Nonstochastic reprogramming from a privileged somatic cell state.

Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ∼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PAPERCLIP:

HEXIM1 is an essential transcription regulator during human erythropoiesis.

ABSTRACT: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor ß. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the ß-globin loci, where there was loss of RNAPII and GATA1 at ß-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the ß-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.

Signatures of tunable superconductivity in a trilayer graphene moiré superlattice.

Understanding the mechanism of high-transition-temperature (high-Tc) superconductivity is a central problem in condensed matter physics. It is often speculated that high-Tc superconductivity arises in a doped Mott insulator1 as described by the Hubbard model2-4. An exact solution of the Hubbard model, however, is extremely challenging owing to the strong electron-electron correlation in Mott insulators. Therefore, it is highly desirable to study a tunable Hubbard system, in which systematic investigations of the unconventional superconductivity and its evolution with the Hubbard parameters can deepen our understanding of the Hubbard model. Here we report signatures of tunable superconductivity in an ABC-trilayer graphene (TLG) and hexagonal boron nitride (hBN) moiré superlattice. Unlike in 'magic angle' twisted bilayer graphene, theoretical calculations show that under a vertical displacement field, the ABC-TLG/hBN heterostructure features an isolated flat valence miniband associated with a Hubbard model on a triangular superlattice5,6 where the bandwidth can be tuned continuously with the vertical displacement field. Upon applying such a displacement field we find experimentally that the ABC-TLG/hBN superlattice displays Mott insulating states below 20 kelvin at one-quarter and one-half fillings of the states, corresponding to one and two holes per unit cell, respectively. Upon further cooling, signatures of superconductivity ('domes') emerge below 1 kelvin for the electron- and hole-doped sides of the one-quarter-filling Mott state. The electronic behaviour in the ABC-TLG/hBN superlattice is expected to depend sensitively on the interplay between the electron-electron interaction and the miniband bandwidth. By varying the vertical displacement field, we demonstrate transitions from the candidate superconductor to Mott insulator and metallic phases. Our study shows that ABC-TLG/hBN heterostructures offer attractive model systems in which to explore rich correlated behaviour emerging in the tunable triangular Hubbard model.

Anemia in the pediatric patient.

The World Health Organization estimates that approximately a quarter of the world's population suffers from anemia, including almost half of preschool-age children. Globally, iron deficiency anemia is the most common cause of anemia. Other important causes of anemia in children are hemoglobinopathies, infection, and other chronic diseases. Anemia is associated with increased morbidity, including neurologic complications, increased risk of low birth weight, infection, and heart failure, as well as increased mortality. When approaching a child with anemia, detailed historical information, particularly diet, environmental exposures, and family history, often yield important clues to the diagnosis. Dysmorphic features on physical examination may indicate syndromic causes of anemia. Diagnostic testing involves a stepwise approach utilizing various laboratory techniques. The increasing availability of genetic testing is providing new mechanistic insights into inherited anemias and allowing diagnosis in many previously undiagnosed cases. Population-based approaches are being taken to address nutritional anemias. Novel pharmacologic agents and advances in gene therapy-based therapeutics have the potential to ameliorate anemia-associated disease and provide treatment strategies even in the most difficult and complex cases.

HMGB1-mediated restriction of EPO signaling contributes to anemia of inflammation.

Anemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways. Genetic ablation of receptor for advanced glycation end products, the only known HMGB1 receptor expressed by erythroid precursors, does not rescue the deleterious effects of HMGB1 on EPO signaling, either in human or murine precursors. Furthermore, surface plasmon resonance studies highlight the ability of HMGB1 to interfere with the binding between EPO and the EPOR. Administration of a monoclonal anti-HMGB1 antibody after sepsis onset in mice partially restores EPO signaling in vivo. Thus, HMGB1-mediated restriction of EPO signaling contributes to the chronic phase of anemia of inflammation.

Joint bleeds in mild hemophilia: Prevalence and clinical characteristics.

