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Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head‐to‐head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell‐free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome‐wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P

Abstract Lower risk (LR) myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem and progenitor disorders caused by the accumulation of somatic mutations in various genes including epigenetic regulators that may produce convergent DNA methylation patterns driving specific gene expression profiles. The integration of genomic, epigenomic, and transcriptomic profiling has the potential to spotlight distinct LR‐MDS categories on the basis of pathophysiological mechanisms. We performed a comprehensive study of somatic mutations and DNA methylation in a large and clinically well‐annotated cohort of treatment‐naive patients with LR‐MDS at diagnosis from the EUMDS registry (ClinicalTrials.gov.NCT00600860). Unsupervised clustering analyses identified six clusters based on genetic profiling that concentrate into four clusters on the basis of genome‐wide methylation profiling with significant overlap between the two clustering modes. The four methylation clusters showed distinct clinical and genetic features and distinct methylation landscape. All clusters shared hypermethylated enhancers enriched in binding motifs for ETS and bZIP (C/EBP) transcription factor families, involved in the regulation of myeloid cell differentiation. By contrast, one cluster gathering patients with early leukemic evolution exhibited a specific pattern of hypermethylated promoters and, distinctly from other clusters, the upregulation of AP‐1 complex members FOS/FOSL2 together with the absence of hypermethylation of their binding motif at target gene enhancers, which is of relevance for leukemic initiation. Among MDS patients with lower‐risk IPSS‐M, this cluster displayed a significantly inferior overall survival (p

Abstract Escherichia coli A0 34/86 (EcO83) is a probiotic strain used in newborns to prevent nosocomial infections and diarrhoea. This bacterium stimulates both pro‐ and anti‐inflammatory cytokine production and its intranasal administration reduces allergic airway inflammation in mice. Despite its benefits, there are concerns about the use of live probiotic bacteria due to potential systemic infections and gene transfer. Extracellular vesicles (EVs) derived from EcO83 (EcO83‐EVs) might offer a safer alternative to live bacteria. This study characterizes EcO83‐EVs and investigates their interaction with host cells, highlighting their potential as postbiotic therapeutics. EcO83‐EVs were isolated, purified, and characterised following the Minimal Information of Studies of Extracellular Vesicles (MISEV) guidelines. Ex vivo studies conducted in human nasal epithelial cells showed that EcO83‐EVs increased the expression of proteins linked to oxidative stress and inflammation, indicating an effective interaction between EVs and the host cells. Further in vivo studies in mice demonstrated that EcO83‐EVs interact with nasal‐associated lymphoid tissue, are internalised by airway macrophages, and stimulate neutrophil recruitment in the lung. Mechanistically, EcO83‐EVs activate the NF‐κΒ signalling pathway, resulting in the nitric oxide production. EcO83‐EVs demonstrate significant potential as a postbiotic alternative to live bacteria, offering a safer option for therapeutic applications. Further research is required to explore their clinical use, particularly in mucosal vaccination and targeted immunotherapy strategies.

Abstract Picophytoplankton are important primary producers, but not always adequately recognized, for example, due to methodological limitations. In this study, we combined flow cytometry and metabarcoding to investigate seasonal and spatial patterns of picophytoplankton abundance and community composition in the Elbe estuary. Due to the mixing of freshwater and seawater and the tidal currents this ecosystem is characterized by typical estuarine features such as salinity gradients and high turbidity. Picophytoplankton (mostly picoeukaryotes such as Mychonastes and Minidiscus) contributed on average 70% (SD = 14%) to the total phytoplankton counts. In summer picocyanobacteria (e.g., Synechococcus) played a more significant role. The contributions of picophytoplankton to the total phytoplankton were particularly high from summer to winter as well as in the mid estuary. However, at salinities of around 10 PSU in the mixing area of freshwater and seawater, the proportion of picophytoplankton was comparably low (average 49%, SD = 13%). Our results indicate that picophytoplankton prevail in the Elbe estuary year‐round with respect to cell counts. Picophytoplankton could occupy important niche positions to maintain primary production under extreme conditions where larger phytoplankton might struggle (e.g., at high or low temperature, high turbidity, and in areas with high grazing pressure) and also benefit from high nutrient availability here. However, we did not find evidence that they played a particularly significant role at the salinity interface. Our study highlights the importance of including picophytoplankton when assessing estuarine phytoplankton as has been suggested for other ecosystems such as oceans.

