Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

Perceptions and attitudes regarding complementary, alternative, and integrative medicine among published neurology authors: a large-scale, international cross-sectional survey.

BACKGROUND: While many patients with neurological disorders and conditions use complementary, alternative, and integrative medicine (CAIM), little is known about the use, perceptions, and attitudes regarding CAIM among published neurology authors. With the increasing popularity of CAIM, our objective was to assess practices, perceptions, and attitudes towards CAIM among published neurology authors. METHODS: We conducted an anonymous online survey of authors who had published articles in neurology journals indexed in MEDLINE. We emailed potential participants our cross-sectional electronic survey after extracting their email addresses from one of their publications in our sample of journals. Basic descriptive statistics were drawn from quantitative data, and thematic content analysis was used to analyse qualitative data from any open-ended questions. RESULTS: The survey was completed by 783 published neurology authors (1.7% response rate, 83.9% completion rate). Overall, respondents perceived CAIM to be promising in preventing, treating, and/or managing neurological diseases. Mind-body therapies received the most positive responses, indicated by over half of respondents cumulatively agreeing that they are promising (n = 368, 59.0%) and safe (n = 280, 50.3%). Whole medical systems and biofield therapy were less favourable. Most neurology clinicians reported a lack of formal (n = 211, 70.3%) and supplementary training (n = 158, 52.5%) on CAIM. Nearly half of clinicians did not feel comfortable counselling patients about CAIM therapies (n = 121, 44.5%), and over half did not feel comfortable recommending them (n = 161, 59.3%). A lack of scientific evidence for CAIM's safety and efficacy was reported as the greatest challenge to CAIM (n = 515, 92.5%). The majority of respondents believed there is value to conducting research on this topic (n = 461, 82.0%) and supported increasing allocation of research funding towards CAIM (n = 241, 58.9%). CONCLUSIONS: Although many participants found CAIM to be promising to the field of neurology, the vast majority did not feel open to integrating CAIM into mainstream medical practices on account of a perceived lack of scientific evidence for its safety and efficacy. Future studies can use our findings to gather more detailed insights, improve educational resources on CAIM within neurology, as well as examine what effects a tailored CAIM education has on the perceptions and attitudes of published neurology authors towards CAIM.

Reported definitions of intraoperative hypotension in adults undergoing non-cardiac surgery under general anaesthesia: a review.

BACKGROUND: Intraoperative hypotension (IOH) during non-cardiac surgery is common and associated with major adverse kidney, neurological and cardiac events and even death. Given that IOH is a modifiable risk factor for the mitigation of postoperative complications, it is imperative to generate a precise definition for IOH to facilitate strategies for avoiding or treating its occurrence. Moreover, a universal and consensus definition of IOH may also facilitate the application of novel and emerging therapeutic interventions in treating IOH. We conducted a review to systematically record the reported definitions of intraoperative hypotension in adults undergoing non-cardiac surgery under general anaesthesia. METHODS: In accordance with Cochrane guidelines, we searched three online databases (OVID [Medline], Embase and Cochrane Library) for all studies published from 1 January 2000 to 6 September 2020. We evaluated the number of studies that reported the absolute or relative threshold values for defining blood pressure. Secondary aims included evaluation of the threshold values for defining IOH, the methodology for accounting for the severity of hypotension, whether the type of surgical procedure influenced the definition of IOH, and whether a study whose definition of IOH aligned with the Perioperative Quality Initiative-3 workgroup (POQI) consensus statement for defining was more likely to be associated with determining an adverse postoperative outcome. RESULTS: A total of 318 studies were included in the final qualitative synthesis. Most studies (n = 249; 78.3%) used an absolute threshold to define hypotension; 150 (60.5%) reported SBP, 117 (47.2%) reported MAP, and 12 (4.8%) reported diastolic blood pressure (DBP). 126 (39.6%) used a relative threshold to define hypotension. Of the included studies, 153 (48.1%) did not include any duration variable in their definition of hypotension. Among the selected 318 studies 148 (46.5%) studies defined IOH according to the POQI statement. When studies used a "relative blood pressure change" to define IOH, there was a weaker association in detecting adverse postoperative outcomes compared to studies who reported "absolute blood pressure change" (χ2(2) = 10.508, P = 0.005, Cramér's V = 0.182). When studies used the POQI statement definition of hypotension or defined IOH by values higher than the POQI statement definition there were statistical differences observed between IOH and adverse postoperative outcomes (χ2(1) = 6.581, P = 0.037, Cramér's V = 0.144). When both the duration of IOH or the numbers of hypotensive epochs were evaluated, we observed a significantly stronger relationship between the definition of IOH use the development of adverse postoperative outcomes. (χ2(1) = 4.860, P = 0.027, Cramér's V = 0.124). CONCLUSIONS: Most studies defined IOH by absolute or relative changes from baseline values. There are substantial inconsistencies in how IOH was reported. Further, definitions differed across different surgical specialities. Our findings further suggest that IOH should be defined using the absolute values stated in the POQI statement i.e., MAP < 60-70 mmHg or SBP < 100 mmHg. Finally, the number of hypotensive epochs or time-weighted duration of IOH should also be reported.