INTRODUCTION: Joint bleeds are a common and frequent complication associated with hemophilia, increasing the risk of hemophilic arthropathy. It is important to define and characterize the presence of joint complications in mild hemophilia to develop strategies to mitigate disease burden. AIMS: To characterize the prevalence, clinical characteristics of joint bleeds, and risk factors that may lead to hemarthrosis in people with mild hemophilia. METHODS: Following Institutional Review Board approval, a retrospective chart review was conducted for patients with mild hemophilia seen at the Yale Hemophilia Treatment Center or Classical Hematology Program. RESULTS: The medical records of 70 patients were reviewed. Eighty one percent were male and 19 percent were female. Twenty individuals with mild hemophilia had a history of joint bleeding, 13 were traumatic bleeds, 7 were spontaneous. The age of first joint bleed ranged from 4 to 58 years old, with an average age of 20.8-years old. Ten patients developed joint bleeds between the ages of 10 and 20 years old. The most common locations of joint bleeding were the knee (n = 11) and ankle (n = 7). Eight of 70 patients had hepatitis C (HCV), 6 experienced joint bleeding. CONCLUSIONS: In this study, almost one third of patients with mild hemophilia experienced joint bleeding, often without history of trauma. Joint range of motion was abnormal in more than a third of the patients with mild hemophilia regardless. These data highlight the need for ongoing evaluation and characterization of joint health in individuals with mild hemophilia. HIGHLIGHTS: Twenty-nine percent of individuals with mild hemophilia had history of joint bleed. PwH and mild diseases with previous or current hepatitis C had higher likelihood of joint bleeding. Approximately 15% of PwH and mild diseases had abnormal joint examinations without a confirmed history of joint bleeding.

Phenotypic and proteomic characterization of the human erythroid progenitor continuum reveal dynamic changes in cell cycle and in metabolic pathways.

Human erythropoiesis is a complex process leading to the production of 2.5 million red blood cells per second. Following commitment of hematopoietic stem cells to the erythroid lineage, this process can be divided into three distinct stages: erythroid progenitor differentiation, terminal erythropoiesis, and reticulocyte maturation. We recently resolved the heterogeneity of erythroid progenitors into four different subpopulations termed EP1-EP4. Here, we characterized the growth factor(s) responsiveness of these four progenitor populations in terms of proliferation and differentiation. Using mass spectrometry-based proteomics on sorted erythroid progenitors, we quantified the absolute expression of ~5500 proteins from EP1 to EP4. Further functional analyses highlighted dynamic changes in cell cycle in these populations with an acceleration of the cell cycle during erythroid progenitor differentiation. The finding that E2F4 expression was increased from EP1 to EP4 is consistent with the noted changes in cell cycle. Finally, our proteomic data suggest that the protein machinery necessary for both oxidative phosphorylation and glycolysis is present in these progenitor cells. Together, our data provide comprehensive insights into growth factor-dependence of erythroid progenitor proliferation and the proteome of four distinct populations of human erythroid progenitors which will be a useful framework for the study of erythroid disorders.

Sites of regulated phosphorylation that control K-Cl cotransporter activity.

Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.

Histone Acetyltransferases p300 and CBP Coordinate Distinct Chromatin Remodeling Programs in Vascular Smooth Muscle Plasticity.

BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. RESULTS: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

Considerations for Expanding Antimicrobial Resistance Surveillance Networks.

Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) gained insight into the range of national antimicrobial resistance (AMR) stakeholders' long-term visions for AMR surveillance networks. As national AMR networks mature, stakeholders often contemplate adding laboratories to the network to achieve greater representativeness, boost data quantity, or meet other goals. Therefore, stakeholders should carefully select laboratories for expansion based on their goals and several practical criteria. Based on CAPTURA experience, the key criteria a national network may consider when expanding its AMR surveillance network include location, laboratory ownership, access to linked clinical and prescription databases, logistical ease, a laboratory's collaborative spirit, laboratory practices and equipment, laboratory staffing and quality assessments, laboratory methods and specimen types, data cleanliness and completeness, and the quantity of AMR data.

Recording and Reporting of Antimicrobial Resistance (AMR) Priority Variables and Its Implication on Expanding Surveillance Sites in Nepal: A CAPTURA Experience.

