Discovery of cell type classification marker genes from single cell RNA sequencing data using NS-Forest.

The use of single-cell transcriptomic technologies that quantitively describe cell transcriptional phenotypes using single cell/nucleus RNA sequencing (scRNA-seq) is revolutionizing our understanding of cell biology, leading to new insights in cell type identification, disease mechanisms, and drug development. The tremendous growth in scRNA-seq data has posed new challenges in efficiently characterizing data-driven cell types and identifying quantifiable marker genes for cell type classification. The use of machine learning and explainable artificial intelligence has emerged as an effective approach to study large-scale scRNA-seq data. NS-Forest is a random forest machine learning-based algorithm that aims to provide a scalable data-driven solution to identify minimum combinations of necessary and sufficient marker genes that capture cell type identity with maximum classification accuracy. Here, we describe the latest version, NS-Forest version 4.0 and its companion Python package (https://github.com/JCVenterInstitute/NSForest), with several enhancements, to select marker gene combinations that exhibit selective expression patterns among closely related cell types and more efficiently perform marker gene selection for large-scale scRNA-seq data atlases with millions of cells. By modularizing the final decision tree step, NS-Forest v4.0 can be used to compare the performance of user-defined marker genes with the NS-Forest computationally-derived marker genes based on the decision tree classifiers. To quantify how well the identified markers exhibit the desired pattern of being exclusively expressed at high levels within their target cell types, we introduce the On-Target Fraction metric that ranges from 0 to1, with a metric of 1 given to markers that are only expressed within their target cell types and not in cells of any other cell types. We have applied NS-Forest v4.0 on scRNA-seq datasets from three human organs, including the brain, kidney, and lung. We observe that NS-Forest v4.0 outperforms previous versions on its ability to identify markers with higher On-Target Fraction values for closely related cell types and outperforms other marker gene selection approaches on the classification performance with significantly higher F-beta scores.

Plasma free hemoglobin is associated with LDH, AST, total bilirubin, reticulocyte count, and the hemolysis score in patients with sickle cell anemia.

Plasma free hemoglobin (PFH) is a direct biomarker for hemolysis that has been associated with clinical complications such as pulmonary hypertension and death in patients with sickle cell disease (SCD). We sought to characterize the relationship between PFH and more clinically available hemolytic markers including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), bilirubin, reticulocyte percentage and to derive a composite hemolysis score derived from principal component analysis (PCA) of these biomarkers. In 68 adult patients (median age 31 years old, IQR 25-39) with HbSS or HbSß0-thalassemia enrolled in the IMPROVE II study, median PFH was elevated at 21.9 mg/dL (IQR 9.9-44.9 mg/dL). Using Pearson correlation analysis, PFH had a stronger relationship to LDH (R=0.699), AST (R=0.587), and total bilirubin (R=0.475), compared to reticulocyte count (R=0.316). The hemolysis score was significantly associated with PFH (R=0.677). When compared with other laboratory measures, PFH correlated with hemoglobin (R= -0.275) and HbS (R=0.277),but did not correlate with white blood cell count (WBC) or HbF. The hemolysis score was significantly associated with WBC (R=0.307), hemoglobin (R = -0.393), HbF (R=- 0.424), and HbS (R=0.423). This study confirms that the conventional hemolytic biomarkers LDH, AST, bilirubin, and reticulocyte percentage correlate with PFH. Additionally, the hemolysis score is a valid tool to measure hemolysis and that it may be a marker of global hemolysis as opposed to PFH, which quantifies intravascular hemolysis. Further studies will be needed to elucidate the role of PFH and intravascular hemolysis in the development of clinical complications of sickle cell disease.

Equivalent thrombotic risk with Warfarin, Dabigatran, or Enoxaparin after failure of initial direct oral anticoagulation (DOAC) therapy.