Abstract Aims Current guidelines suggest calcium channel blockers (CCBs) as the second or third option for blood pressure management in patients with left ventricular assist device (LVAD). However, the clinical outcomes of patients with LVAD who receive CCBs remain unclear. Our study aims to analyse the association of CCBs with clinical outcomes in patients after LVAD implantation. Methods and results This is a retrospective analysis based on the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) from 2006 to 2017, and adult patients who were alive with LVAD and CCB treatment information at 6 months after implantation were included. Among 10 717 patients, 1369 received CCBs 6 months after implantation, and there was an increasing trend of CCB use after LVAD. Patients receiving CCB therapy at 6 months had a similar 5 year survival rate to those not receiving CCB [49.6%, 95% confidence interval (CI): 47.5–51.7% vs. 51.1%, 95% CI: 45.3–56.7%]. In both Cox and competing risk regressions after adjusting for confounding factors, CCB treatment at 6 months after implantation was not associated with long‐term mortality [hazard ratio (HR): 1.03, 95% CI: 0.91–1.17, P = 0.624 and subdistribution HR (SHR): 1.07, 95% CI: 0.95–1.22, P = 0.260]. Consistently, in time‐varying models, CCB treatment was not linked to long‐term mortality (HR: 0.97, 95% CI: 0.87–1.09, P = 0.682 and SHR: 1.05, 95% CI: 0.94–1.18, P = 0.359). This null association remained in subgroup analysis according to device strategy and propensity‐matching analyses. Neurological dysfunction, stroke, bleeding, rehospitalization, and renal dysfunction were more likely to occur among those with CCB when compared with those without CCB treatment. Conclusions In patients with LVAD, CCB therapy fails to show benefits in long‐term survival and is associated with increased incidences of neurological dysfunction, bleeding, renal dysfunction, and rehospitalization.

Abstract Objectives The Mental Health Inventory (MHI‐5) is frequently used as a screener for mood and anxiety disorders. However, few population‐based studies have validated it against a diagnostic instrument assessing disorders following current diagnostic criteria. Methods Within the third Netherlands Mental Health Survey and Incidence Study (NEMESIS‐3), a representative population‐based study of adults (N = 6194; age: 18–75 years), the MHI‐5 was used to measure general mental ill‐health in the past month. Presence of mood (major depressive disorder, persistent depressive disorder, or bipolar disorder) and anxiety disorders (panic disorder, agoraphobia, social phobia, or generalized anxiety disorder) in the past month was assessed with a slightly modified version of the Composite International Diagnostic Interview 3.0 per the Diagnostic and Statistical Manual of Mental disorders‐5. Results The MHI‐5 was good to excellent at distinguishing people with and without a mood disorder, an anxiety disorder, and any mood or anxiety disorder. The cut‐off value associated with the highest sensitivity and highest specificity for mood disorder was ≤68, and ≤76 for an anxiety disorder or any mood or anxiety disorder. Conclusions The MHI‐5 can identify individuals at high risk of a current mood or anxiety disorder in the general population when diagnostic interviews are too time consuming.

Abstract Atmospheric rivers (ARs) deliver significant and essential precipitation to the western United States (US) with consequential interannual variability. The intensity and frequency of ARs strongly influence reservoir levels, mountain snowpack, and groundwater recharge, which are key drivers of water‐resource availability and natural hazards. Between October 2022 and April 2023, western states experienced exceptionally heavy precipitation from several families of powerful ARs. Using observations of surface‐loading deformation from Global Navigation Satellite Systems, we find that terrestrial water‐storage gains exceeded 100% of normal within vital California watersheds. Independent water‐storage solutions derived from different data‐analysis and inversion methods provide an important measure of precision. The sustained storage increases, which we show are closely associated with ARs at daily‐to‐weekly timescales, alleviated both meteorological and hydrological drought conditions in the region, with a lag in hydrological‐drought improvements. Quantifying water‐storage recovery associated with extreme precipitation after drought advances understanding of an increasingly variable hydrologic cycle.

Multiple prognostic biomarkers, including circulating tumour cell (CTC) counts, exist in metastatic castration‐resistant prostate cancer (mCRPC) patients, but none of them have been implemented into daily clinical care. The modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS), which yields a genome‐wide aneuploidy score, is able to reflect the fraction of cell‐free tumour DNA (ctDNA) within cell‐free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (

Abstract Same‐sex sexual behavior occurs in diverse animal taxa, yet its evolutionary maintenance is poorly understood as such behavior seems to be costly and does not directly increase reproductive success. We used male Tribolium castaneum beetles, which frequently engage in same‐sex copulations, to test if same‐sex sexual behavior influences future male mating behavior and reproductive success of males. Furthermore, we tested whether same‐sex sexual behavior has benefits via indirect sperm translocation. We conducted a series of mating trials demonstrating that males exposed to same‐sex behavior did not sire less offspring compared to control males that did not engage in same‐sex behavior. This suggests that same‐sex copulations did not lead to fitness costs in subsequent mating interactions. In addition, we found no evidence that indirect sperm translocation via an intermediate male occurs in T. castaneum. Taken together, these results imply that same‐sex sexual behavior in males is associated with no costs in terms of lower mating rate and reduced siring success and does not seem to entail benefits. Moreover, our data conform to the hypothesis that sexual indiscrimination is prevalent in this species, which may explain the relatively high frequency of same‐sex sexual behavior in T. castaneum.

