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Emily C Barker,1 Julie Flygare,2 Shalini Paruthi,3,4 Katherine M Sharkey5– 7 1Case Western Reserve University School of Medicine, Department of Pediatrics, Cleveland, OH, USA; 2Project Sleep, Los Angeles, CA, USA; 3Sleep Medicine and Research Center, St. Luke’s Hospital, St. Louis, MO, USA; 4Saint Louis University School of Medicine, Departments of Medicine and Pediatrics, St. Louis, MO, USA; 5Department of Medicine, The Warren Alpert Medical School of Brown University, Providence, RI, USA; 6Department of Psychiatry & Human Behavior, The Warren Alpert Medical School of Brown University, Providence, RI, USA; 7Rhode Island Hospital, Providence, RI, USACorrespondence : Katherine M SharkeyAlpert Medical School, Brown University, 233 Richmond Street, Providence, RI 02906 Tel +1 401-863-2450Fax +1 401-863-2940Email Katherine_Sharkey@brown.eduAbstract: Narcolepsy is a neurological disorder of the sleep-wake cycle characterized by excessive daytime sleepiness (EDS), cataplexy, nighttime sleep disturbances, and REM-sleep-related phenomena (sleep paralysis, hallucinations) that intrude into wakefulness. Dysfunction of the hypocretin/orexin system has been implicated as the underlying cause of narcolepsy with cataplexy. In most people with narcolepsy, symptom onset occurs between the ages of 10 and 35 years, but because the disorder is underrecognized and testing is complex, delays in diagnosis and treatment are common. Narcolepsy is treated with a combination of lifestyle modifications and medications that promote wakefulness and suppress cataplexy. Treatments are often effective in improving daytime functioning for individuals with narcolepsy, but side effects and/or lack of efficacy can result in suboptimal management of symptoms and, in many cases, significant residual impairment. Additionally, the psychosocial ramifications of narcolepsy are often neglected. Recently two new pharmacologic treatment options, solriamfetol and pitolisant, have been approved for adults, and the indication for sodium oxybate in narcolepsy has been expanded to include children. In recent years, there has been an uptick in patient-centered research, and promising new diagnostic and therapeutic options are in development. This paper summarizes current and prospective pharmacological therapies for treating both EDS and cataplexy, discusses concerns specific to children and reproductive-age women with narcolepsy, and reviews the negative impact of health-related stigma and efforts to address narcolepsy stigma.Keywords: narcolepsy, cataplexy, sleepiness, hypersomnia, solriamfetol, pitolisant, sodium oxybate, children, pregnancy, lactation, stigma

The WebQuest as an instructional tool has recently been widely adopted in K-16 education. However, its underlying principles and functionality are not well understood, which has resulted in an inconsistency in practice. This study identifies the underlying constructs of WebQuests as perceived by teachers and variables affecting their perceptions on WebQuests. A survey was conducted on teachers (n = 226) recruited from one large research university in the USA and a professional listserv. The findings reveal three constructs perceived by teachers as critical to WebQuests: constructivist problem solving, social interaction and scaffolded learning. Results also show that variables like purpose of WebQuest use, years of teaching, years of WebQuest use and gender predict, at various degrees, teachers' perceptions on WebQuests. Discussions are made on how the constructs identified can be used to improve online teaching and learning. Suggestions for future study are included by examining the influences of social, psychological and affective factors on learners' learning in WebQuests.

A new receiver for the Onsala 20 m antenna with the possibility of being equipped with 3 mm and 4 mm bands has been built and the 3 mm channel has been commissioned during the Spring 2014. For single-dish operation, the receiver uses an innovative onsource /o -source optical switch. In combination with additional optical components and within the same optical layout, the switch provides two calibration loads (for the 3 mm and 4 mm channels), sideband rejection measurement, and tuning possibilities. The optical layout of the receiver employs all cold (4 K) o set elliptical mirrors for both channels, whereas the on-o switch employs flat mirrors only. The 3 mm channel employs a sideband separation (2SB) dual polarization receiver with orthomode transducer (OMT), 4-8 GHz intermediate frequency (IF), ⨰ 2pol ⨰ upper and lower sidebands (USB + LSB). The cryostat has four optical windows made of high density polyethylene (HDPE) with anti-reflection corrugations, two for the signal and two for each frequency band cold load. The cryostat uses a two-stage cryocooler produced by Sumitomo HIRDK408D2 with anti-vibration suspension of the cold-head to minimize impact of the vibrations on the receiver stability. The local oscillator (LO) system is based on a Gunn oscillator with a phase lock loop (PLL) and four mechanical tuners for broadband operation, providing independently tunable LO power for each polarization. This paper provides a technical description of the receiver and its technology and could be useful for instrumentation engineers and observers using the Onsala 20 m telescope. [ABSTRACT FROM AUTHOR]

