucosal lining of the gut consists of a single layer of cells constituting the mechanical component of the gut barrier. Bacterial translocation can occur through the paracellular route, which is regulated by tight junctions (TJs) or the transcellular route. Damage to the barrier function is reflected in increased permeability, which can be evidenced via dual sugar absorption tests. Specifically obesity has been associated with increased gut permeability , which cBeyond inflammation in the gut, obesity, insulin resistance, and subsequent metabolic diseases are frequently preceded and presumably precipitated by inflammation and dysfunction in visceral adipose tissue, liver, muscle, pancreas, and the brain. To connect intestinal health and microbiome profiles with metabolic health, most studies have focused on the study of surrogate parameters to evidence enteric permeability changes instead of performing functional tests .Cani et al. have coined the term “metabolic endotoxemia”, which describes an increased systemic exposure to bacterial LPS in obesity and insulin resistance . The assUntil now, most research investigating bacterial translocation and metabolic disease has focused on LPS measurements, leading to a large body of evidence. LPS are components of the outer membrane of Gram-negative bacteria and are shed into the circulation mostly once a bacterium has been neutralized. Interestingly, fat intake has been associated with increased postprandial LPS, distributed in the circulation via formed chylomicrons , and dirBacterial products are not markers of bacterial translocation per se, but their shifts have been associated repeatedly with the concept of leaky gut and increased intestinal permeability as they are central to maintaining the intestinal barrier  and funcAmong other surrogate parameters, increased concentrations of circulating calprotectin are reported in T2D, which were associated with obesity, metabolic syndrome status, as well as myocardial infarction without being an independent predictor for cardiovascular disease (CVD) . CalprotMarkers such as endotoxin core antibodies (EndoCAb) have also been used and shown to be superior to endotoxin in one studied cohort, although only IgM antibodies were significantly different between lean subjects, patients with obesity, and T2D . ConsideOther widely used surrogate markers such as zonulin, which increases intestinal permeability by dismantling intercellular intestinal tight junctions , are basA few studies have implemented functional testing of intestinal permeability using dual sugar absorption tests. There is robust data supporting the use of such tests . LactuloPreclinical methods such as Ussing chambers, histology, electron microscopy, and the gold standard method of measurement of transepithelial electrical resistance  are limiIn summary, the line between increased gut permeability and bacterial translocation seems to be a hazy one, where sometimes neither increased gut permeability nor actual increased bacterial translocation is evidenced under the convenient title of leaky gut in the realm of permeability-induced metabolic impairment.Bacterial signals can influence systemic inflammatory tone by increasing local inflammation in the gut, therefore leading to a widespread inflammatory response reaching several organs, or could potentially alter local signals in peripheral organs, leading to an overreaching inflammation and exponentiating insulin resistance. It is now recognized that several tissues play important roles in the advent of insulin resistance and related sequelae. Specifically, adipose tissue, the liver, muscle, gastrointestinal tract, the pancreas (β- and α- cells), kidney, and the brain collectively comprise DeFronzo’s ominous octet and contribute to the development of glucose intolerance and insulin resistance in T2D . ConsideTNFA and Interleukin 1B (IL1B). Deletion of macrophages recruiting chemokine Ccl2 and its receptor in a knockout mouse model led to a decreased infiltration of colonic macrophages and inflammasome activation in the colon. This was associated with a decreased adipose tissue inflammation along with improved glucose and insulin tolerance compared with wild-type mice, albeit having similar body weight [Chronic inflammation of adipose tissue has been suggested as an underlying factor in obesity and insulin resistance. Kawano et al. have identified the colon as the first organ to respond to a HFD and contribute subsequently to adipose tissue inflammation and insulin resistance. HFD led to several morphologic as well as immunologic changes in the colon, including increased infiltration of macrophages accompanied by increased proinflammatory gene expression including y weight . These ry weight , possibly weight  and incry weight ,143.E. coli (ETEC) colostrum led to an improvement of insulin resistance and overall metabolic markers such as lowered lipid levels [The body of work connecting liver disease with bacterial translocation is striking and has been elegantly reviewed in several works focusing either on steatohepatitis  or on lid levels .Leptin, an adipokine predominantly secreted by adipose tissue, enhances TNF-α production in LPS-stimulated Kupffer cells and its administration prompted elevated LPS-induced hepatic TNF-α production in wild-type rats. TNF-α in the liver of leptin-deficient Zucker rats, on the other hand, was unaltered after LPS stimulation , furtherTranslocation of bacterial and endotoxic bacterial components into the pancreas has also been related to changed function of this highly metabolic active tissue. In models of acute pancreatitis, impairment of small intestine mucosa evident from increased mucosal permeability to fluorescent latex microspheres and apical villi damage has been observed. Interestingly, labeled bacteria from the intestine were traced from the gut to mesenteric lymph nodes and to the pancreas, further pointing to a gut–pancreas inflammatory axis .Lactobacillus strains led to reduced body fat alongside increased circulating levels of Angptl4, suggesting a relevant contribution of a specific microbial population to the expression of Angptl4 [Although involvement of the pancreas in T2D has been thought of as a strictly endocrine functional loss of the organ, exocrine activity of the pancreas is paramount to normal digestive function and fat storage. Enterocyte-produced Angiopoietin-like 4 (Angptl4) is a potent inhibitor of gut luminal and pancreatic lipoprotein lipase (LPL) activity  and is i Angptl4 . The end Angptl4 . Nonobes Angptl4 . This wa Angptl4 .A direct modulation of pancreatic cell function has been mostly observed in experimental models, where LPS impaired insulin gene expression via TLR4-dependent NF-kB signaling .Akkermansia muciniphila in the gut [The intestine plays a pivotal role in T2D, and therapeutic approaches based on intestinal mechanisms have shown very convincing results related not only to weight loss but also overall cardiovascular health . The int the gut .Although GLP-1 receptor agonists show promising health effects in T2D, an idiosyncratic lack of response has been evidenced in some patients, suggesting GLP-1 resistance of unclear origins. Grasset et al. identified a set of bacteria in the ileum , which impaired the GLP-1-activated gut–brain axis, impacting gastric emptying and insulin secretion . MoreoveBifidobacteria and Lactobacillus and was shown to be GLP-2-dependent [In another study, microbially derived tryptophan metabolite indole led to an increase of GLP-1 production, which ceased after prolonged exposure to the metabolite, supporting the role of the microbiome in modulating L-cell function and therefore subsequently connecting host responses to the environment . GLP-2, ependent . Howeverependent .The average human body consists of approximately 42% and 36% of muscle in males and females, respectively. More importantly, insulin resistance in the skeletal muscle is considered the primary injury in T2D, observable decades before hyperglycemia and β-cell dysfunction were noted . SubclinThe increased recognition of a putative gut–brain