Universal screening for social determinants of health in pediatric sickle cell disease: A quality-improvement initiative.

BACKGROUND: Social determinants of health (SDoH) are socioeconomic factors that influence health outcomes. Guidelines recommend universal screening for SDoH at clinic visits; however, models that do not require additional resources are limited in subspecialty clinics. Individuals with sickle cell disease (SCD) face the burdens of chronic illness and often racial disparities, both of which may increase their vulnerability to adverse SDoH. Hematologists can impact both quality of life and clinical outcomes for their patients by implementing screening and referral programs addressing SDoH. METHODS: Through prospective, quality-improvement methods, we introduced universal screening for SDoH into our pediatric hematology clinic. The intervention was a paper screener followed by a referral to local community organizations for the specific needs endorsed. The aims of this study were to determine the feasibility of universal screening for SDoH in a busy subspeciality clinic using pre-existing resources to identify the needs of our patients and to facilitate referrals between our patients and community organizations via this low touch intervention. RESULTS: Between August 2017 and November 2018, 156 screens were completed. Sixty-six percent were positive for at least one unmet social need for which 80% were referred to a relevant community organization. Forty-five percent of patients available via follow-up phone call reached out to the community organization. CONCLUSIONS: There is a high burden of SDoH in families of children with SCD. Universal screening at a pediatric hematology clinic with the subsequent connection of patients with SCD to community resources is feasible using existing clinic resources.

An Approach to Minimising Risk of Adrenal Insufficiency When Discontinuing Oral Glucocorticoids.

Oral glucocorticoids are commonly used across every field of medicine; however, discontinuing them in patients can be challenging. The risk of acute adrenal crises secondary to glucocorticoid withdrawal can be fatal and arises from chronic suppression of the adrenal glands. Identifying risk factors for adrenal suppression in dermatological patients, such as doses greater than 5 to 7.5 mg of prednisone equivalent, duration of glucocorticoid use greater than 3 weeks, certain medications, and comorbidities, can help risk-stratify patients. The use of adrenal gland testing such as basal cortisol levels and adrenocorticotropic hormone stimulation tests can confirm adrenal suppression in patients. This review article provides an approach that dermatologists can use to minimise the risk of adrenal insufficiency in patients discontinuing glucocorticoids and when it may be appropriate to use adrenal gland testing.

Cell type-specific deletion in mice reveals roles for PAS kinase in insulin and glucagon production.

AIMS/HYPOTHESIS: Per-Arnt-Sim kinase (PASK) is a nutrient-regulated domain-containing protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of PASK in the control of islet hormone release, by generating mice with selective deletion of the Pask gene in pancreatic beta or alpha cells. METHODS: Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1 (Cre); PaskBKO) or alpha (Ppg (Cre) [also known as Gcg]; PaskAKO) cell-selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression and islet cell mass were measured using standard techniques. RESULTS: Ins1 (Cre)-based recombination led to efficient beta cell-targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p < 0.05) compared with controls in PaskBKO mice, as well as in global Pask-null mice (38%, p < 0.05). PaskBKO mice displayed normal body weight and fasting glycaemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high-fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycaemic clamps. Although alpha cell mass was increased (21.9%, p < 0.05), glucagon release at low glucose was impaired (p < 0.05) in PaskAKO islets. CONCLUSIONS/INTERPRETATION: The findings demonstrate cell-autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differences between the glycaemic phenotype of global vs cell type-specific null mice suggest important roles for tissue interactions in the control of glycaemia by PASK.

The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion.

SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet ß- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP(+): glucagon(+) cells from KO mice, revealed recombination in ∼ 30% of α-cells, of which ∼ 50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn(2+) levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca(2+) increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca(2+)-independent mechanisms.

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis.

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy.

Adiponectin attenuates lipopolysaccharide-induced acute lung injury through suppression of endothelial cell activation.