Data on antimicrobial resistance (AMR) from sites not participating in the National AMR surveillance network, conducted by National Public Health Laboratory (NPHL), remain largely unknown in Nepal. The "Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia" (CAPTURA) assessed AMR data from previously untapped data sources in Nepal. A retrospective cross-sectional data review was carried out for the AMR data recorded between January 2017 and December 2019 to analyze AMR data from 26 hospital-based laboratories and 2 diagnostic laboratories in Nepal. Of the 56 health facilities initially contacted to participate in this project activity, 50.0% (28/56) signed a data-sharing agreement with CAPTURA. Eleven of the 28 hospitals were AMR surveillance sites, whereas the other 17, although not part of the National AMR surveillance network, recorded AMR-related data. Data for 663 602 isolates obtained from 580 038 patients were analyzed. A complete record of the 11 CAPTURA priority variables was obtained from 45.5% (5/11) of government hospitals, 63.6% (7/11) of private hospitals, and 54.6% (6/11) of public-private hospitals networked with NPHL for AMR surveillance. Similarly, 80% (8/10) of clinics and 54.6% (6/11) of laboratories outside the NPHL network recorded complete data for the 10 Global Antimicrobial Resistance and Use Surveillance System (GLASS) priority variables and 11/14 CAPTURA priority variables. Retrospective review of the data identified areas requiring additional resources and interventions to improve the quality of data on AMR in Nepal. Furthermore, we observed no difference in the priority variables reported by sites within or outside the NPHL network, thus suggesting that policies could be made to expand the surveillance system to include these sites without substantially affecting the government's budget.

Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA).

BACKGROUND: In 2015, the UK government established the Fleming Fund with the aim to address critical gaps in surveillance of antimicrobial resistance (AMR) in low- and middle-income countries in Asia and Africa. Among a large portfolio of grants, the Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project was awarded with the specific objective of expanding the volume of historical data on AMR, consumption (AMC), and use (AMU) in the human healthcare sector across 12 countries in South and Southeast Asia. METHODS: Starting in early 2019, the CAPTURA consortium began working with local governments and >100 relevant data-holding facilities across the region to identify, assess for quality, prioritize, and subsequently retrieve data on AMR, AMC, and AMU. Relevant and shared data were collated and analyzed to provide local overviews for national stakeholders as well as regional context, wherever possible. RESULTS: From the vast information resource generated on current surveillance capacity and data availability, the project has highlighted gaps and areas for quality improvement and supported comprehensive capacity-building activities to optimize local data-collection and -management practices. CONCLUSIONS: The project has paved the way for expansion of surveillance networks to include both the academic and private sector in several countries and has actively engaged in discussions to promote data sharing at the local, national, and regional levels. This paper describes the overarching approach to, and emerging lessons from, the CAPTURA project, and how it contributes to other ongoing efforts to strengthen national AMR surveillance in the region and globally.

Methodological Approach to Identify and Expand the Volume of Antimicrobial Resistance (AMR) Data in the Human Health Sector in Low- and Middle-Income Countries in Asia: Implications for Local and Regional AMR Surveillance Systems Strengthening.

Antimicrobial resistance (AMR) is a multifaceted global health problem disproportionately affecting low- and middle-income countries (LMICs). The Capturing data on Antimicrobial resistance Patterns and Trends in Use in Regions of Asia (CAPTURA) project was tasked to expand the volume of AMR and antimicrobial use data in Asia. The CAPTURA project used 2 data-collection streams: facility data and project metadata. Project metadata constituted information collected to map out data sources and assess data quality, while facility data referred to the retrospective data collected from healthcare facilities. A down-selection process, labelled "the funnel approach" by the project, was adopted to use the project metadata in prioritizing and selecting laboratories for retrospective AMR data collection. Moreover, the metadata served as a guide for understanding the AMR data once they were collected. The findings from CAPTURA's metadata add to the current discourse on the limitation of AMR data in LMICs. There is generally a low volume of AMR data generated as there is a lack of microbiology laboratories with sufficient antimicrobial susceptibility testing capacity. Many laboratories in Asia are still capturing data on paper, resulting in scattered or unused data not readily accessible or shareable for analyses. There is also a lack of clinical and epidemiological data captured, impeding interpretation and in-depth understanding of the AMR data. CAPTURA's experience in Asia suggests that there is a wide spectrum of capacity and capability of microbiology laboratories within a country and region. As local AMR surveillance is a crucial instrument to inform context-specific measures to combat AMR, it is important to understand and assess current capacity-building needs while implementing activities to enhance surveillance systems.

Regulation of RNA polymerase II activity is essential for terminal erythroid maturation.

The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.

Impairment of human terminal erythroid differentiation by histone deacetylase 5 deficiency.