axis partly modulated by the gut microbiome and its links with metabolic, neurodegenerative , functioAlthough the largest body of evidence has been put forward for the gut microbiome, many tissues accommodate adapted microbial consortia, which are finally accessible with culture-independent techniques. While several studies have focused on surrogate parameters or the evidence of bacterial components in the circulation to link intestinal permeability with metabolic disease, there is scarce research investigating the presence of bacterial components or bacteria in metabolically active tissues, their putative contribution to microenvironment changes and their relationship with tissue dysfunction and local inflammation. Moreover, little is known about tissue selectivity of bacteria, their source of origin, whether they are alive and occupying a relevant ecological niche, or whether there exist disease-specific extra-intestinal bacterial signatures .To this aspect, a limited number of studies have investigated the presence and composition of bacterial DNA in regard to its relationship with metabolic risk or disease in several tissues. While the evidence of the presence of bacteria in blood of even healthy individuals is accumulating ,199, eviA first study reporting a link between circulatory bacterial load and metabolic disease was published in 2011. In a cohort of 3280 subjects followed for 9 years, 16S rRNA gene concentration was significantly increased in subjects who developed T2D over time  and was E. coli in mesenteric adipose tissue of high-fat diet-fed mice. Similarly, Burcelin et al. have evidenced the presence of bacterial DNA in human adipose tissue [Clostridium histolyticum only, which serves as a source for the collagenase used to isolate mature adipocytes [Ralstonia a year later [The evidence for bacteria in other tissues related to the ominous octet has been practically nonexistent till of late. There is a large body of evidence for bacterial infections of the pancreas in acute pancreatitis but no data supporting contamination of the pancreas without acute inflammation. An exception is the recent evidence of oral bacteria in intraductal papillary mucinous neoplasms (IPMNs) preceding invasive pancreatic cancer . Underste tissue . In succe tissue , especiaipocytes . Culturiar later . Bacteriar later . The autThe lack of representative negative controls in these particular studies is the Achilles’ heel of all studies searching for compartment-specific bacterial signatures in human tissues, where negligible or very small amounts of bacterial DNA are expected.in situ hybridization (FISH). We moreover quantified and sequenced 16S rRNA gene content in 75 patients with obesity and with or without T2D and could show that both bacterial quantity and taxonomy were associated with markers of inflammation and insulin resistance. This was further corroborated with functional tests in immortalized human subcutaneous preadipocytes where bacterial DNA challenge led to a bacterial DNA concentration-dependent stimulation of TNFA and Interleukin 6 (IL6) [Very recently, Anhê, Jensen et al. published data comparing the bacterial composition and load of liver, subcutaneous, visceral, and mesenteric adipose tissues as well as plasma samples. They noted the highest abundance for bacterial DNA in visceral adipose tissue and liver samples, whereas subcutaneous and mesenteric adipose tissue samples had similarly reduced amounts of bacterial DNA. Plasma samples did not contain significantly more bacterial DNA than negative controls. The authors were able to delineate preferential compartmentalization of eight specific genera in adipose tissue, whereas plasma samples differed in two tissue-specific genera. Interestingly, mesenteric adipose tissue displayed pronounced taxonomial differences as compared with other fat depots and an increased relative abundance of gut colonizers, consistent with the natural anatomical route of a presumed gut–liver axis. Whereas no differences in bacterial load were found within tissues between subjects with and without T2D, subjects without T2D displayed a significantly increased bacterial diversity in bacterial signature of mesenteric adipose tissue, pointing to a link between tissue-specific bacterial signature and glucose tolerance similar to observations of microbial diversity in gut microbiome studies. A specific strength of this study is the extensive inclusion of negative controls at each step of the preanalytical and experimental procedure accounting for operation field contamination at tissue collection, environmental contamination during tissue manipulation including air samples from surroundings and swab controls for used surfaces, as well as negative controls for DNA extraction, amplification, and sequencing, making it one of the first studies to present contamination-aware evidence of tissue-specific bacterial compartmentalization with a T2D extra-intestinal microbial signature, which was independent of obesity . This ev 6 (IL6) . Evidence in this area has not been without controversy. Recently, Schierwagen et al. published a letter of response noting the important challenges to tackle when working with low-microbial-biomass samples . Beyond Several publications with converging lines of evidence support increased intestinal permeability and bacterial translocation as perpetrators in the development of metabolic disease. This evidence has linked obesity and T2D with altered qualitative and quantitative gut microbiome changes, increased permeability, and ensuing local inflammation in metabolically active remote organs as well as systemic inflammation accounting for an increased systemic insulin resistance. In the near future however, this area of research needs to move beyond association studies towards functional approaches to understand potential directionalities and mechanisms involved. Additionally, there is a need to elaborate hypotheses in preclinical and clinical settings including intervention studies, which actively modulate the gut microbiome and intestinal barrier to establish the relevance of gut permeability in human metabolism. The reason for this being that changes in the gut microbiome as well as intestinal permeability can both lead to inflammation, which itself can modulate these two adjacent axes, making establishing a clear sequence of events almost unmanageable.In addition to bacteria, the intestine is also populated by other organisms such as archaea, yeast, and fungi as well as viruses, phages which can contribute similarly to host–microorganisms interactions ,222,223.Moreover, there is a need to define “bacterial translocation”: whereas some authors equate increased intestinal permeability with bacterial translocation, others have used unreliable surrogate parameters, which reflect increased permeability to bacterial products such as LPS or host markers conferring a possible reaction to bacterial products. They all heavily depend on healthy subjects as a point of reference, making conveying absolute values and states of health and disease impossible, thereby impeding the standardization of the definition for “leaky gut” or bacterial translocation.To this extent, the evidence of bacteria in the circulation and metabolically active organs should be the most direct proof for bacterial translocation. While the body of work in this area of research has been steadily increasing, with several works painting a coherent picture, it has not been without criticism, particularly in light of the controversy surrounding the presence of a placenta microbiome . This crRecent developments including multitechnical approaches  and moreTo this extent, the potential inherent to this field supports coordinated efforts to revisit many of the concepts introduced in the framework of this review. The aim would be to initiate clear, comprehensive, and nuanced approaches, which reflect our times and the immense development achieved in this area of research, in order to establish the clinical significance of these concepts and tap their therapeutic and preventative potential.


