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Diffuse intrinsic pontine glioma (DIPG) is a fatal central nervous system (CNS) tumor that confers a median survival of 11 months. As B7-H3 is expressed on pediatric CNS tumors, we conducted BrainChild-03, a single-center, dose-escalation phase 1 clinical trial of repetitive intracerebroventricular (ICV) dosing of B7-H3-targeting chimeric antigen receptor T cells (B7-H3 CAR T cells) for children with recurrent or refractory CNS tumors and DIPG. Here we report results from Arm C, restricted to patients with DIPG. The primary objectives were to assess feasibility and tolerability, which were both met. Secondary objectives included assessments of CAR T cell distribution and survival. A total of 23 patients with DIPG enrolled, and 21 were treated with repeated doses of ICV B7-H3 CAR T cells using intra-patient dose-escalation regimens without previous lymphodepletion. Concurrent tumor-directed therapy, including re-irradiation, was not allowed while on protocol therapy. We delivered a total of 253 ICV doses and established the highest planned dose regimen, DR4, which escalated up to 10 × 107cells per dose, as the maximally tolerated dose regimen. Common adverse events included headache, fatigue and fever. There was one dose-limiting toxicity (intratumoral hemorrhage) during DR2. For all treated patients (n= 21), the median survival from their initial CAR T cell infusion was 10.7 months and the median survival from diagnosis was 19.8 months with 3 patients still alive at 44, 45 and 52 months from diagnosis. Ultimately, this completed first-in-human trial shows that repetitive ICV dosing of B7-H3 CAR T cells in pediatric and young adult patients with DIPG is tolerable, including multiyear repeated dosing, and may have clinical efficacy that warrants further investigation on a multisite phase 2 trial. ClinicalTrials.gov registration: NCT04185038.

Extensive patient heterogeneity is a challenge in the management of inflammatory bowel disease (IBD). Sex and gender, as well as the interaction of sex and gender with other social identities, referred to as intersectionality, contribute to this heterogeneity and might affect IBD outcomes. An interdisciplinary team of clinicians, researchers, patients, and sex and gender experts reviewed current literature on the effect of sex and gender dimensions on IBD outcomes. The team also investigated the role that stakeholders have in advancing sex-based and gender-based IBD knowledge, as comprehensive studies are scarce. Acknowledging and integrating sex and gender into the organisation and content of research (eg, study design, participant recruitment, data analysis, data interpretation, data dissemination, and impact evaluation) could enhance the validity, relevance, and applicability of research. Such gendered innovation has potential for advancing personalised medicine and improving the quality of life for people with IBD.

Analyses of transcriptional bursting from single-cell RNA-sequencing data have revealed patterns of variation and regulation in the kinetic parameters that could be inferred. Here we profiled newly transcribed (4-thiouridine-labelled) RNA across 10,000 individual primary mouse fibroblasts to more broadly infer bursting kinetics and coordination. We demonstrate that inference from new RNA profiles could separate the kinetic parameters that together specify the burst size, and that the synthesis rate (and not the transcriptional off rate) controls the burst size. Importantly, transcriptome-wide inference of transcriptional on and off rates provided conclusive evidence that RNA polymerase II transcribes genes in bursts. Recent reports identified examples of transcriptional co-bursting, yet no global analyses have been performed. The deep new RNA profiles we generated with allelic resolution demonstrated that co-bursting rarely appears more frequently than expected by chance, except for certain gene pairs, notably paralogues located in close genomic proximity. Altogether, new RNA single-cell profiling critically improves the inference of transcriptional bursting and provides strong evidence for independent transcriptional bursting of mammalian genes.