Adiponectin (APN) is an adipose tissue-derived factor with anti-inflammatory and vascular protective properties whose levels paradoxically decrease with increasing body fat. In this study, APN's role in the early development of ALI to LPS was investigated. Intratracheal LPS elicited an exaggerated systemic inflammatory response in APN-deficient (APN(-/-)) mice compared with wild-type (wt) littermates. Increased lung injury and inflammation were observed in APN(-/-) mice as early as 4 h after delivery of LPS. Targeted gene expression profiling performed on immune and endothelial cells isolated from lung digests 4 h after LPS administration showed increased proinflammatory gene expression (e.g., IL-6) only in endothelial cells of APN(-/-) mice when compared with wt mice. Direct effects on lung endothelium were demonstrated by APN's ability to inhibit LPS-induced IL-6 production in primary human endothelial cells in culture. Furthermore, T-cadherin-deficient mice that have significantly reduced lung airspace APN but high serum APN levels had pulmonary inflammatory responses after intratracheal LPS that were similar to those of wt mice. These findings indicate the importance of serum APN in modulating LPS-induced ALI and suggest that conditions leading to hypoadiponectinemia (e.g., obesity) predispose to development of ALI through exaggerated inflammatory response in pulmonary vascular endothelium.

Single-cell and spatial multi-omics highlight effects of anti-integrin therapy across cellular compartments in ulcerative colitis.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we performed single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Single-cell and spatial multi-omics highlight effects of anti-integrin therapy across cellular compartments in ulcerative colitis.

Ulcerative colitis (UC) is driven by immune and stromal subsets, culminating in epithelial injury. Vedolizumab (VDZ) is an anti-integrin antibody that is effective for treating UC. VDZ is known to inhibit lymphocyte trafficking to the intestine, but its broader effects on other cell subsets are less defined. To identify the inflammatory cells that contribute to colitis and are affected by VDZ, we perform single-cell transcriptomic and proteomic analyses of peripheral blood and colonic biopsies in healthy controls and patients with UC on VDZ or other therapies. Here we show that VDZ treatment is associated with alterations in circulating and tissue mononuclear phagocyte (MNP) subsets, along with modest shifts in lymphocytes. Spatial multi-omics of formalin-fixed biopsies demonstrates trends towards increased abundance and proximity of MNP and fibroblast subsets in active colitis. Spatial transcriptomics of archived specimens pre-treatment identifies epithelial-, MNP-, and fibroblast-enriched genes related to VDZ responsiveness, highlighting important roles for these subsets in UC.

Assessment of the impact of a personalised nutrition intervention in impaired glucose regulation over 26 weeks: a randomised controlled trial.

Dietary interventions can reduce progression to type 2 diabetes mellitus (T2DM) in people with non-diabetic hyperglycaemia. In this study we aimed to determine the impact of a DNA-personalised nutrition intervention in people with non-diabetic hyperglycaemia over 26 weeks. ASPIRE-DNA was a pilot study. Participants were randomised into three arms to receive either (i) Control arm: standard care (NICE guidelines) (n = 51), (ii) Intervention arm: DNA-personalised dietary advice (n = 50), or (iii) Exploratory arm: DNA-personalised dietary advice via a self-guided app and wearable device (n = 46). The primary outcome was the difference in fasting plasma glucose (FPG) between the Control and Intervention arms after 6 weeks. 180 people were recruited, of whom 148 people were randomised, mean age of 59 years (SD = 11), 69% of whom were female. There was no significant difference in the FPG change between the Control and Intervention arms at 6 weeks (- 0.13 mmol/L (95% CI [- 0.37, 0.11]), p = 0.29), however, we found that a DNA-personalised dietary intervention led to a significant reduction of FPG at 26 weeks in the Intervention arm when compared to standard care (- 0.019 (SD = 0.008), p = 0.01), as did the Exploratory arm (- 0.021 (SD = 0.008), p = 0.006). HbA1c at 26 weeks was significantly reduced in the Intervention arm when compared to standard care (- 0.038 (SD = 0.018), p = 0.04). There was some evidence suggesting prevention of progression to T2DM across the groups that received a DNA-based intervention (p = 0.06). Personalisation of dietary advice based on DNA did not result in glucose changes within the first 6 weeks but was associated with significant reduction of FPG and HbA1c at 26 weeks when compared to standard care. The DNA-based diet was effective regardless of intervention type, though results should be interpreted with caution due to the low sample size. These findings suggest that DNA-based dietary guidance is an effective intervention compared to standard care, but there is still a minimum timeframe of adherence to the intervention before changes in clinical outcomes become apparent.Trial Registration: www.clinicaltrials.gov.uk Ref: NCT03702465.

The Tyrosine Kinase Inhibitor Lenvatinib Inhibits Anaplastic Thyroid Carcinoma Growth by Targeting Pericytes in the Tumor Microenvironment.