Ion channels/pumps are essential regulators of innate immune cell function. Macrophages have been increasingly recognized to have phenotypic plasticity and location-specific functions in the lung. Transient receptor potential vanilloid 4 (TRPV4) function in lung injury has been shown to be stimulus- and cell-type specific. In the current review, we discuss the importance of TRPV4 in macrophages and its role in phagocytosis and cytokine secretion in acute lung injury/acute respiratory distress syndrome (ARDS). Furthermore, TRPV4 controls a MAPK molecular switch from predominately c-Jun N-terminal kinase, JNK activation, to that of p38 activation, that mediates phagocytosis and cytokine secretion in a matrix stiffness-dependent manner. Expanding knowledge regarding the downstream mechanisms by which TRPV4 acts to tailor macrophage function in pulmonary inflammatory diseases will allow for formulation of novel therapeutics. Ion channels and transporters are rapidly being recognized as essential for basic physiological functions of immune cells , 2. Howein vivo biologic functions of both resident/recruited alveolar and interstitial macrophage populations are not fully understood. The microenvironment plays a key role in reprograming the monocyte/macrophage phenotypic response to lung injury  and carboxy-(COOH) terminal tails  channels are a family of 6 transmembrane domain proteins that are permeable to multiple cations including calcium . TRP chaal tails –47.It has been increasingly recognized that cellular responses depend on the biophysical properties of the surrounding lung tissue environment , 49. Thuin vitro and protects against infection-associated lung injury in vivo , IgG-coated latex beads, in vitro in response to LPS  express equal amounts of TRPV4 that was functionally active with or without LPS. TRPV4 in macrophages functions to effectively phagocytize both non-opsonized, e to LPS . The LPSe to LPS . TRPV4 h in vivo . Taken t in vivo .in vivo relevance to human disease, and molecular mechanism by which TRPV4 mediates its phagocytic and cytokine secretory effects was determined. TRPV4 was found to function to protect the lung from injury in an experimental model of Pseudomonas aeruginosa pneumonia in intact mice  and carboxy (COOH) terminal intracellular tails, the key signaling molecules that interact with TRPV4 to enhance macrophage phagocytosis and limit cytokine secretion is an active area of investigation. It remains possible that the TRPV4 interacting partners are not TLR4-dependent, as other data suggests that TRPV4 interacts with PI3K to mediate pulmonary fibrosis  other types of infectious stimuli , (b) sex differences, and (c) transcriptional/epigenetic mechanisms. An important goal of future work is to integrate mouse and human macrophage experiments, however there is limitation to this approach. It is well-known that mouse models do not fully recapitulate human disease, and the mouse immune system is programmed differently than that of humans  functionally active in macrophages, (b) required for effective non-opsonized and opsonized phagocytosis in vitro and in vivo, and (c) required for secretion of an anti-inflammatory cytokine profile by macrophages. These phagocytic and cytokine effects in macrophages were both dependent on matrix stiffness in the range of injured or fibrotic lung ((a) protects the lung from injury after P. aeruginosa pneumonia, (b) mediates the lung injury effects through MAPK molecular switching, and (c) is required for effective macrophage phagocytosis in human macrophages. This MAPK switching effect in macrophages was also dependent on matrix stiffness in the range of injured or fibrotic lung (In summary, macrophages function in the lung to maintain homeostasis and clear environmental particles and pathogens. Extensive macrophage heterogeneity and plasticity allows for fine-tuning of the inflammatory response upon inflammation or infection. Ion channels have been shown to play a key role in regulating innate immune function and contribute to the pathogenesis of inflammatory/infectious lung diseases. The cation channel TRPV4 has been implicated in lung diseases associated with parenchymal stretch and inflammation or infection. The data outlined in this review show the importance of macrophage TRPV4 in response to infection and lung injury. The data shows that TRPV4 is tic lung . In additic lung . CollectRS, BS, LG, and MO reviewed the literature and wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The NLRP3 inflammasome represents a critical inflammatory machinery driving pathology in many acute  and chronic  human disorders linked to the activity of IL-1 cytokines. Although the therapeutic potential of NLRP3 is undisputed, currently no clinically approved therapies exist to target the NLRP3 inflammasome directly. The recent discovery of BTK as a direct and positive regulator of the NLRP3 inflammasome has, however, raised the intriguing possibility of targeting the NLRP3 inflammasome via existing or future BTK inhibitors. Here, I review the mechanistic basis for this notion and discuss the molecular and cellular role of BTK in the inflammasome process. Specific attention will be given to cell-type dependent characteristics and differences that may be relevant for targeting approaches. Furthermore, I review recent (pre-)clinical evidence for effects of BTK inhibitors on NLRP3 activity and highlight and discuss open questions and future research directions. Collectively, the concept of targeting BTK to target NLRP3-dependent inflammation will be explored comprehensively at the molecular, cellular and therapeutic levels. Compared to anti-IL-1 therapy, inhibiting BTK probably would still target NLRP3 proximally enough to block all known NLRP3-dependent effects, both in terms of alarmins and pyroptosis. Of course similar indirect NLRP3 targeting approaches could be envisaged for other known direct NLRP3 regulators [e.g., protein kinase D (Zhang et al., S. aureus in vivo infection model, where ibrutinib administration led to reduced bacterial control (Liu et al., Btk-deficient Xid mice were also protected in a polymicrobial sepsis model, with reduced NLRP3 activation contributing to the phenotype (O'Riordan et al., in vivo could counteract the seemingly negative regulatory role of BTK proposed (Mao et al., Insights into the plausibility of the targeting NLRP3 via BTK firstly came from our description of a reduced NLRP3 activity in patients on ibrutinib therapy (Liu et al., in vivo models conditional BTK alleles that circumvent the simultaneous ablation of B cell function characteristic for BTK KO mice will be highly desirable. These should be complemented by ex vivo studies of NLRP3 activity in cancer patients treated with the increasingly specific BTK inhibitors or biomaterial from healthy volunteers and XLA patients. Furthermore, one needs to bear in mind that BTK is only a partial NLRP3 regulator and that some IL-1 can still be released in the absence of BTK, albeit at drastically lower levels (Liu et al., Given the effect of BTK inhibition on B cell development and function, the applicability of targeting NLRP3 via current BTK inhibitors at this stage appears limited to and most suitable for acute settings like myocardial infarction, stroke or COVID-19 rather than chronic NLRP3-mediated diseases such as Alzheimer's or Parkinson's disease. If strategies could be found that can discriminate between BTK function in B cells and macrophages, e.g., by targeted delivery or inhibitors based on protein-protein interactions, more long-term conditions may also come into view therapeutically. On the back of the promising studies described above, further mechanistic work seems mandatory. Additionally, in pre-clinical The author confirms being the sole contributor of this work and has approved it for publication.The author declares that his laboratory received funding from Novartis during the course of the generation of this review. The funder was not involved in the collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.