Background Sarcopenia and hypoalbuminemia have been identified as independent predictors of increased adverse outcomes, including mortality and readmissions, in hospitalized older adults with acute decompensated heart failure (ADHF). However, the impact of coexisting sarcopenia and hypoalbuminemia on morbidity and death in adults with ADHF has not yet been investigated. We aimed to investigate the combined effects of lower muscle mass (LMM) as a surrogate for sarcopenia and hypoalbuminemia on in‐hospital and postdischarge outcomes of patients hospitalized for ADHF. Methods and Results A total of 385 patients admitted for ADHF between 2017 and 2020 at a single institution were retrospectively identified. Demographic and clinical data were collected, including serum albumin levels at admission and discharge. Skeletal muscle indices were derived from semi‐automated segmentation software analysis on axial chest computed tomography at the twelfth vertebral level. Our analysis revealed that patients who had LMM with admission hypoalbuminemia experienced increased diagnoses of infection and delirium with longer hospital length of stay and more frequent discharge to a facility. Upon discharge, 27.9% of patients had higher muscle mass without discharge hypoalbuminemia (reference group), 9.7% had LMM without discharge hypoalbuminemia, 38.4% had higher muscle mass with discharge hypoalbuminemia, and 24.0% had LMM with discharge hypoalbuminemia; mortality rates were 37.6%, 51.4%, 48.9%, and 63.2%, respectively. 1‐ and 3‐year mortality risks were highest in those with LMM and discharge hypoalbuminemia; this relationship remained significant over a median 23.6 (3.1–33.8) months follow‐up time despite multivariable adjustments (hazard ratio, 2.03 [95% CI, 1.31–3.16]; P=0.002). Conclusions Hospitalization with ADHF, LMM, and hypoalbuminemia portend heightened mortality risk.

Abstract Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year‐on‐year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non‐vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Abstract This study presents a methodological advancement in the field of clumped‐isotope (∆47) thermometry, specifically tailored for application to freshwater ostracods. The novel ostracod clumped isotope approach enables quantitative temperature and hydrological reconstruction in lacustrine records. The relationship between ∆47 and the temperature at which ostracod shell mineralized is determined by measuring ∆47 on different species grown under controlled temperatures, ranging from 4 ± 0.8 to 23 ± 0.5ºC. The excellent agreement between the presented ∆47 ostracod data and the monitored temperatures confirms that ∆47 can be applied to ostracod shells and that a vital effect is absent outside the uncertainty of measurements. Results are consistent with the carbonate clumped‐isotope unified calibration (Anderson et al., 2021, https://doi.org/10.1029/2020gl092069), therefore, an ostracod‐specific calibration is not needed. The ostracod clumped‐isotope thermometer represents a powerful tool for terrestrial paleoclimate studies all around the world, as lakes and ostracods are found in all climatic belts.

Abstract High blood pressure (BP) and type‐2 diabetes (T2DM) are forerunners of chronic kidney disease and left ventricular dysfunction. Home BP telemonitoring (HTM) and urinary peptidomic profiling (UPP) are technologies enabling risk stratification and personalized prevention. UPRIGHT‐HTM (NCT04299529) is an investigator‐initiated, multicenter, open‐label, randomized trial with blinded endpoint evaluation designed to assess the efficacy of HTM plus UPP (experimental group) over HTM alone (control group) in guiding treatment in asymptomatic patients, aged 55–75 years, with ≥5 cardiovascular risk factors. From screening onwards, HTM data can be freely accessed by all patients and their caregivers; UPP results are communicated early during follow‐up to patients and caregivers in the intervention group, but at trial closure in the control group. From May 2021 until January 2023, 235 patients were screened, of whom 53 were still progressing through the run‐in period and 144 were randomized. Both groups had similar characteristics, including average age (62.0 years) and the proportions of African Blacks (81.9%), White Europeans (16.7%), women 56.2%, home (31.2%), and office (50.0%) hypertension, T2DM (36.4%), micro‐albuminuria (29.4%), and ECG (9.7%) and echocardiographic (11.5%) left ventricular hypertrophy. Home and office BP were 128.8/79.2 mm Hg and 137.1/82.7 mm Hg, respectively, resulting in a prevalence of white‐coat, masked and sustained hypertension of 40.3%, 11.1%, and 25.7%. HTM persisted after randomization (48 681 readings up to 15 January 2023). In conclusion, results predominantly from low‐resource sub‐Saharan centers proved the feasibility of this multi‐ethnic trial. The COVID‐19 pandemic caused delays and differential recruitment rates across centers.

Abstract Heterotopic heart transplantation (HHT) is an alternative to the orthotopic technique in selected patients with terminal heart failure. We report the case of the longest survival after HHT, with an uneventful follow‐up for over three decades after transplantation. At the age of 25 years, endomyocardial fibrosis following myocarditis rendered the patient's native heart unable to maintain the body's needs. An allograft provided a second chance at life. The HHT technique was favoured due to severe pulmonary hypertension. The patient had an uneventful follow‐up since then. The scarcity of donors and the revolutionary advances in the mechanical circulatory device field restricted the utilization of the HHT technique, but it has the potential to provide an excellent prognosis with a good quality of life.