BACKGROUND: The direct oral anticoagulants (DOACs) are now commonly regarded as first line anticoagulants in most cases of venous thromboembolism (VTE). However, the optimal choice of subsequent anticoagulant in instances of first line DOAC failure is unclear. OBJECTIVES: To describe and compare outcomes with second line anticoagulants used after DOAC failure. METHODS: Patients seen at an urban hospital system for an episode of acute VTE initially treated with either apixaban or rivaroxaban who experienced a subsequent recurrent thrombosis while on anticoagulation (1st recurrent thrombosis) were included. RESULTS: In total, 166 patients after apixaban or rivaroxaban failure were included. Following DOAC failure (1st recurrent thrombosis), the subsequent anticoagulant was warfarin in 60 patients (36%), dabigatran in 42 patients (25%), and enoxaparin in 64 patients (39%). Enoxaparin was preferentially prescribed in patients with a malignancy-associated etiology for 1st recurrent thrombosis (p < 0.01). The median follow-up time in our cohort was 16 months. There was no difference in 2nd recurrent thrombosis-free survival (p = 0.72) or risk for major bleeding event (p = 0.30) among patients treated with dabigatran, warfarin, or enoxaparin. CONCLUSIONS: In this retrospective analysis of patients failing first line DOAC therapy, rates of 2nd recurrent thrombosis and bleeding did not differ among subsequently chosen anticoagulants. Our study provides evidence that the optimal 2nd anticoagulant is not clear, and the choice of 2nd anticoagulant should continue to balance patient preference, cost, and provider experience.

Myeloid neoplasms in inflammatory bowel disease: A case series and review of the literature.

Patients with inflammatory bowel disease (IBD) are exposed to chronic systemic inflammation and are at risk for secondary malignancies. Here we review the literature on the risk of myeloid neoplasms (MN) in IBD and present the disease profiles of patients at a single institution with IBD who later developed MN, comparing them to those in the literature. No IBD characteristic was found to associate with MN disease severity, including the previously-identified association between MNs and thiopurine exposure. Of the somatic mutations identified in out cohort's MN, mutations in TET2 were most prevalent, followed by FLT3-ITD, BCR-ABL, and NPM1 mutations.

Filgrastim and infliximab biosimilar uptake in Medicare Advantage compared with Traditional Medicare, 2016-2019.

BACKGROUND: Medicare Advantage (MA) and Traditional Medicare face different financing structures and incentives and may implement different strategies to encourage biosimilar uptake. Strategies used by health insurers can influence biosimilar uptake, which can in turn promote savings to insurers and patients. OBJECTIVE: To compare filgrastim and infliximab biosimilar uptake between MA and Traditional Medicare from 2016 to 2019 and examine biosimilar uptake by different MA carriers and plan types (Health Maintenance Organization [HMO] or Preferred Provider Organization). METHODS: We use a 2016-2019 nationally representative random 20% sample of the carrier (physician) and outpatient paid claims for Traditional Medicare data and final-action carrier and outpatient records for MA data. We compare quarterly biosimilar uptake from 2016 to 2019 for the first 2 drugs with biosimilar competition: (1) filgrastim, (Neupogen, originator), and biosimilars tbo-filgrastim (GRANIX) and filgrastim-sndz (ZARXIO), and (2) infliximab (Remicade, originator), and biosimilars infliximab-dyyb (Inflectra) and infliximab-abda (Renflexis). RESULTS: From their introduction, there was consistently greater uptake of filgrastim and infliximab biosimilars in MA compared with Traditional Medicare. By Q4 2019, filgrastim biosimilar uptake was 7.6 percentage points higher in MA (80.3%) than Traditional Medicare (72.7%). By Q4 2019, infliximab biosimilar uptake was 28.7% and 15.4% in MA and Traditional Medicare, respectively. Kaiser HMO plans were primarily responsible for the higher uptake of biosimilars in MA; in Q4 2019, filgrastim and infliximab biosimilar uptake was 98.8% and 78.8%, respectively. CONCLUSIONS: Our findings suggest that filgrastim and infliximab biosimilar uptake is greater in MA compared with Traditional Medicare, which is driven in part by particularly high uptake of biosimilars in MA Kaiser HMO plans. This highlights the need for future work to examine specific strategies and levers employed by MA Kaiser HMO plans and other insurers to increase biosimilar uptake, which can lead to cost savings for physician-administered drugs.