Genome-wide association studies (GWAS) have led to the identification of hundreds of susceptibility loci across cancers, but the impact of further studies remains uncertain. Here we analyse summary-level data from GWAS of European ancestry across fourteen cancer sites to estimate the number of common susceptibility variants (polygenicity) and underlying effect-size distribution. All cancers show a high degree of polygenicity, involving at a minimum of thousands of loci. We project that sample sizes required to explain 80% of GWAS heritability vary from 60,000 cases for testicular to over 1,000,000 cases for lung cancer. The maximum relative risk achievable for subjects at the 99th risk percentile of underlying polygenic risk scores (PRS), compared to average risk, ranges from 12 for testicular to 2.5 for ovarian cancer. We show that PRS have potential for risk stratification for cancers of breast, colon and prostate, but less so for others because of modest heritability and lower incidence. In cancer many gene variants may contribute to disease etiology, but the impact of a given gene variant may have varied effect size. Here, the authors analyse summary statistics of genome-wide association studies from fourteen cancers, and show the utility of polygenic risk scores may vary depending on cancer type. The number of discoveries varies widely across cancers, largely driven by available sample size, which reflects, in part, disease incidence in the general population. However, specific cancers, e.g., chronic lymphoid leukemia (CLL)3 and testicular cancer4, are notable for unexpectedly high numbers of genome-wide significant discoveries from GWASs of relatively small sample size. Previous studies have also reported that these two cancers have high heritability5. Across cancer types, polygenic risk scores (PRSs) show varying levels of risk stratification depending on the heritability explained by the identified variants and the disease incidence rates in the population12. Their potential clinical utility would depend not only on the level of risk stratification but also on other factors such as the availability of appropriate risk-reducing interventions for those identified as at high risk.Genome-wide association studies (GWASs) have led to the identification of hundreds of independent cancer susceptibility loci containing common, low-risk variants13, referred to as GWAS heritability in this article, have shown that common variants have substantial potential to identify individuals at different levels of risk for many cancer types14. It remains, however, unclear how large the sample sizes of GWAS need to be to reap the full potential of PRS-based risk prediction. Herein we apply our recently published method15 to estimate the degree of polygenicity and the effect-size distribution associated with common variants  > 0.05) across 14 different cancer types, based on summary-level association statistics from available GWASs28 from populations of European ancestry  included in GWAS arrays31. Estimates of the number of susceptibility variants with independent risk associations vary from ~1000 to 7500 between the 14 cancer sites , melanoma is noteworthy because it is associated with a wider effect size distribution than other group 2 cancers. The estimated number of susceptibility variants in this group ranges from 1000 to 2000. GWAS heritability estimates indicate that aggregated common variants make a relatively small contribution to ovarian and endometrial cancer susceptibility. Finally, for the 3 GWAS with >25,000 cases each (group 3), prostate cancer is remarkable for having more variants with large effect sizes, namely, the underlying effect-size distribution has a heavier tail, compared with cancers of the breast and lung   would improve performance of PRS-based risk prediction versus using the stringent genome-wide significance level, but the anticipated gains would be generally modest  consist of etiologically heterogeneous subtypes that were not considered in our analyses due to lack of adequate sample sizes for appropriate subtypes for most of these cancer sites. Further studies of ancestry- and subtype-specific genetic architectures are needed to address these limitations.Our study is subject to several limitations. We may have underestimated the number of underlying common susceptibility loci, especially for those cancers for which current GWAS have small sample sizes36 and genetic risk prediction37. We have evaluated the merit of future GWAS only in terms of their ability to explain heritability and improve risk prediction. However, current and future discoveries have other major implications, including provident insights to biological pathways and mechanisms, potential gene–environment interactions, and understanding causal relationships through Mendelian Randomization analyses38. A number of these cancers are known to have rare high-penetrant risk variants, but for this study we have focused on estimating effect-size distribution associated with common variants. Furthermore, heritability analysis indicate that uncommon and rare variants could explain a substantial fraction of the variation of complex traits39, and thus it is likely that there are many unknown uncommon and rare variants associated with these cancers as well. In the future, characterization of heritability and effect-size distribution associated with the full spectrum of allele frequencies will require individual-level sequencing data on a substantially larger number of cases and controls.In our projections, we assume standard agnostic association analysis of SNPs without incorporating any external information on population genetics or functional characteristics of SNPs. It is, however, possible to incorporate various types of external information to improve power for discovery of associations40, tissue-specific genetic regulation of gene expression41, cell of origin42, the number of biological steps needed to transition from normal to malignant tissue43, mediation of genetic effects by underlying environmental exposures44, and the presence of heterogeneous cancer-specific subtypes28. A number of cancer types, including those of lung, oropharynx, and esophagus, which were associated with large numbers of SNPs with small average effect sizes, have known strong environmental risk factors and distinct etiologic subtypes. It is also noteworthy that testicular cancer also stands out for a large number of discoveries in cross-tissue expression quantitative trait loci analyses, likely indicating a stronger association of SNPs on gene expression levels for this tissue compared to others41.The observed differences in the underlying genetic architecture of susceptibility across cancers could be due to various factors, including the effect of negative selectionIn conclusion, our comprehensive analysis of 14 cancer sites in adults of European ancestry reveals that, while all sites have polygenic influences, there is substantial diversity observed in their underlying genetic architectures, which reflects important biology and also influences the utility of polygenic risk prediction for individual cancers. Our projections for future yields of GWAS across these cancers provide a roadmap for important returns from future investment in research, including the potential clinical utility of polygenic risk prediction for stratification of absolute risks in the population.28, we downloaded publicly available genome-wide summary-level statistics from the latest consortium-based analyses. For others, we obtained access to data through collaborative efforts with individual consortia. Details about individual studies, including the number of cases and controls, are provided in Supplementary Table We analyzed summary data from GWAS studies across 14 cancer types. For select cancer sitesWe consider a reference panel with ~1.07 million SNPs included in the HapMap3 and that had MAF > 0.05 in the 1000 Genome European Ancestry sample. Based on known LD among common variants, we expect these set of variants to provide high coverage for all common variants for European ancestry population and thus loss of information due to imperfect tagging of causal variants to be fairly minimal.46 and effect-size distribution using summary-level data15. First, we restricted analysis to SNPs within a set of reference ~1.07 million SNPs included in the HapMap3 and that had MAF > 0.05 in the 1000 Genome European Ancestry sample. Second, we excluded SNPs having substantial amounts of missing genotype data: sample sizes <0.67 times the 90th percentile of the distribution of sample sizes across all SNPs. Third, we excluded SNPs within the major histocompatibility complex region , which is known to have very complex allelic architecture and can have uncharacteristically large effects on some traits. Fourth, we removed regions that have SNPs with extremely large effect sizes to reduce possible undue influence of them on estimation of parameters associated with