Histone deacetylases (HDACs) are a group of enzymes that catalyze the removal of acetyl groups from histone and nonhistone proteins. HDACs have been shown to have diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. This study showed that, of the 11 classic HDAC family members, 6 (HDAC1, -2, -3, and HDAC5, -6, -7) are expressed in human erythroid cells, with HDAC5 most significantly upregulated during terminal erythroid differentiation. Knockdown of HDAC5 by either short hairpin RNA or small interfering RNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, although acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late-stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA sequencing analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome-wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation, and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.

The congenital dyserythropoieitic anemias: genetics and pathophysiology.

PURPOSE OF REVIEW: The congenital dyserythropoietic anemias (CDA) are hereditary disorders characterized by ineffective erythropoiesis. This review evaluates newly developed CDA disease models, the latest advances in understanding the pathogenesis of the CDAs, and recently identified CDA genes. RECENT FINDINGS: Mice exhibiting features of CDAI were recently generated, demonstrating that Codanin-1 (encoded by Cdan1) is essential for primitive erythropoiesis. Additionally, Codanin-1 was found to physically interact with CDIN1, suggesting that mutations in CDAN1 and CDIN1 result in CDAI via a common mechanism. Recent advances in CDAII (which results from SEC23B mutations) have also been made. SEC23B was found to functionally overlap with its paralogous protein, SEC23A, likely explaining the absence of CDAII in SEC23B-deficient mice. In contrast, mice with erythroid-specific deletion of 3 or 4 of the Sec23 alleles exhibited features of CDAII. Increased SEC23A expression rescued the CDAII erythroid defect, suggesting a novel therapeutic strategy for the disease. Additional recent advances included the identification of new CDA genes, RACGAP1 and VPS4A, in CDAIII and a syndromic CDA type, respectively. SUMMARY: Establishing cellular and animal models of CDA is expected to result in improved understanding of the pathogenesis of these disorders, which may ultimately lead to the development of new therapies.

Phase 3, multicentre, randomised, placebo-controlled study evaluating the efficacy and safety of ustekinumab in patients with systemic lupus erythematosus.

OBJECTIVE: Evaluate the efficacy and safety of ustekinumab, an anti-interleukin-12/23 p40 antibody, in a phase 3, randomised, placebo-controlled study of patients with active systemic lupus erythematosus (SLE) despite receiving standard-of-care. METHODS: Active SLE patients (SLE Disease Activity Index 2000 (SLEDAI-2K) ≥6 during screening and SLEDAI-2K ≥4 for clinical features at week 0) despite receiving oral glucocorticoids, antimalarials, or immunomodulatory drugs were randomised (3:2) to receive ustekinumab (intravenous infusion ~6 mg/kg at week 0, followed by subcutaneous injections of ustekinumab 90 mg at week 8 and every 8 weeks) or placebo through week 48. The primary endpoint was SLE Responder Index (SRI)-4 at week 52, and major secondary endpoints included time to flare through week 52 and SRI-4 at week 24. RESULTS: At baseline, 516 patients were randomised to placebo (n=208) or ustekinumab (n=308). Following the planned interim analysis, the sponsor discontinued the study due to lack of efficacy but no safety concerns. Efficacy analyses included 289 patients (placebo, n=116; ustekinumab, n=173) who completed or would have had a week 52 visit at study discontinuation. At week 52, 44% of ustekinumab patients and 56% of placebo patients had an SRI-4 response; there were no appreciable differences between the treatment groups in the major secondary endpoints. Through week 52, 28% of ustekinumab patients and 32% of placebo patients had a British Isles Lupus Assessment Group flare, with a mean time to first flare of 204.7 and 200.4 days, respectively. Through week 52, 70% of ustekinumab patients and 74% of placebo patients had ≥1 adverse event. CONCLUSIONS: Ustekinumab did not demonstrate superiority over placebo in this population of adults with active SLE; adverse events were consistent with the known safety profile of ustekinumab. TRIAL REGISTRATION NUMBER: NCT03517722.

D-bifunctional protein deficiency caused by splicing variants in a neonate with severe peroxisomal dysfunction and persistent hypoglycemia.