Role of Patient Sorting in Avoidable Hospital Stays in Medicare Advantage vs Traditional Medicare.

Importance: Unlike traditional Medicare (TM), Medicare Advantage (MA) plans limit in-network care to a specific network of Medicare clinicians. MA plans thus play a role in sorting patients to a subset of clinicians. It is unknown whether the performance of physicians who treat MA and TM beneficiaries is different. Objective: To examine whether avoidable hospital stay differences between MA and TM can be explained by the primary care clinicians who treat MA and TM beneficiaries. Design, Setting, and Participants: This was a cross-sectional study of a nationally representative sample of MA and TM beneficiaries in 2019 with any of 5 chronic ambulatory care-sensitive conditions (ACSCs). The relative risk (RR) of avoidable hospital stays in MA compared with TM was estimated with inverse probability of treatment-weighted Poisson regression, both without and with clinician fixed effects. The degree to which the estimated MA vs TM difference could be explained by patient sorting was calculated by comparing the 2 RR estimates. Data were analyzed between February 2022 and April 2023. Exposure: Enrollment in MA. Main Outcome and Measures: Whether a beneficiary had avoidable hospital stays in 2019 due to any of the ACSCs. Avoidable hospital stays included both hospitalizations and observation stays. Results: The study sample comprised 1 323 481 MA beneficiaries (mean [SD] age, 75.4 [7.0] years; 56.9% women; 69.3% White) and 1 965 863 TM beneficiaries (mean [SD] age, 75.9 [7.4] years; 57.1% women; 82.5% White). When controlling for the primary care clinician, the RR of avoidable hospital stays in MA vs TM changed by 2.6 percentage points (95% CI, 1.72-3.50; P < .001), suggesting that compared with TM beneficiaries, MA beneficiaries saw clinicians with lower rates of avoidable hospital stays. This effect size was statistically significant to explain the 2% lower rate of avoidable hospital stays in MA than in TM. Conclusions and Relevance: In this cross-sectional study of MA and TM beneficiaries, the lower rate of avoidable hospital stays among MA beneficiaries than TM beneficiaries was attributable to MA beneficiaries visiting clinicians with lower rates of avoidable hospital stays. The patient sorting that occurs in MA plays a critical role in the lower rates of avoidable hospital stays compared with TM.

Leukotriene A4 hydrolase inhibition improves age-related cognitive decline via modulation of synaptic function.

Leukotrienes, a class of inflammatory bioactive lipids, are well studied in the periphery, but less is known of their importance in the brain. We identified that the enzyme leukotriene A4 hydrolase (LTA4H) is expressed in healthy mouse neurons, and inhibition of LTA4H in aged mice improves hippocampal dependent memory. Single-cell nuclear RNA sequencing of hippocampal neurons after inhibition reveals major changes to genes important for synaptic organization, structure, and activity. We propose that LTA4H inhibition may act to improve cognition by directly inhibiting the enzymatic activity in neurons, leading to improved synaptic function. In addition, LTA4H plasma levels are increased in both aging and Alzheimer's disease and correlated with cognitive impairment. These results identify a role for LTA4H in the brain, and we propose that LTA4H inhibition may be a promising therapeutic strategy to treat cognitive decline in aging related diseases.

Teaching antenatal hand expression: a feasibility study in an inner urban U.S. hospital.