Abstract Background B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL) is presumed to derive predominantly from naïve, pre-germinal centre (pre-GC) B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods Subpopulations of B cells representing the germinal centre (GC), the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS) of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results Quantitative MS data of significant protein peaks (p-value < 0.05) separating the three B-cell subpopulations were generated. Together, hierarchical clustering and principal component analysis (PCA) showed that the primary MCL samples clustered together with the pre- and post-GC subpopulations. Both primary MCL cells and MCL cell lines were clearly separated from the B cells representing the GC compartment. Conclusion AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.

Abstract Background Human B lymphocytes can produce leukotriene B4 but the biological function of the 5-lipoxygenase (5-LO) pathway in B cells is unclear. In order to better understand and define the role of 5-LO in B cells, we investigated the expression of 5-LO mRNA and protein in subsets of B cells from human tonsils and different types of B cell lymphoma. Results Based on RT-PCR and western blot/immunohistochemical staining, with a polyclonal antibody raised against 5-LO, high expression of 5-LO was found in mantle zone B cells from tonsils. By contrast, only a weak expression of 5-LO was detected in germinal centre cells and no expression in plasma cells from tonsils. This pattern of 5-LO expression was preserved in malignant lymphoma with high expression in mantle B cell lymphoma (MCL) and weak or no expression in follicular lymphoma. Primary leukemized MCL, so called B-prolymphocytic leukaemia cells, and MCL cell lines also expressed 5-LO and readily produced LTB4 after activation. Conclusion The present report demonstrates the expression of 5-LO mainly in normal and malignant mantle zone B cells while the expression is low or absent in germinal centre B cells and plasma cells, indicating a role of the 5-LO pathway in B cells before the cells finally differentiate to plasma cells.