D-bifunctional protein (DBP) deficiency is a rare, autosomal recessive peroxisomal enzyme deficiency resulting in a high burden of morbidity and early mortality. Patients with DBP deficiency resemble those with a severe Zellweger phenotype, with neonatal hypotonia, seizures, craniofacial dysmorphisms, psychomotor delay, deafness, blindness, and death typically within the first 2 years of life, although patients with residual enzyme function can survive longer. The clinical severity of the disease depends on the degree of enzyme deficiency. Loss-of-function variants typically result in no residual enzyme activity; however, splice variants may result in protein with residual function. We describe a full-term newborn presenting with hypotonia, seizures, and unexplained hypoglycemia, who was later found to have rickets at follow up. Rapid whole genome sequencing identified two HSD17B4 variants in trans; one likely pathogenic variant and one variant of uncertain significance (VUS) located in the polypyrimidine tract of intron 13. To determine the functional consequence of the VUS, we analyzed RNA from the patient's father with RNA-seq which showed skipping of Exon 14, resulting in a frameshift mutation three amino acids from the new reading frame. This RNA-seq analysis was correlated with virtually absent enzyme activity, elevated very-long-chain fatty acids in fibroblasts, and a clinically severe phenotype. Both variants are reclassified as pathogenic. Due to the clinical spectrum of DBP deficiency, this provides important prognostic information, including early mortality. Furthermore, we add persistent hypoglycemia to the clinical spectrum of the disease, and advocate for the early management of fat-soluble vitamin deficiencies to reduce complications.

Identification and transcriptome analysis of erythroblastic island macrophages.

The erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The identity of EBI macrophages has thus far remained elusive. Given that Epo is essential for erythropoiesis and that Epor is expressed in numerous nonerythroid cells, we hypothesized that EBI macrophages express Epor so that Epo can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis. To test this notion, we used Epor-eGFPcre knockin mouse model. We show that in bone marrow (BM) and fetal liver, a subset of macrophages express Epor-eGFP. Imaging flow cytometry analyses revealed that >90% of native EBIs comprised F4/80+Epor-eGFP+ macrophages. Human fetal liver EBIs also comprised EPOR+ macrophages. Gene expression profiles of BM F4/80+Epor-eGFP+ macrophages suggest a specialized function in supporting erythropoiesis. Molecules known to be important for EBI macrophage function such as Vcam1, CD169, Mertk, and Dnase2α were highly expressed in F4/80+Epor-eGFP+ macrophages compared with F4/80+Epor-eGFP- macrophages. Key molecules involved in iron recycling were also highly expressed in BM F4/80+Epor-eGFP+ macrophages, suggesting that EBI macrophages may provide an iron source for erythropoiesis within this niche. Thus, we have characterized EBI macrophages in mouse and man. Our findings provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such as thalassemia, polycythemia vera, and myelodysplastic syndromes.

Comprehensive phenotyping of erythropoiesis in human bone marrow: Evaluation of normal and ineffective erythropoiesis.

Identification of stage-specific erythroid cells is critical for studies of normal and disordered human erythropoiesis. While immunophenotypic strategies have previously been developed to identify cells at each stage of terminal erythroid differentiation, erythroid progenitors are currently defined very broadly. Refined strategies to identify and characterize BFU-E and CFU-E subsets are critically needed. To address this unmet need, a flow cytometry-based technique was developed that combines the established surface markers CD34 and CD36 with CD117, CD71, and CD105. This combination allowed for the separation of erythroid progenitor cells into four discrete populations along a continuum of progressive maturation, with increasing cell size and decreasing nuclear/cytoplasmic ratio, proliferative capacity and stem cell factor responsiveness. This strategy was validated in uncultured, primary erythroid cells isolated from bone marrow of healthy individuals. Functional colony assays of these progenitor populations revealed enrichment of BFU-E only in the earliest population, transitioning to cells yielding BFU-E and CFU-E, then CFU-E only. Utilizing CD34/CD105 and GPA/CD105 profiles, all four progenitor stages and all five stages of terminal erythroid differentiation could be identified. Applying this immunophenotyping strategy to primary bone marrow cells from patients with myelodysplastic syndrome, identified defects in erythroid progenitors and in terminal erythroid differentiation. This novel immunophenotyping technique will be a valuable tool for studies of normal and perturbed human erythropoiesis. It will allow for the discovery of stage-specific molecular and functional insights into normal erythropoiesis as well as for identification and characterization of stage-specific defects in inherited and acquired disorders of erythropoiesis.