Myocarditis is most recognized in patients with moderate to severe, recent‐onset heart failure. However, less typical presentations including myocardial infarction with normal coronary arteries and arrhythmias are important manifestations but less commonly recognized to be caused by myocarditis. Most cases of myocarditis can be self‐limiting without specific treatment; however, appropriate identification of risk during the diagnostic process of myocarditis and once a diagnosis is established is of primordial importance to identify patients in need for more specific follow‐up and management. We propose a flexible, multitiered approach to the diagnostic process, allowing for capturing of the spectrum of myocarditis at an early time‐point, individualized use of diagnostic resources through disease severity phenotyping, and providing structured follow‐up care once myocarditis is confirmed. Such diagnostic processes allow for identification of specific etiologies with potential therapeutic consequences or allows for the comprehension of disease chronicity by understanding genetic contributions or elements of persistent immune dysregulation and degree of cardiac damage. The article highlights the evolving field of immunophenotyping in myocarditis, generating a potential for the development of targeted therapeutic approaches. Currently long‐term follow‐up should be titrated to the refined risk assessments of patients with a diagnosis of myocarditis and includes arrhythmia monitoring and imaging when the results will likely impact management. Genetic testing should be considered in selected cases, and histologic diagnosis may be considered in nonresponders even at later stages.

Abstract Amino acids (AAs) and ammonia are metabolic markers essential for nitrogen metabolism and cell regulation in both plants and humans. NMR provides interesting opportunities to investigate these metabolic pathways, yet lacks sensitivity, especially in case of 15N. In this study, spin order embedded in p‐H2 is used to produce on‐demand reversible hyperpolarization in 15N of pristine alanine and ammonia under ambient protic conditions directly in the NMR spectrometer. This is made possible by designing a mixed‐ligand Ir‐catalyst, selectively ligating the amino group of AA by exploiting ammonia as a strongly competitive co‐ligand and preventing deactivation of Ir by bidentate ligation of AA. The stereoisomerism of the catalyst complexes is determined by hydride fingerprinting using 1H/D scrambling of the associated N‐functional groups on the catalyst (i.e., isotopological fingerprinting), and unravelled by 2D‐ZQ‐NMR. Monitoring the transfer of spin order from p‐H2 to 15N nuclei of ligated and free alanine and ammonia targets using SABRE‐INEPT with variable exchange delays pinpoints the monodentate elucidated catalyst complexes to be most SABRE active. Also RF‐spin locking (SABRE‐SLIC) enables transfer of hyperpolarization to 15N. The presented high‐field approach can be a valuable alternative to SABRE‐SHEATH techniques since the obtained catalytic insights (stereochemistry and kinetics) will remain valid at ultra‐low magnetic fields.

Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike‐in experiments using MCF‐7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI‐g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient‐derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient‐derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

Second‐line treatment with immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate (~ 20%). Circulating tumour‐derived DNA (ctDNA) levels may guide patient stratification, provided that an affordable and robust assay is available. Here, we investigate whether the modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS) may provide such an assay. To this end, mFast‐SeqS was performed on cell‐free DNA (cfDNA) from 74 patients with mUC prior to treatment with pembrolizumab. Results were associated with corresponding tissue‐based profiles, plasma‐based variant allele frequencies (VAFs) and clinical response. We found that plasma‐derived mFast‐SeqS‐based aneuploidy scores significantly correlated with those observed in the corresponding tumour tissue as well as with the ctDNA level in the plasma. In multivariate logistic regression analysis, a high aneuploidy score was independently associated with lack of clinical benefit from treatment with pembrolizumab. In conclusion, mFast‐SeqS provides a patient‐friendly, high‐throughput and affordable method to estimate ctDNA level. Following independent validation, this test could be used to stratify mUC patients for response prior to the initiation of treatment with pembrolizumab.

The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid profiling (LP), its acceptance by clinicians, and its integration into clinical workflows in real‐world settings remain limited. Here, LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at

Background Insight into outcome variation between hospitals could help to improve quality of care. We aimed to assess the validity of early outcomes as quality indicators for acute ischemic stroke care for patients treated with endovascular therapy (EVT). Methods and Results We used data from the MR CLEAN (Multicenter Randomized Controlled Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) Registry, a large multicenter prospective cohort study including 3279 patients with acute ischemic stroke undergoing EVT. Random effect linear and proportional odds regression were used to analyze the effect of case mix on between‐hospital differences in 2 early outcomes: the National Institutes of Health Stroke Scale (NIHSS) score at 24 to 48 hours and the expanded thrombolysis in cerebral infarction score. Between‐hospital variation in outcomes was assessed using the variance of random hospital effects (tau2). In addition, we estimated the correlation between hospitals' EVT‐patient volume and (case‐mix–adjusted) outcomes. Both early outcomes and case‐mix characteristics varied significantly across hospitals. Between‐hospital variation in the expanded thrombolysis in cerebral infarction score was not influenced by case‐mix adjustment (tau 2=0.17 in both models). In contrast, for the NIHSS score at 24 to 48 hours, case‐mix adjustment led to a decrease in variation between hospitals (tau 2 decreases from 0.19 to 0.17). Hospitals' EVT‐patient volume was strongly correlated with higher expanded thrombolysis in cerebral infarction scores (r=0.48) and weakly with lower NIHSS score at 24 to 48 hours (r=0.15). Conclusions Between‐hospital variation in NIHSS score at 24 to 48 hours is significantly influenced by case‐mix but not by patient volume. In contrast, between‐hospital variation in expanded thrombolysis in cerebral infarction score is strongly influenced by EVT‐patient volume but not by case‐mix. Both outcomes may be suitable for comparing hospitals on quality of care, provided that adequate adjustment for case‐mix is applied for NIHSS score.