overall effect-size distributions. Using PLINK --clump, we identify all top SNPs that have associated chi-square statistics >80  >2.19) and removed all SNPs that were within 1-MB distance of or had an estimated squared LD >0.1 with those top SNPs. We added back the contribution of these top independent SNPs in the final reporting of the total number of susceptibility SNPs, estimates of total heritability, and various projections we made as a function of sample size of the GWAS.Across all cancers, we applied several filtering steps analogous to those used earlier for estimation of heritability15, a method we recently developed to characterize underlying effect-size distributions in terms of the total number of susceptibility SNPs (polygenicity) and a normal mixture model for the distribution of their effects. Specifically, it is assumed that standardized effects of common SNPs in an underlying logistic regression model on the risk of a cancer can be specified in the mixture distribution in the form We inferred common variant genetic architecture of the different cancers using GENESISαth quantile for the standard normal distribution. Similar to heritability calculations, we added back the contributions of independent top SNPs with very large effects to the number of expected discoveries and associated variances explained by the quantities Given the estimated effect-size distribution, we calculated expected discoveries and genetic variance explained using 48p value <548. The relative risk for those estimated to be at the 99th percentile or higher of the distribution of a PRS (compared to the average risk of the population) was calculated using the formula12As we quantify heritability in terms of the variability of the underlying “true” PRS, we used the formula29. The iCARE tool uses projected standard deviations of PRS at different GWAS sample sizes and age-specific cancer incidence rates available from the US National Cancer Institute-Surveillance, Epidemiology, and End Results Program (NCI-SEER) (2015) to obtain absolute risk distributions. In deriving absolute risks, we adjusted for competing risk of mortality due to other causes using the age-specific mortality rates from the Center for Disease Control WONDER database (2016). We then weighted the projected residual lifetime risk distribution at different baseline ages (in 5-year categories) based on the US population distribution of ages within 30–75 years, as observed in the estimated 2016 US Census. For cancers of the reproductive system, weights were based on the age distributions among males or females, as appropriate.For each cancer site, we projected the distribution of residual lifetime risk (up to age 80 years) for non-Hispanic white individuals in the general US population according to PRSs, which could be built from GWASs of different sample sizes. For any given age, we first obtain the distribution of residual lifetime risks based on a model for absolute risks developed using the iCARE tool that we have described earlierFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The IDF  Diabetes Atlas Committee has recently published the global estimates of diabetes prevalence for 2019 [Type 2 diabetes is a societal chronic disease associated with several macrovascular and microvascular problems, with cardiovascular complications being the leading cause of morbidity and mortality in these patients . HoweverAlthough treating type 2 diabetes complications is of utmost importance to protect organ damage, it is now increasingly recognized that even the more ambitious goal of diabetes remission may be possible . Thus, wThis review  also eleFinally, the last approach discussed in the narrative review of Hallberg  was low-However, one aspect of diabetes management, remission and prevention has not received enough attention in this nice article: physical activity/exercise. Physical activity and exercise are not only effective to prevent the development of type 2 diabetes in high-risk patients, but these behaviours are also useful to treat patients with type 2 diabetes and even lead to remission in some cases. Through several molecular mechanisms, regular physical activity/exercise can delay or prevent the development of type 2 diabetes ,19,20,21In conclusion, a diversity of personalized solutions, which must include a high-quality nutritional diet and a physically active lifestyle, along with other important lifestyle determinants such as alcohol consumption, smoking and sleeping habits, should be promoted in order to curb the type 2 diabetes epidemic sweeping the world. Together with population-based solutions that will lead to the development of environments promoting healthy behaviours, lifestyle medicine  should h


Phospho-lipid bilayer nanodiscs have gathered much scientific interest as a stable and tunable membrane mimetic for the study of membrane proteins. Until recently the size of the nanodiscs that could be produced was limited to ~ 16 nm. Recent advances in nanodisc engineering such as covalently circularized nanodiscs (cND) and DNA corralled nanodiscs (DCND) have opened up the possibility of engineering nanodiscs of size up to 90 nm. This enables widening the application of nanodiscs from single membrane proteins to investigating large protein complexes and biological processes such as virus-membrane fusion and synaptic vesicle fusion. Another aspect of exploiting the large available surface area of these novel nanodiscs could be to engineer more realistic membrane mimetic systems with features such as membrane asymmetry and curvature. In this review, we discuss the recent technical developments in nanodisc technology leading to construction of large nanodiscs and examine some of the implicit applications. Membrane proteins (MPs) play critical roles in health, disease and hence in drug design. A majority of approved systemic drugs target membrane proteins due to their involvement in a variety of cellular processes such as signal transduction, transport of ions and molecules across the cell membrane, and cell adhesion to surfaces  of size up to 50 nm without compromising on the sample homogeneity  helix has a diameter ranging from 0.6 to 1 nm, taking up around 8 nm2. This means a seven TM-helical protein, like a GPCR, would require around 56 nm2. These quick calculations could save time during the screening for the optimal ratio between proteins and lipids.The optimal ratio between the scaffold protein and lipid must be determined by a series of test assembly reactions and screening for sample homogeneity  into DCND and present these proteins at a very high density and forming multimeric assemblies. This protocol could be further developed to form 2D crystals of membrane proteins which may be useful for membrane protein structure determination using techniques such as X-ray free electron laser or cryo-electron tomography  and the Thermus thermophilus V/A-ATPase, in multiple conformations in nanodisc studied by Cryo-EM indicated flexibility between V1 and Vo in a working enzyme, resulting in competition between central shaft rotation and resistance from the peripheral stalks  which shed light into several critical biological processes. Transient receptor potential cation channel subfamily V (TRPV-1) is shown to be interacting with the lipids in the nanodisc causing the regulation of its ligand binding , vesicles (v)-SNARE (VAMP2/Synaptobrevin) folds with a plasma membrane-situated target (t)-SNARE to form SNARE pin which is a four-helical bundle  and phosphatidylcholine (PC) in the outer membrane monolayer and phosphatidyl ethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) on inner monolayer (Verkleij, This review provides a brief insight of how introduction of cNDs and DCNDs has enabled the possibility to construct very large nanodiscs, which has extended the application of nanodisc technology from structural biology of small membrane proteins to study of large membrane protein complexes and membrane interaction of viruses. We highlighted some of the still unexploited potentials of these stable large tunable bilayers, which could be applied to several branches of cellular structural biology. Large nanodiscs offer a versatile tool to study membrane interaction of viruses which can solve several questions about virus entry such as mechanism of membrane penetration, steps in disassembly of virus particles, conformational changes, and the regulatory role played by host cell surface receptors. Large nanodiscs can also aid in understanding several biological processes such as membrane and vesicular fusion, and can be further developed to engineer asymmetric nanodiscs which will provide a more realistic membrane mimetic.KP, WS, GW, and MN wrote the manuscript. MN and KP drew the figures. All authors approved the final manuscript.GW and MN have founded NOW Scientific to allow purchase of covalently circularized scaffold proteins. Plasmids are available for a nominal fee, and detailed protocols have been published.