Objective: To evaluate factors associated with feeling supported in adjusting to life when diagnosed with narcolepsy or idiopathic hypersomnia., Methods: An online survey was disseminated by nonprofit organization Project Sleep via email and social networks. Intended recipients were adults (≥18 years old) diagnosed with narcolepsy or idiopathic hypersomnia. Multivariable regression was used to assess predictors of feeling supported in adjusting to life with these conditions., Results: The survey was completed by 1308 individuals (narcolepsy type 1, 48.4 %; narcolepsy type 2, 28.7 %; idiopathic hypersomnia, 22.9 %). Overall, 68.7 % of respondents were ≤40 years of age; 87.7 % were female; and 88.8 % were White. Respondents lived in 38 different countries (65.5 % in the United States). At time of diagnosis, most (91.1 %) did not know anyone with the same diagnosis. At time of the survey, 486 respondents (37.2 %) still did not know anyone with their diagnosis. Only 32.3 % of respondents felt supported in adjusting to life with narcolepsy or idiopathic hypersomnia. Knowing someone with the same diagnosis at the time of the survey was a significant predictor of perceived support. Younger age (18-30 years) and being married were also associated with greater perceived support, whereas a diagnosis of narcolepsy type 2 or idiopathic hypersomnia (versus narcolepsy type 1) and living outside the US were associated with less perceived support., Conclusions: People with narcolepsy or idiopathic hypersomnia often do not know others living with the same condition, even though this is associated with feeling supported. Clinicians should assess for social support and assist with resource identification., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julie Flygare reports financial support was provided by Avadel Pharmaceuticals PLC. Julie Flygare reports financial support was provided by Harmony Biosciences. Julie Flygare reports a relationship with Alkermes that includes: consulting or advisory. Julie Flygare reports a relationship with Harmony Biosciences that includes: consulting or advisory. Julie Flygare reports a relationship with Project Sleep that includes: employment. Lauren Oglesby reports a relationship with Project Sleep that includes: employment. Sairam Parthasarathy reports a relationship with Jazz Pharmaceuticals Inc that includes: consulting or advisory. Sairam Parthasarathy reports a relationship with AbbVie Inc that includes: consulting or advisory. Sairam Parthasarathy reports a relationship with Apria Healthcare Group LLC that includes: consulting or advisory. Sairam Parthasarathy reports a relationship with Verily Life Sciences Inc that includes: funding grants. Sairam Parthasarathy reports a relationship with Philips, Inc that includes: funding grants. Sairam Parthasarathy reports a relationship with Sommetrics, Inc that includes: funding grants. Sairam Parthasarathy reports a relationship with WHOOP, Inc that includes: funding grants. Sairam Parthasarathy reports a relationship with Regeneron Pharmaceuticals Inc that includes: funding grants. Sairam Parthasarathy reports a relationship with US Biotest, Inc that includes: funding grants. Sairam Parthasarathy reports a relationship with The National Institutes of Health that includes: funding grants. Sairam Parthasarathy reports a relationship with USDepartment of Defense that includes: funding grants. Sairam Parthasarathy reports a relationship with Pima County Health Department that includes: funding grants. Sairam Parthasarathy reports a relationship with Arizona Commerce Authority that includes: funding grants. Sairam Parthasarathy reports a relationship with Sergey Brin Family Foundation that includes: funding grants. Michael J. Thorpy reports a relationship with Axsome Therapeutics Inc that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Balance Therapeutics that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Eisai Co, Ltd that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Avadel Pharmaceuticals that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Harmony Biosciences that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Jazz Pharmaceuticals Inc that includes: consulting or advisory. Michael J. Thorpy reports a relationship with NLS Pharmaceuticals that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Suven Life Sciences Limited that includes: consulting or advisory. Michael J. Thorpy reports a relationship with Takeda Pharmaceutical Company Limited that includes: consulting or advisory. Emmanuel Mignot reports a relationship with Centessa Pharmaceuticals that includes: consulting or advisory. Emmanuel Mignot reports a relationship with Takeda Pharmaceutical Company Limited that includes: consulting or advisory. Emmanuel Mignot reports a relationship with Harmony Biosciences that includes: consulting or advisory and funding grants. Emmanuel Mignot reports a relationship with Merck & Co Inc that includes: consulting or advisory. Emmanuel Mignot reports a relationship with National Institutes of Health that includes: funding grants. Emmanuel Mignot reports a relationship with Alkermes that includes: funding grants. Emmanuel Mignot reports a relationship with Apple that includes: funding grants. Emmanuel Mignot reports a relationship with Avadel Pharmaceuticals that includes: funding grants. Emmanuel Mignot reports a relationship with Eisai Co, Ltd that includes: funding grants. Emmanuel Mignot reports a relationship with Jazz Pharmaceuticals Inc that includes: funding grants. Emmanuel Mignot reports a relationship with The ResMed Foundation that includes: funding grants. Eileen B. Leary reports a relationship with Axsome Therapeutics that includes: employment. Anne Marie Morse reports a relationship with Avadel Pharmaceuticals that includes: consulting or advisory and speaking and lecture fees. Anne Marie Morse reports a relationship with Eisai Co, Ltd that includes: consulting or advisory. Anne Marie Morse reports a relationship with Harmony Biosciences that includes: consulting or advisory and speaking and lecture fees. Anne Marie Morse reports a relationship with Jazz Pharmaceuticals that includes: consulting or advisory, funding grants, and speaking and lecture fees. Anne Marie Morse reports a relationship with NLS Pharmaceuticals that includes: consulting or advisory. Anne Marie Morse reports a relationship with Alkermes that includes: consulting or advisory. Anne Marie Morse reports a relationship with Takeda Pharmaceutical Company Limited that includes: consulting or advisory and speaking and lecture fees. Anne Marie Morse reports a relationship with Neura Health that includes: consulting or advisory. Anne Marie Morse reports a relationship with National Institutes of Health that includes: funding grants. Anne Marie Morse reports a relationship with UCB Pharmaceuticals that includes: funding grants. Anne Marie Morse reports a relationship with The ResMed Foundation that includes: funding grants. Anne Marie Morse reports a relationship with Coverys Community Healthcare Foundation that includes: funding grants. Anne Marie Morse reports a relationship with Geisinger Health Plan that includes: funding grants. Anne Marie Morse reports a relationship with DAMM Good Sleep, LLC that includes: employment. Sairam Parthasarathy, co-author, receives royalties from UpToDate, Inc., for writing a book chapter., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Tuberculosis is the leading cause of death from an infectious disease, and is caused by Mycobacterium tuberculosis (M.tb). More than 1 billion people worldwide are thought to harbor an M.tb infection. The multidrug therapy that represents the current standard of care requires a minimum of four months of dosing and drug resistant Mycobacterium tuberculosis treatment regimens are significantly longer. Inosine-5'-monophosphate dehydrogenase (GuaB) is the enzyme that performs the rate-limiting step in de novo guanine nucleotide biosynthesis that is critical for growth and viability of bacteria including M.tb. The development of a novel antibiotic that inhibits GuaB could combine with existing therapies in novel ways and thereby contribute to effective therapeutic regimens for the treatment of tuberculosis. Here we describe the discovery of structurally distinct small molecule GuaB inhibitors that are potent against M.tb H37Ra and H37Rv strains and have desirable safety and AMDE profiles., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Y.Z., I. A., S. W., C. B., D. J. B., K. C., T.D., J. F., A. G., H. G., E. H., S. F. H., C. H., S. K., M. K., J. L., X. L., F. M., J. M., J. N., J. S., S. U., G. W., P. W., Y. X., C. W. Y., T. F. E., M. W. T., E. M. K., T. D. C., are either current or former employees of Genentech or Roche and may hold stock in Roche Holding AG. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Hematopoietic stem cells (HSCs) react to various stress conditions. However, it is unclear whether and how HSCs respond to severe anemia. Here, we demonstrate that upon induction of acute anemia, HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia, lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs, transcription levels of lipid metabolism-related genes, such as very low-density lipoprotein receptor (Vldlr), are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential, whereas HSCs in Apoe knockout mice do not respond to anemia induction. Vldlr high HSCs show higher erythroid potential, which is enhanced after acute anemia induction. Vldlr high HSCs are epigenetically distinct because of their low chromatin accessibility, and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions., (© 2024. The Author(s).)