IMPORTANCE: Increases in pulmonary capillary wedge pressure (PCWP) during exercise reduce pulmonary artery (PA) compliance, increase pulsatile right ventricular (RV) afterload, and impair RV-PA coupling in patients with heart failure with preserved ejection fraction (HFpEF). The effects of the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin on pulmonary vascular properties and RV-PA coupling are unknown. OBJECTIVE: To test the effect of dapagliflozin on right ventricular performance and pulmonary vascular load during exertion in HFpEF. DESIGN, SETTING, AND PARTICIPANTS: Evaluation of the Cardiac and Metabolic Effects of Dapagliflozin in Heart Failure With Preserved Ejection Fraction (CAMEO-DAPA) randomized clinical trial demonstrated improvement in PCWP at rest and exercise over 24 weeks with dapagliflozin compared with placebo with participants recruited between February 2021 and May 2022. This secondary analysis evaluates the effects of dapagliflozin on pulsatile pulmonary vascular load and RV-PA coupling using simultaneous echocardiography and high-fidelity invasive hemodynamic testing with exercise. This was a single-center study including patients with hemodynamically confirmed HFpEF with exercise PCWP of 25 mm Hg or greater. INTERVENTIONS: Dapagliflozin or placebo for 24 weeks. MAIN OUTCOMES AND MEASURES: Pulsatile pulmonary vascular load (PA compliance and elastance) and right ventricular performance (PA pulsatility index, RV systolic velocity [s′]/PA mean) during rest and exercise. RESULTS: Among 37 randomized participants (mean [SD] age, 67.4 [8.5] years; 25 female [65%]; mean [SD] body mass index, 34.9 [6.7]; calculated as weight in kilograms divided by height in meters squared), there was no effect of dapagliflozin on PA loading or RV-PA interaction at rest. However, with exercise, dapagliflozin improved PA compliance (placebo-corrected mean difference, 0.57 mL/mm Hg; 95% CI, 0.11-1.03 mL/mm Hg; P = .02) and decreased PA elastance (stiffness; −0.17 mm Hg/mL; 95% CI, −0.28 to −0.07 mm Hg/mL; P = .001). RV function during exercise improved, with increase in PA pulsatility index (0.33; 95% CI, 0.08-0.59; P = .01) and increase in exercise RV s′ indexed to PA pressure (0.09 cm·s−1/mm Hg; 95% CI, 0.02-0.16 cm·s−1/mm Hg; P = .01). Improvements in pulsatile RV load and RV-PA coupling were correlated with reduction in right atrial (RA) pressure (PA elastance Pearson r = 0.55; P =.008; RV s′/PA elastance Pearson r = −0.60; P =.002) and PCWP (PA elastance Pearson r = 0.58; P <.001; RV s′/PA elastance Pearson r = −0.47; P = .02). Dapagliflozin increased resistance-compliance time (dapagliflozin, median [IQR] change, 0.06 [0.03-0.15] seconds; placebo, median [IQR] change, 0.01 [−0.02 to 0.05] seconds; P =.046), resulting in higher PA compliance for any exercise pulmonary vascular resistance. CONCLUSIONS AND RELEVANCE: Results of this randomized clinical trial reveal that treatment with dapagliflozin for 24 weeks reduced pulsatile pulmonary vascular load and enhanced dynamic RV-PA interaction during exercise in patients with HFpEF, findings that are related to the magnitude of PCWP reduction. Benefits on dynamic right ventricular–pulmonary vascular coupling may partially explain the benefits of SGLT2 inhibitors in HFpEF. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04730947

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

BackgroundNational guidelines recommend that waist circumference (WC) be measured in patients with a body mass index (BMI) 27–35 kg/m2. Unfortunately, perhaps due to logistical reasons, WC is seldom measured in clinical settings. Herein, we describe the performance of a self-operated waist measurement device (SOWMD) as a potential means to overcome barriers to measuring WC.Materials and methodsTen volunteers underwent WC measures by professionals and SOWMD on 5 separate days to assess the reproducibility and accuracy. We then compared SOWMD measures with CT-derived fat content by recruiting 81 patients scheduled for a diagnostic abdominal CT scan.ResultsThere was no difference between professionally measured and SOWMD-measured WC; the intraindividual coefficient of variation over the 5 days was between 0.4% and 2.2%. The WC measured manually, by SOWMD and CT scan were highly correlated (r=0.90–0.92, all p<0.001). The minimal WC measured by SOWMD was a better predictor (r=0.81 for all patients, r=0.87 for men, both p<0.001) of CT-measured visceral adipose tissue volume than other approaches. The minimal WC measured by SOWMD was correlated with fasting plasma glucose (r=0.40, p<0.05), triglyceride (r=0.41, p<0.01) and high-density cholesterol (r=−0.49, p<0.001) concentrations.ConclusionSOWMD is a reproducible, accurate and convenient way to measure WC that can provide added value for health care providers when combined with BMI information.