Alpha-1 antitrypsin deficiency (AATD) is a genetic condition characterised by low circulating levels of alpha-1 antitrypsin (AAT), a serine proteinase inhibitor. The most common deficiency variants are the S and Z mutations, which cause the accumulation of misfolded AAT in hepatocytes resulting in endoplasmic reticular stress and insufficient release of AAT into the circulation (<11μmol/L). This leads to liver disease, as well as an increased risk of emphysema due to unopposed proteolytic activity of neutrophil-derived serine proteinases in the lungs. AATD has been traditionally viewed as an inflammatory disorder caused directly by a proteinase-antiproteinase imbalance in the lung, but increasing evidence suggests that low AAT levels may affect other cellular functions. Recently, AAT polymers have been identified in both monocytes and macrophages from AATD patients and evidence is building that these cells may also play a role in the development of AATD lung disease. Alveolar macrophages are phagocytic cells that are important in the lung immune response but are also implicated in driving inflammation. This review explores the potential implications of monocyte and macrophage involvement in non-liver AAT synthesis and the pathophysiology of AATD lung disease. Alpha-1 antitrypsin deficiency (AATD) is an autosomal co-dominant genetic condition that is characterised by low circulating levels of alpha-1 antitrypsin (AAT) protein, a serine proteinase inhibitor synthesised and secreted mainly by hepatocytes, but also by immune and other cells.5AATD is caused by a genetic mutation on the SERPINA1 gene that encodes the AAT protein and results in low circulating levels of the AAT protein in the ZZ variant 2–8µmol/L). An estimated 3.4 million people worldwide are affected; however, under diagnosis means many individuals remain undetected so this number is likely to be higher, highlighting the need for screening programmes in at risk individuals.µmol/L. A12Although AAT is produced mainly by hepatocytes,++ CD16−, intermediate CD14+ CD16+ and non-classical CD14−, CD16+.Human monocytes account for 5–10% of circulating blood leukocytes. Monocytes are equipped with chemokine and adhesion receptors which mediate their migration from the blood to sites of infection or injury. Once migrated, activated monocytes release pro-inflammatory cytokines such as IL-1β, IL-6 and TNFα as well as other inflammatory mediators which induce the recruitment of further immune cell types, and moderates inflammation  secrete little AAT due to the accumulation of polymers within the cell.Alveolar macrophages line the alveoli and the airways of the lungs where they provide an important part of innate immunity, including phagocytosis of inhaled pathogens and releasing mediators to modulate the physiological immune response if required. The macrophage pool is long lived but also supplemented through the recruitment of monocytes, especially during acute and chronic episodes of inflammation, generating monocyte-derived macrophages with similar activity. Macrophages exhibit plasticity and are able to alter their phenotype and function in response to the local environmental requirements – from pro-inflammatory during infection, to anti-inflammatory during resolution of inflammation.42Both peripheral lung macrophages isolated from bronchoalveolar lavage and monocyte-derived macrophages (MDM) transcribe the AAT gene, and secrete the protein.AAT has several other effects on macrophages unrelated to its direct anti-proteinase activity. Neutrophil elastase binding to macrophages induces the release of LTB4,Mycobacterium abscessus suggesting a potential to prevent bacterial infection in the lungs by acting directly on the macrophage.Exogenous AAT has also been shown to restore efferocytosis and phagocytosis by macrophages exposed to cigarette smoke, due to a reduction in shedding of CD206 and phosphatidylserine receptors.1/FVC ratio, small airways dysfunction, CD8 T cell and neutrophil numbers in the alveolar walls, suggesting that polymer accumulation could be important in both AATD and non-deficient COPD. This observation is important as it is the first to show polymer accumulation within the macrophage, and it would be important to determine whether this relates to all macrophages within the slices and that the antibody used was specific to polymeric AAT.Macrophages from PiZZ patients express normal levels of the AAT gene but release 10 times less AAT than normalIn non-AATD COPD, macrophages are believed to be the key drivers of processes leading to disease initiation and progression.The role of the neutrophil in AATD has been extensively reviewed;4 and CXCL-8. Neutrophils are equally capable of recruiting monocytes to the lungs via the release of cathepsin G and azurocidin, which upregulates E-selectin and VCAM-1 on the vascular endothelium, promoting monocyte rolling, sticking and recruitment into the parenchyma.Macrophages recruit neutrophils to the lungs via the release of LTBNeutrophils are able to modify macrophage phenotype, and thereby modulate their function, through the release of reactive oxygen species, which activates macrophage AMPK, suppresses NFkB and induces an anti-inflammatory phenotype.65In AATD, polymers have been shown to increased degranulation in airway neutrophil, resulting in increased apoptosis, possibly due to high levels of endoplasmic reticulum stress, and activation of the internal death pathways induced by polymers.48Augmentation therapy is currently the only therapy specific for AATD approved in the US and Europe . However, there are issues of cost-effectiveness and benefits to the patient which currently questions its use, particularly in the UK. Although augmentation can modulate (though not prevent) the progression of emphysema,Mycobacterium intracellulare due to greater phagosome-lysosome fusion and increased autophagosome formation/maturation.Augmentation restores AAT, which in turn reduces LTB4 production in airways due to the prevention of neutrophil elastase binding to macrophage surfaces.Alternative approaches to treating AATD are being developed, which target the polymeric AAT protein itself. These include chemical chaperones, which reverse cellular mislocalisation and misfolding of the protein, such as 4-phenylbutyric acid (PBA). PBA which has been shown to increase the secretion of functional Z-AAT in human skin fibroblasts expressing Z-AAT, and produced a 20–50% increase in serum AAT level in PiZZ mice.Alternatively, small molecule correctors facilitate proper folding of the AAT protein within affected cells, and thus may increase systemic (via hepatocytes) and local release of AAT (by macrophage/monocytes). These include small peptides that fit into the open beta sheet of abnormal AAT, to block insertion of the inhibitory site loop of one AAT into the sheet of the next,75AATD is typically considered a disease driven by the proteolytic nature of neutrophil enzymes, but evidence is increasing that other cells of the innate and secondary immune system are also implicated. Fully understanding the interactive role and function of monocytes and macrophages in AATD may help explain the partial response to augmentation therapy and identify important alternative targets for treatment, and determine whether drugs that remove polymers from within the cells is essential for effective disease treatment.