BACKGROUND: Many women have low confidence in breastfeeding and have concerns regarding low milk volume or discomfort with breastfeeding. Antenatal hand expression may be an opportunity to help women feel more comfortable with breastfeeding and help promote exclusive breastfeeding. A study at a hospital in Philadelphia, Pennsylvania, U.S. assessed the feasibility of teaching antenatal hand expression at 39 weeks among socioeconomically disadvantaged populations, overall participant satisfaction and adoption of hand expression and breastfeeding. METHODS: From March 2020 to June 2021, women recruited at 34-39 weeks were taught to hand express, collect, and store colostrum. Starting from 39 weeks, participants were asked to practice hand expression 1-3 times / day until delivery, log their experiences, and store colostrum expressed. Women were contacted to encourage continued hand expression and answer any questions. Postpartum, a survey assessed satisfaction with hand expression and issues encountered. The survey also inquired about breastfeeding plans and barriers, and whether women were exclusively breastfeeding (defined as infants who received only breastmilk from the time of birth). Chart review of postpartum or well-baby visit notes determined whether women continued breastfeeding. RESULTS: Of the 29 participants, 72% (21/29) reported hand expressing at home, and no women reported contractions when hand expressing. Participants rated mean satisfaction of 8.1/10 (SD = 1.62) with antenatal hand expression, mean satisfaction of 9.4/10 (SD = 0.90) toward hand expression education, mean likelihood of 9.4/10 (SD = 1.24) recommending hand expression to others, and a mean score of 8.1/10 (SD = 1.69) on how helpful hand expression was in breastfeeding initiation. 90% (26/29) of women initiated breastfeeding after birth and 72% (21/29) exclusively breastfed on discharge, but only 11/29 (38%) continued exclusively breastfeeding when re-assessed 4-6 weeks postpartum. Barriers included maternal discomfort, low milk supply, and maternal or infant illness. CONCLUSIONS: This study suggests that women in an urban setting would be willing to practice antenatal hand expression. A larger and adequately powered study could be feasible to determine associations between antenatal hand expression and breastfeeding rates and confidence.

Novel Frizzled-specific antibody-based Wnt mimetics and Wnt superagonists selectively activate WNT/ß-catenin signaling in target tissues.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/ß-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development.

Incorporating Added Therapeutic Benefit and Domestic Reference Pricing Into Medicare Payment for Expensive Part B Drugs.

OBJECTIVES: Identify expensive Part B drugs and evidence for each drug's added benefit and model a reimbursement policy for Medicare that integrates added benefit assessment and domestic reference pricing. METHODS: A retrospective analysis using a 20% nationally representative sample of 2015 to 2019 traditional Medicare Part B claims. Expensive drugs were defined as having average annual spending per beneficiary exceeding the average annual social security benefit ($17 532 in 2019). For expensive drugs identified in 2019, added benefit assessments conducted by the French Haute Autorité de Santé were collected. For expensive drugs with a low added benefit rating, comparator drugs were identified in French Haute Autorité de Santé reports. For each comparator, average annual spending per beneficiary in Part B was computed. Potential savings from 2 reference pricing scenarios were calculated: reimbursing expensive Part B drugs with low added benefit at the level of each drug's (1) lowest cost comparator and (2) beneficiary-weighted-average cost of all comparators. RESULTS: The number of expensive Part B drugs grew from 56 in 2015 to 92 in 2019. Of the 92 expensive drugs in 2019, 34 offer low added benefit. Implementing reference pricing for these expensive drugs with low added benefit could have saved an estimated $2.1 billion if prices were set based on spending for their lowest cost comparator, or $1 billion if prices were set based on the weighted average of spending for comparators. CONCLUSION: Reference pricing based on added benefit assessment could be used to address the launch prices for expensive Part B drugs with low added benefit.

Safety, tolerability, pharmacokinetics, and immunogenicity of an anti-SARS-CoV-2 monoclonal antibody HFB30132A after single dose intravenous administration in healthy Chinese subjects: a phase 1, randomized, double-blind, placebo-controlled study.