Interleukin (IL)-6 is a central inflammatory mediator and potential therapeutic target in heart failure (HF). Prior studies have shown that IL-6 concentrations are elevated in patients with HF, but much fewer data are available in heart failure with preserved ejection fraction (HFpEF).

The reconfigurable over-the-air (OTA) chamber (ROTAC) is a reverberation chamber (RC) that, when used for OTA testing of mobile devices, offers enhanced control of the field incident on a device under test (DUT). This article presents a model based on the method of moments (MoM) electromagnetic analysis and network theory that offers an efficient characterization of the ROTAC, making it useful for ROTAC design and optimization. The development includes the application of the Ewald transformation to accelerate the computation of the slowly convergent cavity Green’s function. Comparisons with measured data from a prototype ROTAC demonstrate the model’s accuracy. The article concludes by using the tools to optimize the ROTAC physical probe arrangement and explore its ability to synthesize specified fields as well as its sensitivity to excitation errors as a function of the probe arrangement.

Cells in the precentral gyrus directly send signals to the periphery to generate movement and are principally organized as a topological map of the body. We find that movement-induced electrophysiological responses from depth electrodes extend this map three-dimensionally throughout the gyrus. Unexpectedly, this organization is interrupted by a previously undescribed motor association area in the depths of the midlateral aspect of the central sulcus. This ‘Rolandic motor association’ (RMA) area is active during movements of different body parts from both sides of the body and may be important for coordinating complex behaviors.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker’s yeast Saccharomyces cerevisiaeand deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

The reconfigurable over-the-air (OTA) chamber (ROTAC) is a reverberation chamber with antennas positioned along the chamber walls. By controlling the signals driving a small number of these antennas and tunable impedances terminating the ports of the remaining antennas, the ROTAC offers dynamical control of the field incident on a mobile device during OTA testing. This article uses finite-difference time-domain electromagnetic simulations and measurements from a ROTAC prototype in combination with a network model to characterize the fields incident on the device as a function of the excitations and terminations. A gradient search optimization is formulated to determine the excitations and terminations required to approximate specified field characteristics in the chamber test zone, including multipath direction of arrival, signal correlation, and fading statistics. The article further explores the relationship between the field spatial sampling within the test zone and the ability to synthesize a field with the specified characteristics.

Magnetic resonance enterocolonography (MREC) and pan‐enteric capsule endoscopy (CE) offers visualization of the entire gastrointestinal tract in a single examination. We examined the diagnostic accuracy of MREC and CE in patients with suspected Crohn's disease (CD). In a prospective, blinded, multicenter study, we included patients with clinically suspected CD. Patients were examined with MREC, CE, and ileocolonoscopy (IC) within 2 weeks. The primary outcome was per patient sensitivity, specificity, and diagnostic accuracy for ileocolonic CD. IC served as reference standard. 153 patients were included in the study and IC, MREC, and CE was performed in 152, 151, 133 patients, respectively. CD was diagnosed with IC in 59 (39%) patients (terminal ileum (TI) 22, colon 20, TI and colon 17). The sensitivity and specificity for diagnosing ileocolonic CD with MREC was 67.9% (CI 53.7–80.1) and 76.3% (CI 65.2–85.3) (TI 76.9% and 85.6%; colon 27% and 93%) compared to 87.5% (CI 73.2–95.8) and 87.8% (CI 78.2–94.3) with CE (TI 96.6% and 87.5%; colon 75.0% and 93.0%). The sensitivity of CE was superior to that of MREC (p= 0.02). The patient experienced discomfort was equal with CE and MREC and significantly less than with IC. In patients with suspected CD, CE has a high sensitivity for diagnosing CD in the TI and colon, which is superior to that of MREC. The sensitivity of MREC for diagnosing CD in the colon is poor. CE could be a patient‐friendly alternative to IC in selected patients with suspected CD. Registered at ClinicalTrials.gov: NCT03134586.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient’s apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novomodel building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