Thymus involution occurs in all vertebrates. It is thought to impact on immune responses in the aged, and in other clinical circumstances such as bone marrow transplantation. Determinants of thymus growth and size are beginning to be identified. Ectopic expression of factors like cyclin D1 and Myc in thymic epithelial cells (TEC)s results in considerable increase in thymus size. These models provide useful experimental tools that allow thymus function to be understood. In future, understanding TEC-specific controllers of growth will provide new approaches to thymus regeneration. The thymus is the primary site of T cell development. It is a specialized environment that fosters the production of a diverse T cell repertoire, allowing T cells to recognize and eliminate foreign antigen but remain tolerant to self. The thymic epithelial cells (TEC)s educate developing T cells, where cortical (c)TECs select for those expressing functional receptors, whilst medullary (m)TECs deplete those with potential specificity to the body's own cells . TECs prThe thymus is the first organ to undergo aged-related involution and at an accelerated rate relative to other tissue . The proStressed-induced thymic involution results in decreased naïve T cell output and compromised host immunity, and is commonly reversible, with recovery of size and function after the insult is removed , 14, 15.Signals between TEC and thymocytes are bi-directional, with defects and alterations in the haemopoietic component of the thymus also having dramatic consequences on TEC development and maintenance. The importance of lymphoid-epithelial cell interactions for thymic architecture was established in mice lacking mature T cells, that present with severely impaired mTEC development . Early sMany agents have been identified to be involved in acute stress-induced thymic atrophy, including proinflammatory cytokines, steroids, and hormones. Inhibition of such agents can prevent this acute involution . The driAlthough aging results in widespread immunodeficiency, the direct consequences of age-related thymic involution on impaired immune function in the elderly is poorly understood. Old people are more susceptible to infection, and infections in the elderly often have higher severity compared to the young . FurtherAge-related thymic involution in mouse limits the numbers of recent thymic emigrants in the periphery . MoreoveThere are examples of viruses that aged mice are more susceptible to, as a consequence of age-related alteration to T cell responses. These include West Nile Virus, where old mice have reduced rates of survival compared to young. This increased susceptibility is a consequence of age-related defects in T cell immunity, involving both CD8 and CD4 T cell responses . InfluenRejuvenation of the involuted thymus restores peripheral T cell function in aged mice and humans , 35. ManOverall the immunological consequences of age-related thymic involution on the peripheral T cell pool potentially leaves the elderly with compromised protective immunity against pathogens. Thus, a better understanding of the molecular and cellular mechanisms underlining the causes of age-related thymic involution should offer benefits to the aging population in multiple clinical settings. It also needs to be considered, however, that age associated immune system defects may also be due to the aging of the hematopoietic cells, and not all a consequence of reduced thymic function. The general physiological consequences of aging to the host also needs to be considered when exploring the attributes of compromised protection against infectious agents in the elderly.It has been reported that the transfer of bone marrow cells or early T cell progenitors isolated from young mice does not restore thymic function in aged recipients, whereas the transplantation of fetal thymi into aged hosts exhibits thymocyte development equivalent to a young host. Thus, indicating that thymic stromal cells, rather than hematopoietic cells, drive thymic involution , 39–41. It has been demonstrated that genetic ablation of cTECs alone has profound effects on thymic size, and severely impaired thymocyte development . This coIn addition to morphological changes with age in cTECs, their expression of Catalase, an antioxidative enzyme, is lower compared to mTECs and lymphocytes, which results in their increased susceptibility to oxidative damage . Indeed,Transcriptional profiling of TECs in young and aged thymi has revealed dynamic gene profiles during the initial involution process in both cTECs and mTECs, aiding the understanding of the mechanisms that govern the decline of TEC numbers with age. A transcriptional hallmark of the initiation of involution in TECs was the downregulation of genes involved in cell cycle, specifically diminished E2F3 activity, suggesting possible reductions to cell cycle progression in all aging TECs . The samThe underlying mechanisms of the age-related decline in TEC numbers and function that mediate thymic atrophy remain ambiguous, although many changes to TEC biology throughout life have been documented. The pivotal role of TECs in the process of thymic atrophy identify them as an attractive therapeutic target to counter thymic involution . AlthougIn addition to controlling thymic atrophy, TECs regulate all changes in thymic size observed throughout life. This includes the dramatic increase seen during mouse embryogenesis, when the thymus doubles in size daily, and the continued expansion, although at a reduced rate, until 4 weeks of age, when thymic cellularity peaks. TECs also maintain this maximal size until around 8 weeks of age, when involution is initiated . DefectsThe rapid expansion in thymic size and TEC number during fetal and neonatal life is supported by TECs having unique properties that distinguish them from postnatal populations. This includes higher rates of proliferation , 51 and Understanding the transcriptional controllers of fetal TECs and how they support the rapid expansion in thymic size during embryogenesis offers insight into mechanisms underlying thymic function and regeneration. Recently, transcriptional assessment of TECs through development at a single cell resolution has revealed distinct transcriptional programs of TECs at specific stages of life , 63–65. The dramatic thymic growth conferred by the Myc transgene did not jeopardize function, similar to previous reports with cyclin D1 transgenic mice , 54, andCollectively, these results identify Myc as a major regulator of a fetal specific transcriptional program of TECs. They establish a role for Myc activity in rapid thymic growth during development; provide evidence Myc expression can confer at least one functional distinct property of fetal TECs into adulthood and furthermore, confirm that Myc activity is limiting for thymic size and function . UpstreaIn addition to unique transcriptional profiles of fetal TECs, the adult TECs were highly enriched for genes involved in antigen processing and presentation, along with regulation of the actin cytoskeleton and the lysosomes . Our resCyclin family molecules are key regulators for cell cycle progression that control the number of cells and the size of organs. Cyclin proteins associate and activate cyclin-dependent kinases (CDKs), serine/threonine