Objective: The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) still protracts worldwide. HFB30132A is an anti- SARS-CoV-2 monoclonal antibody purposely engineered for an extended half-life with neutralizing activity against majority of the virus variants identified so far. The aim of this study was to evaluate the safety, tolerability, pharmacokinetics (PK), and immunogenicity of HFB30132A in healthy Chinese subjects. Methods: A phase 1, randomized, double-blind, placebo-controlled, single ascending dose clinical trial was designed. Twenty subjects were enrolled to Cohort 1 (1,000 mg dose level, 10 subjects) or Cohort 2 (2,000 mg dose level, 10 subjects). Subjects in each cohort were assigned randomly to receive a single intravenous (IV) dose of HFB30132A or placebo at a ratio of 8:2. Safety was assessed in terms of treatment emergent adverse events (TEAEs), vital signs, physical examination, laboratory tests, and ECG findings. PK parameters were measured and calculated appropriately. Anti-drug antibody (ADA) test was performed to detect anti-HFB30132A antibodies. Results: All subjects completed the study. Overall, 13 (65%) of the 20 subjects experienced TEAEs. The most common TEAEs were laboratory abnormalities (12 subjects [60%]), gastrointestinal disorders (6 subjects [30%]), and dizziness (4 subjects [20%]). All TEAEs were Grade 1 or Grade 2 in severity based on the criteria of Common Terminology Criteria for Adverse Events (CTCAE). Serum exposure (Cmax, AUC0-t, AUC0-∞) of HFB30132A increased with ascending dose. After single dose of 1,000 mg and 2000 mg HFB30132A, the mean Cmax was 570.18 µg/mL and 898.65 µg/mL, the mean AUC0-t value was 644,749.42 h*µg/mL and 1,046,209.06 h*µg/mL, and the mean AUC0-∞ value was 806,127.47 h*µg/mL and 1,299,190.74 h*µg/mL, respectively. HFB30132A showed low clearance ranging from 1.38 to 1.59 mL/h, and a long terminal elimination half-life (t½) of 89-107 days. ADA test did not detect any anti-HFB30132A antibodies Conclusion: HFB30132A was safe and generally well-tolerated after single IV dose of 1,000 mg or 2000 mg in healthy Chinese adults. HFB30132A did not induce immunogenic response in this study. Our data support further clinical development of HFB30132A. Clinical Trial Registration: https://clinicaltrials.gov, identifier: NCT05275660.

Patterns of Manufacturer Coupon Use for Prescription Drugs in the US, 2017-2019.

Importance: Although manufacturer-sponsored coupons are commonly used, little is known about how patients use them within a treatment episode. Objectives: To examine when and how frequently patients use manufacturer coupons during a treatment episode for a chronic condition, and to characterize factors associated with more frequent use. Design, Setting, and Participants: This is a retrospective cohort study of a 5% nationally representative sample of anonymized longitudinal retail pharmacy claims data from October 1, 2017, to September 30, 2019, obtained from IQVIA's Formulary Impact Analyzer. The data were analyzed from September to December 2022. Patients with new treatment episodes using at least 1 manufacturer coupon over a 12-month period were identified. This study focused on patients with 3 or more fills for a given drug and characterized the association of the outcomes of interest with patient, drug, and drug class characteristics. Main Outcomes and Measures: The primary outcomes were (1) the frequency of coupon use, measured as the proportion of prescription fills accompanied by manufacturer coupon within the treatment episode, and (2) the timing of first coupon use relative to the first prescription fill within the treatment episode. Results: A total of 36 951 treatment episodes accounted for 238 474 drug claims and 35 352 unique patients (mean [SD] age, 48.1 [18.2] years; 17 676 women [50.0%]). Among these episodes, nearly all instances (35 103 episodes [95.0%]) of first coupon use occurred within the first 4 prescription fills. Approximately two-thirds of treatment episodes (24 351 episodes [65.9%]) used a coupon for the incident fill. Coupons were used for a median (IQR) of 3 (2-6) fills. The median (IQR) proportion of fills with a coupon was 70.0% (33.3%-100.0%), and many patients discontinued the drug after the last coupon. After adjustment for covariates, there was no significant association between an individual's out-of-pocket costs or neighborhood-level income and the frequency of coupon use. The estimated proportion of fills with a coupon was greater for products in competitive (19.5% increase; 95% CI, 2.1%-36.9%) or oligopolistic (14.5% increase; 95% CI, 3.5%-25.6%) markets than monopoly markets when there is only 1 drug in the therapeutic class. Conclusions and Relevance: In this retrospective cohort analysis of individuals receiving pharmaceutical treatment for chronic diseases, the frequency of manufacturer-sponsored drug coupon use was associated with the degree of market competition, rather than patients' out-of-pocket costs.