Background It has been suggested that selection of a first‐line endovascular thrombectomy device, that is, contact aspiration (CA) or stent retriever (SR) thrombectomy, could be based on thrombus type. Thrombus composition and mechanical behavior can partially be predicted with thrombus computed tomography (CT) characteristics. We aimed to assess the influence of thrombus CT characteristics on the association between first‐line device and outcomes of endovascular thrombectomy. Methods For patients enrolled in the MR CLEAN (Multicenter Randomized Clinical Trial of Endovascular Treatment for Acute Ischemic Stroke in The Netherlands) Registry between March 2014 and November 2017, we assessed thrombus density, thrombus length, and presence of hyperdense artery sign on thin‐slice (≤2.5 mm) admission CT. We used regression models to estimate the relationship between first‐line endovascular thrombectomy device (CA versus stent retriever) and first‐pass reperfusion (FPR, expanded Thrombolysis in Cerebral Infarction score 2C‐3 after first attempt), final reperfusion, procedure duration, 24‐hour National Institutes of Health Stroke Scale, and 90‐day modified Rankin scale score and tested for interaction of thrombus characteristics with first‐line device by adding interaction terms. Results Of 703 included patients, 520 (74%) received first‐line stent retriever and 183 (26%) first‐line CA. Overall, the first‐line device was not associated with FPR (adjusted odds ratio [aOR], 1.32 [95% CI, 0.88–1.98]). In patients with thrombus density below the median (

Abstract In the Himalayas, a number of secondary contact zones have been described for vicariant vertebrate taxa. However, analyses of genetic divergence and admixture are missing for most of these examples. In this study, we provide a population genetic analysis for the coal tit (Periparus ater) hybrid zone in Nepal. Intermediate phenotypes between the distinctive western “spot‐winged tit” (P. a. melanolophus) and Eastern Himalayan coal tits (P. a. aemodius) occur across a narrow range of

Abstract Aims Although acute heart failure (AHF) with volume overload is treated with loop diuretics, their dosing and type of administration are mainly based upon expert opinion. A recent position paper from the Heart Failure Association (HFA) proposed a step‐wise pharmacologic diuretic strategy to increase the diuretic response and to achieve rapid decongestion. However, no study has evaluated this protocol prospectively. Methods and results The Efficacy of a Standardized Diuretic Protocol in Acute Heart Failure (ENACT‐HF) study is an international, multicentre, non‐randomized, open‐label, pragmatic study in AHF patients on chronic loop diuretic therapy, admitted to the hospital for intravenous loop diuretic therapy, aiming to enrol 500 patients. Inclusion criteria are as follows: at least one sign of volume overload (oedema, ascites, or pleural effusion), use ≥ 40 mg of furosemide or equivalent for >1 month, and a BNP > 250 ng/L or an N‐terminal pro‐B‐type natriuretic peptide > 1000 pg/L. The study is designed in two sequential phases. During Phase 1, all centres will treat consecutive patients according to the local standard of care. In the Phase 2 of the study, all centres will implement a standardized diuretic protocol in the next cohort of consecutive patients. The protocol is based upon the recently published HFA algorithm on diuretic use and starts with intravenous administration of two times the oral home dose. It includes early assessment of diuretic response with a spot urinary sodium measurement after 2 h and urine output after 6 h. Diuretics will be tailored further based upon these measurements. The study is powered for its primary endpoint of natriuresis after 1 day and will be able to detect a 15% difference with 80% power. Secondary endpoints are natriuresis and diuresis after 2 days, change in congestion score, change in weight, in‐hospital mortality, and length of hospitalization. Conclusions The ENACT‐HF study will investigate whether a step‐wise diuretic approach, based upon early assessment of urinary sodium and urine output as proposed by the HFA, is feasible and able to improve decongestion in AHF with volume overload.

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.

Abstract Fat embolism is a well‐known and life‐threatening condition that can develop after long bone fractures and lower limb orthopedic surgery. It presents in a wide range of respiratory, hematological, neurological, and cutaneous symptoms and signs of varying severity, resulting from embolic showering. It is important for clinicians to have a high index of suspicion for fat embolism in patients with respiratory compromise postoperatively. Rapid recognition and supportive treatment are key in improving the outcome of these patients. The prognosis is usually good, except in fulminant fat embolism syndrome.