Single-cell sequencing methods rely on molecule-counting strategies to account for amplification biases, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes—RNA spike-ins containing built-in unique molecular identifiers (UMIs) that we use to identify critical experimental and computational conditions for accurate RNA counting in single-cell RNA-sequencing (scRNA-seq). Using molecular spikes, we uncovered impaired RNA counting in methods that were not informative for cellular RNA abundances due to inflated UMI counts. We further leverage molecular spikes to improve estimates of total endogenous RNA amounts in cells, and introduce a strategy to correct experiments with impaired RNA counting. The molecular spikes and the accompanying R package UMIcountR (https://github.com/cziegenhain/UMIcountR) will improve the validation of new methods, better estimate and adjust for cellular mRNA amounts and enable more indepth characterization of RNA counting in scRNA-seq.

An increasing number of long noncoding RNAs (lncRNAs) have experimentally confirmed functions, yet little is known about their transcriptional dynamics and it is challenging to determine their regulatory effects. Here, we used allele-sensitive single-cell RNA sequencing to demonstrate that, compared to messenger RNAs, lncRNAs have twice as long duration between two transcriptional bursts. Additionally, we observed increased cell-to-cell variability in lncRNA expression due to lower frequency bursting producing larger numbers of RNA molecules. Exploiting heterogeneity in asynchronously growing cells, we identified and experimentally validated lncRNAs with cell state-specific functions involved in cell cycle progression and apoptosis. Finally, we identified cis-functioning lncRNAs and showed that knockdown of these lncRNAs modulated the nearby protein-coding gene’s transcriptional burst frequency or size. In summary, we identified distinct transcriptional regulation of lncRNAs and demonstrated a role for lncRNAs in the regulation of mRNA transcriptional bursting.

The preconceptual, intrauterine, and early life environments can have a profound and long-lasting impact on the developmental trajectories and health outcomes of the offspring. Given the relatively low success rates of assisted reproductive technologies (ART; ∼25%), additives and adjuvants, such as glucocorticoids, are used to improve the success rate. Considering the dynamic developmental events that occur during this window, these exposures may alter blastocyst formation at a molecular level, and as such, affect not only the viability of the embryo and the ability of the blastocyst to implant, but also the developmental trajectory of the first three cell lineages, ultimately influencing the physiology of the embryo. In this study, we present a comprehensive single-cell transcriptome, methylome, and small RNA atlas in the day 7 human embryo. We show that, despite no change in morphology and developmental features, preimplantation glucocorticoid exposure reprograms the molecular profile of the trophectoderm (TE) lineage, and these changes are associated with an altered metabolic and inflammatory response. Our data also suggest that glucocorticoids can precociously mature the TE sublineages, supported by the presence of extravillous trophoblast markers in the polar sublineage and presence of X Chromosome dosage compensation. Further, we have elucidated that epigenetic regulation—DNA methylation and microRNAs (miRNAs)—likely underlies the transcriptional changes observed. This study suggests that exposures to exogenous compounds during preimplantation may unintentionally reprogram the human embryo, possibly leading to suboptimal development and longer-term health outcomes.

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 (NCT03500991), an ongoing phase 1 clinical trial at Seattle Children’s evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the cancer treatment landscape, utilizing ex vivomodified autologous T cells to treat relapsed or refractory B-cell leukemias and lymphomas. However, the therapy’s broader impact has been limited, in part, by a complicated, lengthy, and expensive production process. Accordingly, as CAR T-cell therapies are further advanced to treat other cancers, continual innovation in cell manufacturing will be critical to their successful clinical implementation. In this Account, we describe our research efforts using biomaterials to improve the three fundamental steps in CAR T-cell manufacturing: (1) isolation, (2) activation, and (3) genetic modification.