Molecular Tumor Boards: The Next Step towards Precision Therapy in Cancer Care.

The application of molecular tumor profiles in clinical decision making remains a challenge. To aid in the interpretation of complex biomarkers, molecular tumor boards (MTBs) have been established worldwide. In the present study, we show that a multidisciplinary approach is essential to the success of MTBs. Our MTB, consisting of pediatric oncologists, pathologists, and pharmacists, evaluated 115 cases diagnosed between March 2016 and September 2021. If targetable mutations were identified, pharmacists aided in the evaluation of treatment options based on drug accessibility. Treatable genetic alterations detected through molecular testing most frequently involved the cell cycle. For 85% of the cases evaluated, our MTB provided treatment recommendations based on the patient's history and results of molecular tumor testing. Only three patients, however, received MTB-recommended targeted therapy, and only one of these patients demonstrated an improved clinical outcome. For the remaining patients, MTB-recommended treatment often was not administered because molecular tumor profiling was not performed until late in the disease course. For the three patients who did receive MTB-recommended therapy, such treatment was not administered until months after diagnosis due to physician preference. Thus, the education of healthcare providers regarding the benefits of targeted therapy may increase acceptance of these novel agents and subsequently improve patient survival.

Salivary Exosomes in Health and Disease: Future Prospects in the Eye.

Exosomes are a group of vesicles that package and transport DNA, RNA, proteins, and lipids to recipient cells. They can be derived from blood, saliva, urine, and/or other biological tissues. Their impact on several diseases, such as neurodegenerative, autoimmune, and ocular diseases, have been reported, but not fully unraveled. The exosomes that are derived from saliva are less studied, but offer significant advantages over exosomes from other sources, due to their accessibility and ease of collection. Thus, their role in the pathophysiology of diseases is largely unknown. In the context of ocular diseases, salivary exosomes have been under-utilized, thus creating an enormous gap in the literature. The current review discusses the state of exosomes research on systemic and ocular diseases and highlights the role and potential of salivary exosomes as future ocular therapeutic vehicles.

Macroalgae exhibit diverse responses to human disturbances on coral reefs.

Scientists and managers rely on indicator taxa such as coral and macroalgal cover to evaluate the effects of human disturbance on coral reefs, often assuming a universally positive relationship between local human disturbance and macroalgae. Despite evidence that macroalgae respond to local stressors in diverse ways, there have been few efforts to evaluate relationships between specific macroalgae taxa and local human-driven disturbance. Using genus-level monitoring data from 1205 sites in the Indian and Pacific Oceans, we assess whether macroalgae percent cover correlates with local human disturbance while accounting for factors that could obscure or confound relationships. Assessing macroalgae at genus level revealed that no genera were positively correlated with all human disturbance metrics. Instead, we found relationships between the division or genera of algae and specific human disturbances that were not detectable when pooling taxa into a single functional category, which is common to many analyses. The convention to use percent cover of macroalgae as an indication of local human disturbance therefore likely obscures signatures of local anthropogenic threats to reefs. Our limited understanding of relationships between human disturbance, macroalgae taxa, and their responses to human disturbances impedes the ability to diagnose and respond appropriately to these threats.

Immune thrombotic thrombocytopenic purpura: Spotlight on long-term outcomes and survivorship.