Abstract Large herbivore migrations are imperiled globally; however the factors limiting a population across its migratory range are typically poorly understood. Zambia's Greater Liuwa Ecosystem (GLE) contains one of the largest remaining blue wildebeest (Connochaetes taurinus taurinus) migrations, yet the population structure, vital rates, and limiting factors are virtually unknown. We conducted a long‐term demographic study of GLE wildebeest from 2012 to 2019 of 107 collared adult females and their calves, 7352 herd observations, 12 aerial population surveys, and concurrent carnivore studies. We applied methods of vital rate estimation and survival analysis within a Bayesian estimation framework. From herd composition observations, we estimated rates of fecundity, first‐year survival, and recruitment as 68%, 56%, and 38% respectively, with pronounced interannual variation. Similar rates were estimated from calf‐detections with collared cows. Adult survival rates declined steadily from 91% at age 2 years to 61% at age 10 years thereafter dropping more sharply to 2% at age 16 years. Predation, particularly by spotted hyena, was the predominant cause of death for all wildebeest ages and focused on older animals. Starvation only accounted for 0.8% of all unbiased known natural causes of death. Mortality risk differed substantially between wet and dry season ranges, reflecting strong spatio‐temporal differences in habitat and predator densities. There was substantial evidence that mortality risk to adults was 27% higher in the wet season, and strong evidence that it was 45% higher in the migratory range where predator density was highest. The estimated vital rates were internally consistent, predicting a stable population trajectory consistent with aerial estimates. From essentially zero knowledge of GLE wildebeest dynamics, this work provides vital rates, age structure, limiting factors, and a plausible mechanism for the migratory tendency, and a robust model‐based foundation to evaluate the effects of potential restrictions in migratory range, climate change, predator–prey dynamics, and poaching.

Abstract In 2020, the International Association for the Study of Pain (IASP) revised the definition of pain, providing an update to IASP’s original definition in place since 1979. The origins of the field of pediatric pain, born in part in the 1980s from the advocacy of Jill Lawson, mother to Jeffrey Lawson who experienced gross inadequacies in pain care as a premature infant, emphasize the critical need to listen to and act with the voice of people living with pain and their families and caregivers. Furthermore, the field of pediatric pain emerged in the mid‐1980s marking this revised definition of pain as the first opportunity within which the experience and science of pain in childhood has been considered. This invited commentary offers two lived experience reactions to the revised IASP definition of pain, from the perspective of one's own experience with pain during childhood and as parents and family members. Together, they highlight that the value of a revised definition must be judged on its ability to directly benefit children experiencing pain and their families. Their skeptical hope reflects their lived experience backed by empirical evidence demonstrating continued inequities and inadequacies in preventing and managing pediatric pain. We must mobilize together to ensure change in culture, knowledge, and behavior. With the combined efforts of researchers, healthcare professionals, and policymakers, in partnership with diverse people with lived experience, we can ensure that more effective action is taken to rapidly improve pain for children and their families.

Abstract Objectives New strategies for implant surface functionalization in the prevention of peri‐implantitis while not compromising osseointegration are currently explored. The aim of this in vivo study was to assess the osseointegration of a titanium‐silica composite implant, previously shown to enable controlled release of therapeutic concentrations of chlorhexidine, in the Göttingen mini‐pig oral model. Material and Methods Three implant groups were designed: macroporous titanium implants (Ti‐Porous); macroporous titanium implants infiltrated with mesoporous silica (Ti‐Porous + SiO2); and conventional titanium implants (Ti‐control). Mandibular last premolar and first molar teeth were extracted bilaterally and implants were installed. After 1 month healing, the bone in contact with the implant and the bone regeneration in the peri‐implant gap was evaluated histomorphometrically. Results Bone‐to‐implant contact and peri‐implant bone volume for Ti‐Porous versus Ti‐Porous + SiO2 implants did not differ significantly, but were significantly higher in the Ti‐Control group compared with Ti‐Porous + SiO2 implants. Functionalization of titanium implants via infiltration of a SiO2 phase into the titanium macropores does not seem to inhibit implant osseointegration. Yet, the importance of the implant macro‐design, in particular the screw thread design in a marginal gap implant surgery set‐up, was emphasized by the outstanding results of the Ti‐Control implant. Conclusions Next‐generation implants made of macroporous Ti infiltrated with mesoporous SiO2 do not seem to compromise the osseointegration process. Such implant functionalization may be promising for the prevention and treatment of peri‐implantitis given the evidenced potential of mesoporous SiO2 for controlled drug release.

Abstract Objective We report longitudinal data from 144 type III SMA pediatric and adult patients treated with nusinersen as part of an international effort. Methods Patients were assessed using Hammersmith Functional Motor Scale Expanded (HFMSE), Revised Upper Limb Module (RULM), and 6‐Minute Walk Test (6MWT) with a mean follow‐up of 1.83 years after nusinersen treatment. Results Over 75% of the 144 patients had a 12‐month follow‐up. There was an increase in the mean scores from baseline to 12 months on both HFMSE (1.18 points, p = 0.004) and RULM scores (0.58 points, p = 0.014) but not on the 6MWT (mean difference = 6.65 m, p = 0.33). When the 12‐month HFMSE changes in the treated cohort were compared to an external cohort of untreated patients, in all untreated patients older than 7 years, the mean changes were always negative, while always positive in the treated ones. To reduce a selection bias, we also used a multivariable analysis. On the HFMSE scale, age, gender, baseline value, and functional status contributed significantly to the changes, while the number of SMN2 copies did not contribute. The effect of these variables was less obvious on the RULM and 6MWT. Interpretation Our results expand the available data on the effect of Nusinersen on type III patients, so far mostly limited to data from adult type III patients.