Hammock mitral valve (MV) repair is historically technically difficult with a guarded prognosis. Surgical experience is extremely limited and variable outcomes are reported. The perioperative strategy and technical details of hammock MV repair in an infant who presented with severe mitral stenosis are described and review of the existing literature was undertaken.

Although visceral fat is strongly correlated with the metabolic complications of obesity, the existing data indicate it is not the cause of these complications. Excess release of free fatty acids (FFA) from adipose tissue lipolysis can account for a sizable portion of the metabolic complications of obesity. In humans, upper-body subcutaneous adipose tissue accounts for most systemic FFA, whereas visceral fat contributes a modest portion of the excess amount to which the liver is exposed. This pattern is maintained in upper-body/visceral obesity, except that greater amounts of visceral fat expose the liver to more FFA from visceral adipose tissue lipolysis.

Background/Objectives: Because no validated tool exists to assess nutrition knowledge regarding weight management we developed and tested the Weight Management Nutrition Knowledge Questionnaire (WMNKQ). Subjects/Methods: The questionnaire assesses nutrition knowledge in these categories: energy density of food, portion size/serving size, alcohol and sugar sweetened beverages, how food variety affects food intake, and reliable nutrition information sources. In total 60 questions were reviewed by 6 experts for face validity and quantitative analysis was used to assess item difficulty, item discrimination, internal consistency, inter-item-correlation, test-retest reliability, construct validity, criterion validity, and convergent validity. Results: The final WMNKQ contained 43 items. Experts removed 3 of the original 60 questions and modified 41. Eighteen items did not meet criteria for item difficulty, item discrimination, and/or inter-item correlation; 4 were retained. The WMNKQ met criteria for internal consistency (Cronbach's alpha = 0.88), reliability (test-retest correlation ρ = 0.90, P&lt; 0.0001), construct validity (known groups comparison) - dietitians scored 16% better (p&lt; 0.0001) than information technology workers, and criterion validity (pre- to post-intervention improvement in knowledge scores = 11.2% (95% CI 9.8–12.5, p&lt; 0.0001)). Participants younger than age 55 scored significantly better than those over age 55 (convergent validity). Conclusions: The WMNKQ measures how well nutrition principles of weight management are understood.

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)–mediated signaling, but the number and type of protein interaction–mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.

Cell types can be classified according to shared patterns of transcription. Non-genetic variability among individual cells of the same type has been ascribed to stochastic transcriptional bursting and transient cell states. Using high-coverage single-cell RNA profiling, we asked whether long-term, heritable differences in gene expression can impart diversity within cells of the same type. Studying clonal human lymphocytes and mouse brain cells, we uncovered a vast diversity of heritable gene expression patterns among different clones of cells of the same type in vivo. We combined chromatin accessibility and RNA profiling on different lymphocyte clones to reveal thousands of regulatory regions exhibiting interclonal variation, which could be directly linked to interclonal variation in gene expression. Our findings identify a source of cellular diversity, which may have important implications for how cellular populations are shaped by selective processes in development, aging, and disease. A record of this paper’s transparent peer review process is included in the supplemental information.