Background: Anaplastic thyroid carcinoma (ATC) is a rapidly fatal cancer with a median survival of a few months. Enhanced therapeutic options for durable management of ATC will rely on an understanding of genetics and the role of the tumor microenvironment. The prognosis for patients with ATC has not improved despite more detailed scrutiny of underlying tumor genetics. Pericytes in the microenvironment play a key evasive role for thyroid carcinoma (TC) cells. Lenvatinib improves outcomes in patients with radioiodine-refractory well-differentiated TC. In addition to the unclear role of pericytes in ATC, the effect and mechanism of lenvatinib efficacy on ATC have not been sufficiently elucidated. Design: We assessed pericyte enrichment in ATC. We determined the effect of lenvatinib on ATC cell growth cocultured with pericytes and in a xenograft mouse model from human BRAFWT/V600E-ATC-derived cells coimplanted with pericytes. Results: ATC samples were significantly enriched in pericytes compared with normal thyroid samples. BRAFWT/V600E-ATC-derived cells were resistant to lenvatinib treatment shown by a lack of suppression of MAPK and Akt pathways. Moreover, lenvatinib increased CD47 protein (thrombospondin-1 [TSP-1] receptor) levels over time vs. vehicle. TSP-1 levels were downregulated upon lenvatinib at late vs. early time points. Critically, ATC cells, when cocultured with pericytes, showed increased sensitivity to this therapy and ultimately decreased number of cells. The coimplantation in vivo of ATC cells with pericytes increased ATC growth and did not downregulate TSP-1 in the microenvironment in vivo. Conclusions and Implications: Pericytes are enriched in ATC samples. Lenvatinib showed inhibitory effects on BRAFWT/V600E-ATC cells in the presence of pericytes. The presence of pericytes could be crucial for effective lenvatinib treatment in patients with ATC. Degree of pericyte abundance may be an attractive prognostic marker in assessing pharmacotherapeutic options. Effective durable management of ATC will rely on an understanding not only of genetics but also on the role of the tumor microenvironment.

A solution-processed radiative cooling glass.

Passive daytime radiative cooling materials could reduce the energy needed for building cooling up to 60% by reflecting sunlight and emitting long-wave infrared (LWIR) radiation into the cold Universe (~3 kelvin). However, developing passive cooling structures that are both practical to manufacture and apply while also displaying long-term environmental stability is challenging. We developed a randomized photonic composite consisting of a microporous glass framework that features selective LWIR emission along with relatively high solar reflectance and aluminum oxide particles that strongly scatter sunlight and prevent densification of the porous structure during manufacturing. This microporous glass coating enables a temperature drop of ~3.5° and 4°C even under high-humidity conditions (up to 80%) during midday and nighttime, respectively. This radiative "cooling glass" coating maintains high solar reflectance even when exposed to harsh conditions, including water, ultraviolet radiation, soiling, and high temperatures.

Equity, diversity, and inclusion at the Global Alliance for Genomics and Health.

A lack of diversity in genomics for health continues to hinder equitable leadership and access to precision medicine approaches for underrepresented populations. To avoid perpetuating biases within the genomics workforce and genomic data collection practices, equity, diversity, and inclusion (EDI) must be addressed. This paper documents the journey taken by the Global Alliance for Genomics and Health (a genomics-based standard-setting and policy-framing organization) to create a more equitable, diverse, and inclusive environment for its standards and members. Initial steps include the creation of two groups: the Equity, Diversity, and Inclusion Advisory Group and the Regulatory and Ethics Diversity Group. Following a framework that we call "Reflected in our Teams, Reflected in our Standards," both groups address EDI at different stages in their policy development process.

Increasing risk of mortality across the spectrum of aortic stenosis is independent of comorbidity & treatment: An international, parallel cohort study of 248,464 patients.

BACKGROUND: While large scientific and medical evidence has demonstrated the increased risk of death and cardiovascular mortality in patients with severe AS, the independent contribution of moderate AS to an increased risk of death remains uncertain. METHODS AND FINDINGS: We conducted a multicenter study including a cohort of 30,865 US patients and another cohort of 217,599 Australian patients with equivalent echocardiographic and aortic valve profiling over the same period (2003-2017). During a median 5.2 years (US) and 4.4 years (Australian) follow-up, the risk of death (hazard ratio) of patients with moderate AS as compared to those without AS was 1.66 (95%CI 1.52-1.80) and 1.37 (95%CI 1.34-1.41) in the US and Australian cohorts, even after adjusting this analysis for age and sex. This increased risk of death and cardiovascular mortality (odds ratio) in patients with moderate AS was consistent also across subgroups of left ventricular ejection fraction (LVEF) (subgroups of LVEF < 40%, 40-49%, 50-59%, and ≥ 60%: OR of moderate AS for CV mortality 2.0 [95%CI 1.4-2.7], 1.7 [95%CI 1.2-2.4], 1.5 [95%CI 1.1-1.9], and 1.4 [95%CI 1.2-1.6], respectively). CONCLUSIONS: The findings of this study suggest that patients with moderate AS have a potential increased risk of death and cardiovascular mortality, regardless of age, sex, and LVEF. Hence, these data suggest the need to develop specific strategies to detect and treat individuals with moderate AS.