Introduction: Immediate-release sodium oxybate (SXB) has been Food and Drug Administration (FDA)-approved to treat narcolepsy since 2002; in 2020, a mixed-salt oxybates formulation was also approved. Both are taken at bedtime with a second dose taken 2.5-4 h later. A third oxybate option, an investigational extended-release SXB, may soon be available. This study was undertaken to understand clinicians' preferences between these 3 different oxybate treatments., Methods: Clinicians in active clinical practice for 3-35 years and experience treating patients with narcolepsy were recruited. A 30-min web-based survey quantified narcolepsy disease-state attitudes, treatment perceptions, and satisfaction with oxybates on 9-point scales. A discrete choice experiment (DCE) of 12 choice sets, with 2 hypothetical treatment profiles in each, was used to capture clinician preferences about overall oxybate therapy preference, impact on patient quality of life (QoL), and patient anxiety/stress. Attributes associated with current therapies and those expected to be available in the near future were included in the design., Results: The clinicians surveyed (n = 100) indicated that narcolepsy has a negative impact on patient QoL (mean rating, 7.7) and rated impact on QoL and treatment efficacy as the most important aspects of a narcolepsy treatment (mean rating, 7.3-7.7). Clinicians with experience prescribing oxybates had moderately high satisfaction with SXB and mixed-salt oxybates efficacy (mean ratings, 6.5-6.9) and safety (mean ratings, 6.1-6.7) and lower satisfaction with nightly dosing frequency (mean rating, 5.9 and 6.3, respectively). In the DCE, dosing frequency was the most important attribute driving overall product choice, patient QoL, and reducing patient anxiety/stress (relative attribute importance, 46.1, 41.7, and 44.0, respectively), with once nightly preferred over twice nightly., Conclusion: Clinicians indicated a significantly higher preference for the once-at-bedtime dosing schedule versus twice nightly in selecting oxybate therapies overall and when aiming to improve patient QoL or reduce patient anxiety., (© 2023. The Author(s).)