Biliary atresia (BA) is a cholangiopathy characterized by bile flow obstruction due to destruction of the biliary tree. Without surgical correction with Kasai portoenterostomy (KPE), BA leads to death or liver transplant (LTx). Early-onset, progressive liver fibrosis is a defining characteristic of BA. Collagen hybridizing peptide (CHP) is a synthetic peptide which binds to denatured collagen strands allowing quantification of fibrosis. This technique has not been used on human liver tissue. The aim of this pilot study was to evaluate the utility of CHP as a measurement of quantitative fibrosis to allow earlier survival with native liver prognostication. We identified 21 patients with wedge liver biopsies available, of which 14 required LTx. No deaths occurred. Patients requiring LTx tended to be girls with a significantly different mean bilirubin (P= 0.002), albumin (P= 0.001), and alanine aminotransferase (P= 0.03) at 3 months post-KPE. By 1 year post-KPE, 50% of patients in the high CHP intensity group required LTx versus 27% in the low CHP. Overall, fibrosis as quantified by CHP at time of KPE was associated with more than 3 times the risk of requiring LTx by 4 years of age (hazard ratio 3.6, 95% confidence interval 1.15–10.93, P= 0.03). When controlling for sex and total bilirubin >2 mg/dL and albumin at 3 months post-KPE, it predicted nearly 7 times the risk of LTx (hazard ratio 6.89, 95% confidence interval 1.38–34.32, P= 0.02). Our results suggest that quantitative assessment of fibrosis at the time of KPE holds promise as an earlier predictor of LTx requirement in BA. A larger study is justified to assess quantitative fibrosis as a BA prognostic tool.

Immunotherapy with the adoptive transfer of T cells redirected with CD19-specific chimeric antigen receptors (CARs) for B-lineage acute lymphoblastic leukemia (ALL) can salvage >80% of patients having relapsed/refractory disease. The therapeutic index of this emerging modality is attenuated by the occurrence of immunologic toxicity syndromes that occur upon CAR T-cell engraftment. Here, we report on the low incidence of severe cytokine release syndrome (CRS) in a subject treated with a CAR T-cell product composed of a defined ratio CD4:CD8 T-cell composition with a 4-1BB:zeta CAR targeting CD19 who also recieved early intervention treatment. We report that early intervention with tocilizumab and/or corticosteroids may reduce the frequency at which subjects transition from mild CRS to severe CRS. Although early intervention doubled the numbers of subjects dosed with tocilizumab and/or corticosteroids, there was no apparent detrimental effect on minimal residual disease–negative complete remission rates or subsequent persistence of functional CAR T cells compared with subjects who did not receive intervention. Moreover, early intervention therapy did not increase the proportion of subjects who experience neurotoxicity or place subjects at risk for infectious sequelae. These data support the contention that early intervention with tocilizumab and/or corticosteroids in subjects with early signs of CRS is without negative impact on the antitumor potency of CD19 CAR T cells. This intervention serves to enhance the therapeutic index in relapsed/refractory patients and provides the rationale to apply CAR T-cell therapy more broadly in ALL therapy. This trial was registered at www.clinicaltrials.gov as #NCT020284.

Nonalcoholic fatty liver disease can lead to hepatic inflammation/damage. Understanding the physiological mechanisms that contribute to excess hepatic lipid accumulation may help identify effective treatments.

This study contrasts characteristics of mature squall‐line mesoscale convective systems (MCSs) observed by extended ground‐based radar wind profiler (RWP) deployments from the U.S. Department of Energy Atmospheric Radiation Measurement program. This analysis compares the dynamical structure, precipitation, and cold pool properties associated with MCS events over RWP sites in Oklahoma, USA, (midlatitude) to those observed during a 2‐year RWP deployment to Manaus, Brazil, during GoAmazon2014/5 campaign (tropical). The MCSs indicate similar convective line rainfall rates and total rainfall accumulations. However, midlatitude events suggest a larger fractional stratiform contribution to total precipitation. For both regions, convective line cold pools are associated with sharp decreases (approximately 10 K) in the surface equivalent potential temperature (θe) near the time of line passage. Surface θeproperties for both regions suggest a modest relationship between rainfall rate and the probability of observing measurable surface rainfall. The probability of observing convective updrafts in both tropical and midlatitude MCS events is found to be similar as a function of low‐level radar reflectivity. However, midlatitude MCSs are associated with more intense convective updrafts, with upward air motions (mean, maximum) peaking at higher altitude. The most pronounced contrast is the propensity for deeper and more intense downdrafts in midlatitude MCSs. An analysis based on observed downdraft properties is performed using simple mixing assumptions. For these events, the vertical gradient of θein the lower troposphere is relatively consistent between the Amazon and Oklahoma, suggesting similar mixing rates for downdrafts originating below 3 km (0.1 km−1). However, if downdrafts originate nearer to the level of minimum θeat SGP, mixing may be occurring at rates comparable to 0.3 km−1. Mesoscale systems in Amazonia and Oklahoma have similar rain rates, cold pool properties, and inferred mixing rates for low‐level downdraftsMesoscale systems in Oklahoma show intense updrafts peaking at higher altitudes and greater stratiform rain fractions than Amazon systemsOklahoma systems produce strong downdrafts that occur more frequently at higher altitudes (up to 6 km with mixing) than Amazon systems