Advances in diagnosis and treatment have dramatically improved survival of acute immune thrombotic thrombocytopenic purpura (iTTP) and iTTP has evolved from an acute fatal condition to a chronic relapsing disorder. In addition to the risk of iTTP relapse, iTTP survivors are at risk of multiple adverse health outcomes including higher than expected rates of all-cause mortality, increased rates of stroke and other cardiovascular disease, and higher rates of morbidities such as obesity, hypertension, and autoimmune disorders. iTTP survivors also report neurocognitive impairment, depression, and reduced quality of life. Women with iTTP are at risk for recurrent iTTP, preeclampsia, and other maternal and fetal complications in subsequent pregnancies. ADAMTS13 activity during clinical remission has emerged as an important targetable risk factor for iTTP relapse and other outcomes including stroke and all-cause mortality. This review summarizes current literature regarding the epidemiology and potential mechanisms for adverse long-term sequelae of iTTP, outlines current best practices in iTTP survivorship care, and highlights a research agenda to improve long-term iTTP outcomes.

GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development.

Ectodermal dysplasias including skin abnormalities and cleft lip/palate result from improper surface ectoderm (SE) patterning. However, the connection between SE gene regulatory networks and disease remains poorly understood. Here, we dissect human SE differentiation with multiomics and establish GRHL2 as a key mediator of early SE commitment, which acts by skewing cell fate away from the neural lineage. GRHL2 and master SE regulator AP2a balance early cell fate output, with GRHL2 facilitating AP2a binding to SE loci. In turn, AP2a restricts GRHL2 DNA binding away from de novo chromatin contacts. Integration of these regulatory sites with ectodermal dysplasia-associated genomic variants annotated within the Biomedical Data Commons identifies 55 loci previously implicated in craniofacial disorders. These include ABCA4/ARHGAP29 and NOG regulatory regions where disease-linked variants directly affect GRHL2/AP2a binding and gene transcription. These studies elucidate the logic underlying SE commitment and deepen our understanding of human oligogenic disease pathogenesis.

Inhibition of SARS-CoV-2 infection in cellular systems using engineered trimeric receptor-binding domain of spike protein.

Here, we provide a protocol for the design, expression, purification, and functional studies of an engineered trimeric version of the receptor-binding domain (tRBD) of SARS-CoV-2 spike protein. We describe the use of tRBD to block SARS-CoV-2 spike pseudovirus and true virus binding to cellular angiotensin converting enzyme-2 (ACE2), thereby blocking viral infection. This protocol is applicable to generate a trimeric version of any protein of interest. For complete details on the use and execution of this protocol, please refer to Basavarajappa et al. (2022).1.

Factors Affecting Presentation and Treatment of Pediatric/Adolescent Cancer Patients Diagnosed With SARS-CoV-2.

BACKGROUND: It is thought that the clinical course of actively treated pediatric/adolescent cancer patients diagnosed with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is more severe than experienced by the general pediatric population. We describe the clinical course, risk factors affecting presentation, and management of coronavirus disease 2019 (COVID-19) infection for these patients. METHODS: Patients at a single institution receiving cancer therapy while diagnosed with SARS-CoV-2 between January 2020 and June 2021 were retrospectively reviewed. Data collected included age at SARS-CoV-2 diagnosis, sex, ethno-race, adjusted body mass index, and active therapies. RESULTS: Twenty-nine patients met inclusion criteria, with 16 (55.2%) experiencing symptoms. Twenty-three (79.3%) patients required no institutional support; 10 (34.4%) required hospitalization, of which 80.0% required oxygen, 30.0% required intensive care, and 10.0% required intubation. Three (10.3%) patients developed MIS-C. Obesity increased odds of hospitalization (odds ratio=25.5; P =0.002) and oxygenation (odds ratio=14.88; P =0.012). CONCLUSIONS: Hospitalization and MIS-C rates were significantly higher than, whereas mortality rates and symptom presentations were consistent with, rates in the general pediatric population. Obesity was the only risk factor predictive of clinical severity. Cancer treatment modifications and pre-emptive administration of COVID-19 treatment did not modify clinical course.