Abstract Background Deeply divided ideological positions challenge collaboration when engaging youth with mental disorders, caregivers and providers in mental health research. The integrative dynamics (ID) approach can restructure relationships and overcome ‘us vs them’ thinking. Objective To assess the extent to which an experience‐based co‐design (EBCD) approach to patient and family engagement in mental health research aligned with ID processes. Methods A retrospective case study of EBCD data in which transitional‐aged youth (n = 12), caregivers (n = 8) and providers (n = 10) co‐designed prototypes to improve transitions from child to adult services. Transcripts from focus groups and a co‐design event, co‐designed prototypes, the resulting model, evaluation interviews and author reflections were coded deductively based on core ID concepts, while allowing for emergent themes. Analysis was based on pattern matching. Triangulation across data sources, research team, and youth and caregiver reflections enhanced rigour. Findings The EBCD focus group discussions of touchpoints in experiences aligned with ID processes of acknowledging the past, by revealing the perceived identity mythos of each group, and allowing expression of and working through emotional pain. These ID processes were briefly revisited in the co‐design event, where the focus was on the remaining ID processes: building cross‐cutting connections and reconfiguring relationships. The staged EBCD approach may facilitate ID, by working within one's own perspective prior to all perspectives working together in co‐design. Conclusion Researchers can augment patient engagement approaches by applying ID principles with staged integration of groups to improve relations in mental health systems, and EBCD shows promise to operationalize this.

Abstract Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label‐free methods without (bio)‐chemical markers or surface‐engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label‐ and receptor‐free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell‐detachment with sharply defined time‐patterns, a phenomenon unknown from literature. These patterns depend on metabolic activity (controlled through temperature, nutrients, and drugs) and provide a library of cell‐type‐specific indicators, allowing to distinguish several yeast strains as well as cancer cells. Under specific conditions, synchronized glycolytic‐type oscillations are observed during detachment of mammalian and yeast‐cell ensembles, providing additional cell‐specific signatures. These findings suggest potential applications for cell viability analysis and for assessing the collective response of cancer cells to drugs.

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.

Quantification of tumor‐specific variants (TSVs) in cell‐free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next‐generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell‐free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell‐free DNA to its full potential.

Abstract Encompassing some of the major hotspots of biodiversity on Earth, large mountain systems have long held the attention of evolutionary biologists. The region of the Qinghai‐Tibet Plateau (QTP) is considered a biogeographic source for multiple colonization events into adjacent areas including the northern Palearctic. The faunal exchange between the QTP and adjacent regions could thus represent a one‐way street (“out of” the QTP). However, immigration into the QTP region has so far received only little attention, despite its potential to shape faunal and floral communities of the QTP. In this study, we investigated centers of origin and dispersal routes between the QTP, its forested margins and adjacent regions for five clades of alpine and montane birds of the passerine superfamily Passeroidea. We performed an ancestral area reconstruction using BioGeoBEARS and inferred a time‐calibrated backbone phylogeny for 279 taxa of Passeroidea. The oldest endemic species of the QTP was dated to the early Miocene (ca. 20 Ma). Several additional QTP endemics evolved in the mid to late Miocene (12–7 Ma). The inferred centers of origin and diversification for some of our target clades matched the “out of Tibet hypothesis’ or the “out of Himalayas hypothesis” for others they matched the “into Tibet hypothesis.” Three radiations included multiple independent Pleistocene colonization events to regions as distant as the Western Palearctic and the Nearctic. We conclude that faunal exchange between the QTP and adjacent regions was bidirectional through time, and the QTP region has thus harbored both centers of diversification and centers of immigration.

Abstract Objectives To compare the effect of a high‐speed barbecue maneuver with the modified Lempert maneuver and sham in patients with benign paroxysmal positional vertigo (BPPV) of the horizontal canal. Methods Randomized sham‐controlled, single blinded multicenter clinical trial in two university hospitals investigating consecutive patients with horizontal canal BPPV. Patients were randomly assigned to high‐speed barbecue (HSB), modified Lempert maneuver (ML), or sham maneuver (SM). All treatments were performed in a biaxial rotational chair with weekly follow‐up to a maximum of three treatment sessions. The final follow‐up was 3 months after the last treatment. Results Primary outcome: 2‐week recovery rate per protocol. Secondary outcome: Cumulative recovery rate and Dizziness Handicap Inventory (DHI) scores after 3 months per protocol (HSB and ML) and intention to treat (all groups). Fifty‐four patients were analyzed after 2 weeks (HSB = 17; ML = 20; SM = 17). Two‐week recovery rate was 14/17 after HSB, 11/20 after ML, and 4/17 after SM, with significantly better recovery in HSB [OR 15.17, 95% CI (1.85, 124.63), P = .001] using sham as base level. Recovery rate after 3 months was 15/17 after HSB and 15/19 after ML. Cumulative recovery rate showed no significant differences between the two treatment groups [95% CI (0.30, 13.14), P = .46] in cure rate DHI [95% CI (−16.56, 15.02), P = .92]. No unexpected adverse events were observed. Conclusion Velocity change in horizontal canal BPPV treatment gives a faster initial recovery. Rapid recovery could reduce the disease burden. Trial Registration Clinicaltrials.gov. Identifier: NCT01905800. Level of Evidence 1b