Marine low clouds are critical to the climate system because of their extensive coverage and associated controls on boundary layer dynamics and radiative energy balance. The primary foci for this study are marine low cloud observations over a heavily instrumented site on the Azores archipelago in the Eastern North Atlantic and their associated raindrop size distribution (DSD) properties, relative low cloud contributions to the precipitation, and additional sampling (instrument, environmental) considerations. The contribution from low clouds (e.g., cloud top < 4 km) to the overall precipitation over midlatitude oceans is poorly understood, in part because of the lack of coupled, high‐quality measurements of precipitation and low cloud properties. Cloud regime and precipitation breakdowns performed for a multiyear (2014–2017) record emphasize diurnal precipitation and raindrop size distribution characteristics for both low and deeper clouds, as well as differences between the two disdrometer types used. Results demonstrate that marine low clouds over this Eastern North Atlantic location account for a significant (45%) contribution to the total rainfall and exhibit a diurnal cycle in cloud (thickness, top, and base) and precipitation characteristics similar to satellite records. Additional controls on observed surface rainfall characteristics of low clouds allowed by the extended ground‐based facility data sets are also explored. From those analyses, it is suggested that the synoptic state exerts a significant control on low cloud and surface precipitation properties. Low clouds over ENA account for a significant (45%) contribution to the total rainfallLow cloud diurnal cycle suggests a rainfall peak during the overnight hours and a secondary peak after local noonSynoptic state (sfc pressure and 850‐mb winds) exerts a significant control on low cloud properties including rainfall rate and drop size

The correct representation of low‐level midlatitude clouds found in the wake of cold fronts strongly relies on the representation of planetary boundary layer (PBL) and convection processes, which are typically parameterized separately in numerical models. Using the Weather Research and Forecasting Model (WRF), this study investigates how distinct pairs of PBL and convection parameterization schemes represent cloud fraction in the post cold frontal region. The simulations focus on the region of the Department of Energy Atmospheric Radiation Measurement (ARM) Eastern North Atlantic observation site in the Azores Islands in the wake of a cold front that passed on 25 December 2015. Different PBL and convection schemes are combined to create 12 distinct configurations. The main differences between the selected physical parameterizations are the strength of vertical mixing and the entrainment. The simulations produce a wide range of cloud fractions, where some configurations significantly underestimate while others clearly overestimate satellite and surface‐based estimates of cloud fraction. A skill score is used to quantitatively assess the performance of each configuration with respect to ground‐based radar data. The key processes that are found to significantly impact the cloud fraction distribution are the strength of the PBL decoupling, the vertical wind shear, entrainment and detrainment rates in shallow convection, and the occurrence of drizzle. This indicates that to successfully simulate post cold frontal clouds, modeled physics must balance strong internal vertical mixing and weak exchange with the free troposphere. For this case study, cloud fraction was more sensitive to the choice of convection scheme than PBL scheme. The key processes that affect modeled post cold frontal cloud fraction are the boundary layer decoupling, vertical wind shear, entrainment/detrainment, and drizzleModeled physics must balance strong internal vertical mixing and weak exchange with the free troposphereNo single mechanism can explain the differences in cloud fractions across an ensemble of different boundary layer/convection configurations