kinases, important for the progression of cell cycle . ApproxiCyclin D1 is a member of cyclin D family, along with cyclin D2 and cyclin D3. Overexpression of cyclin D1 in cells results in a rapid progression from G1 to S phase, whereas inhibition of cyclin D1 prevents the entry into S phase . The oveCCND1 gene, was driven by bovine keratin 5 promoter  thymuses, and the cellularity of thymocytes in K5D1 mice reaches to ~50-fold larger numbers of that in B6 mice , 84. In −CD8−, CD4+CD8+, CD4+CD8−, and CD4−CD8+ thymocytes in K5D1 mice is almost comparable to that in B6 mice  are required for current mass spectrometry-based technology of proteomic analysis, unlike transcriptomic analysis, which can be carried out from small number of cells. Using the enlarged thymus in K5D1 mice, it is possible to isolate ~2 × 105 cTECs and ~2 × 105 mTECs from one mouse , as well as Aire-dependent and Aire-independent tissue-restricted self-antigen genes are highly detected in mTECs in an indistinguishable manner between K5D1 mTECs and B6 mTECs (Dll4 (encoding DLL4), Psmb11 (encoding β5t), Prss16 (encoding thymus-specific serine protease or TSSP), and Ctsl (encoding cathepsin L), are highly detected in cTECs in an indistinguishable manner between K5D1 cTECs and B6 cTECs  higher in cTECs than mTECs, whereas 1,021 proteins, including Aire, cathepsin S, and CD40, which are known to play an important role in mTECs, are significantly (p < 0.05) higher in mTECs than cTECs (Tandem mass tag (TMT)-based mass spectrometry analysis is a powerful technology for unbiased proteomics that enables multiplex analysis of relative quantification of proteins . It succan cTECs . It was an cTECs .These proteomic profiles for cTECs and mTECs have led to integrated analysis of proteomic and transcriptomic profiles in cTECs or mTECs . ApproxiIt is interesting to note that proteomic profiles and transcriptomic profiles do not provide a proportional correlation between the amount of proteins and mRNAs in cTECs and mTECs . It is pPropagation of mobilized cell lines that maintain functionally relevant molecular expression profiles has not been successful for cTECs or mTECs, so that the enlarged thymus provides a useful source of large numbers of freshly isolated cTECs and mTECs, not only for proteomic analysis but also for other analyses, including metabolomic analysis and other biochemical analyses. It is certainly important to identify MHC-associated self-peptides presented by cTECs and mTECs, which induce positive and negative selection of thymocytes to form an immunocompetent yet self-tolerant TCR repertoire.Although the immunological consequences of thymic involution have potential disadvantages to the elderly, thymic involution may have evolved for the benefit of the aging host. However, such benefits may be less favorable given current lifespans. One hypothesis is that reduced thymic activity would protect against autoimmunity . SeemingIt has also been suggested the involution process may conserve energy. The majority of developing thymocytes die during the stringent selection process in the thymus, where over 90% are estimated to undergo cell death , and theAnother possibility is that the reduced numbers of naïve T cells may also reduce the incidence of leukemias . Others T cell-mediated immunity is essential throughout life. However, the thymus, where T cells are generated, involutes rapidly in early life. Postnatal thymic involution is attributable to TECs rather than thymocytes. In this review, we summarize recent findings that the decline in a fetal-specific transcriptional program of TECs controls the size of the postnatal thymus and that cTEC morphology is altered during postnatal thymic involution. These findings provide novel insights into molecular and cellular mechanisms in TECs that control thymus size during involution. Thymic involution results in reduced thymocyte development and reduced numbers of naïve T cells and so is predicted to result in immunological disadvantages, including increased incidence in infectious disease and delayed T cell reconstitution after hematopoietic stem cell transplantation . TherefoIn this review, we also summarize how the development of large thymus models are useful tools for understanding TEC biology. This includes the versatility of enlarged K5D1 thymi for biochemical analysis, including proteomic profiling of TECs. Using such useful tools, we can examine whether functional senescence is induced in enlarged thymi and how counter-involution in aged mice may impact health. Moreover, such models could give novel targets for manipulation to prevent or reverse thymus atrophy.The review was written by JC and IO and was revised by YT and AB.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.


Potassium voltage-gated channel subfamily q member 4 (KCNQ4) is a voltage-gated potassium channel that plays essential roles in maintaining ion homeostasis and regulating hair cell membrane potential. Reduction of the activity of the KCNQ4 channel owing to genetic mutations is responsible for nonsyndromic hearing loss, a typically late-onset, initially high-frequency loss progressing over time. In addition, variants of KCNQ4 have also been associated with noise-induced hearing loss and age-related hearing loss. Therefore, the discovery of small compounds activating or potentiating KCNQ4 is an important strategy for the curative treatment of hearing loss. In this review, we updated the current concept of the physiological role of KCNQ4 in the inner ear and the pathologic mechanism underlying the role of KCNQ4 variants with regard to hearing loss. Finally, we focused on currently developed KCNQ4 activators and their pros and cons, paving the way for the future development of specific KCNQ4 activators as a remedy for hearing loss. The lensequence  [11]. ThKCNQ1 are known to cause heart diseases, including long QT syndrome (MIM 192500), and Jervell and Lange-Nielsen syndrome (MIM 220400) in autosomal dominant and recessive manners, respectively . In. In pore region mutation, which results in unresponsiveness to KCNQ activators [+ channels or variants in the two cytoplasmic termini of KCNQ4 to investigate the in vivo efficacy of channel openers. In addition, the effectiveness of KCNQ4 activators needs to be examined in mouse models of noise-hearing loss.Although some compounds could activate KCNQ4 in vitro, none of them were potent in inducing the native I in OHCs . The mol in OHCs ; howevertivators ,42. TherKCNQ4 mutation might also be an option for clinical application of KCNQ4 activators in deafness treatment with advantages of reducing the cost and shortening the time when compared to de novo drug discovery. In addition, the half-life duration and bioavailability of drugs targeting the activation of KCNQ4 would also be required to satisfy clinical degrees.More importantly, KCNQ4 activators should be validated in clinical trials, as there is no ongoing clinical trial targeting hearing loss by KCNQ4 activators currently. Target population, such as individuals with genetic hearing loss or noise-induced hearing, should be carefully selected. Both the therapeutic window and convenient application methods are important issues to be discussed in clinical trials. Drug repurposing and optimization for applicable specific