Abstract Estradiol and exercise can decrease risk factors associated with type 2 diabetes (T2D) including total body weight gain and abdominal fat gain. Estradiol functions through estrogen receptor (ER) α and ERβ. Some studies suggest that activation of ERα may provide protection against T2D. Female Wistar rats were ovariectomized and fed a high‐fat diet for 10 weeks and divided into the following 5 experimental groups: (1) no treatment (control), (2) exercise, (3) estradiol, (4) propylpyrazoletriyl (a selective ERα agonist), and (5) diarylpropionitrile (a selective ERβ agonist). ERα activation decreased the abundance of Firmicutes, and ERα and ERβ activation increased the abundance of Bacteroidetes. ERα activation decreased food consumption, and ERα and ERβ activation increased voluntary activity. Exercise was the only treatment to decrease the blood glucose and serum insulin levels. ERα activation, but not ERβ, increased hepatic protein expression of ACC and FAS and decreased hepatic protein expression of LPL. ERα activation also decreased hepatic mRNA expression of PPARα and PPARγ. This study elucidates the functions of estradiol by assessing specific activation of ERα and ERβ. As obesity increases the abundance of Firmicutes and decreases the abundance of Bacteroidetes, our study shows that ERα activation can restore the gut microbiome to non‐obese abundances. This study further provides novel insights into ERα’s role in hepatic fat metabolism via regulation of ACC, FAS, LPL, PPARα, and PPARγ.

Abstract Background Improving the poor prognosis of infant leukaemias remains an unmet clinical need. This disease is a prototypical fusion oncoprotein‐driven paediatric cancer, with MLL (KMT2A)‐fusions present in most cases. Direct targeting of these driving oncoproteins represents a unique therapeutic opportunity. This rationale led us to initiate a drug screening with the aim of discovering drugs that can block MLL‐fusion oncoproteins. Methods A screen for inhibition of MLL‐fusion proteins was developed that overcomes the traditional limitations of targeting transcription factors. This luciferase reporter‐based screen, together with a secondary western blot screen, was used to prioritize compounds. We characterized the lead compound, disulfiram (DSF), based on its efficient ablation of MLL‐fusion proteins. The consequences of drug‐induced MLL‐fusion inhibition were confirmed by cell proliferation, colony formation, apoptosis assays, RT‐qPCR, in vivo assays, RNA‐seq and ChIP‐qPCR and ChIP‐seq analysis. All statistical tests were two‐sided. Results Drug‐induced inhibition of MLL‐fusion proteins by DSF resulted in a specific block of colony formation in MLL‐rearranged cells in vitro, induced differentiation and impeded leukaemia progression in vivo. Mechanistically, DSF abrogates MLL‐fusion protein binding to DNA, resulting in epigenetic changes and down‐regulation of leukaemic programmes setup by the MLL‐fusion protein. Conclusion DSF can directly inhibit MLL‐fusion proteins and demonstrate antitumour activity both in vitro and in vivo, providing, to our knowledge, the first evidence for a therapy that directly targets the initiating oncogenic MLL‐fusion protein.

Background Current American guidelines are uncertain regarding endovascular treatment (EVT) for patients with acute ischemic stroke with an Alberta Stroke Program Early Computed Tomography Score (ASPECTS)

Abstract Reduced food availability during chick raising is a major driver of farmland bird declines. For the Eurasian Skylark (Alauda arvensis), food availability is determined by various factors (i.e., arthropod abundance/diversity, accessibility of the vegetation, distance to foraging sites). In modern farmland, it is supposed to decrease over the breeding season due to less penetrable vegetation. We explored foraging habitat selection by chick‐raising Skylarks with a focus on the seasonal dynamics of habitat use and food availability. We investigated (i) habitat selection concerning prey biomass/diversity, vegetation cover, and distance to foraging sites, (ii) the overall and seasonal habitat use, and (iii) seasonal developments of foraging parameters (e.g., the feeding frequency) as indicators of food availability. We collected data on foraging habitats and foraging parameters of chick‐raising Skylark pairs at 51 nests from a Central European population in 2018 and 2019. Prey biomass/diversity and vegetation cover were measured for all habitats around 42 of these nests. As revealed by multivariate and compositional analyses, Skylarks mainly selected foraging habitats based on the proximity to nests. The most frequent habitats within home ranges could not be ranked according to an overall importance for foraging and their use partially changed over time. The feeding frequency increased throughout the breeding season, while other foraging parameters did not show significant changes. In contrast to our expectations, our data indicated therefore an increase, not a decrease in food availability in the late breeding season. This also implies that the way in which Skylarks used habitats was constantly suitable to raise offspring. We interpret this to be a consequence of the heterogeneous farmland composition of the study area that enabled Skylarks to establish a diverse home range and to benefit from the synergetic effects of neighboring habitat types. Thus, our findings provide support for the high importance of crop diversity in Skylark conservation.