177 Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177 Lu-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. α 1 -microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M's renal protective effect in a mouse 177 Lu-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post- 177 Lu-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with 177 Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177 Lu-DOTATATE.

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity., Competing Interests: Declaration of interests R.D.H. is a Chief Scientific Advisor of Pimera, Inc. (San Diego, CA). All other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1a short promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA., (Copyright © 2021. Published by Elsevier Inc.)

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond-Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1-mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3'-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1-mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3'-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity., (© 2021. The Author(s).)

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success., Competing Interests: Conflict of interest disclosure The authors declare no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α 1 -microglobulin (A1M) is a ubiquitous reductase and radical- and heme-binding protein with antioxidation properties. Although present in the circulation in micromolar concentrations, its function in blood is unclear. Here, we show that A1M provides RBC stability. A1M -/- mice display abnormal RBC morphology, reminiscent of macrocytic anemia conditions, i.e. fewer, larger and more heterogeneous cells. Recombinant human A1M (rA1M) reduced in vitro hemolysis of murine RBC against spontaneous, osmotic and heme-induced stress. Moreover, A1M is taken up by human RBCs both in vitro and in vivo. Similarly, rA1M also protected human RBCs against in vitro spontaneous, osmotic, heme- and radical-induced hemolysis as shown by significantly reduced leakage of hemoglobin and LDH. Addition of rA1M resulted in decreased hemolysis compared to addition of the heme-binding protein hemopexin and the radical-scavenging and reducing agents ascorbic acid and Trolox (vitamin E). Furthermore, rA1M significantly reduced spontaneous and heme-induced fetal RBC cell death. Addition of A1M to human whole blood resulted in a significant reduction of hemolysis, whereas removal of A1M from whole blood resulted in increased hemolysis. We conclude that A1M has a protective function in reducing hemolysis which is neither specific to the origin of hemolytic insult, nor species specific., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diamond-Blackfan anemia (DBA) results from haploinsufficiency of ribosomal protein subunits in hematopoietic progenitors in the earliest stages of committed erythropoiesis. Nemo-like kinase (NLK) is chronically hyperactivated in committed erythroid progenitors and precursors in multiple human and murine models of DBA. Inhibition of NLK activity and suppression of NLK expression both improve erythroid expansion in these models. Metformin is a well-tolerated drug for type 2 diabetes with multiple cellular targets. Here we demonstrate that metformin improves erythropoiesis in human and zebrafish models of DBA. Our data indicate that the effects of metformin on erythroid proliferation and differentiation are mediated by suppression of NLK expression through induction of miR-26a, which recognizes a binding site within the NLK 3' untranslated region (3'UTR) to facilitate transcript degradation. We propose that induction of miR-26a is a potentially novel approach to treatment of DBA and could improve anemia in DBA patients without the potentially adverse side effects of metformin in a DBA patient population., Competing Interests: Conflict of interest disclosure The authors have no competing interests to declare., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Massive expansion of erythroid progenitor cells is essential for surviving anemic stress. Research towards understanding this critical process, referred to as stress-erythropoiesis, has been hampered due to lack of specific marker-combinations enabling analysis of the distinct stress-progenitor cells capable of providing radioprotection and enhanced red blood cell production. Here we present a method for precise identification and in vivo validation of progenitor cells contributing to both steady-state and stress-erythropoiesis, enabling for the first time in-depth molecular characterization of these cells. Differential expression of surface markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors during irradiation-induced stress recovery in mice, and provides high-purity isolation of the functional stress-BFU-Es with a 100-fold improved enrichment compared to state-of-the-art. By transplanting purified stress-progenitors expressing the fluorescent protein Kusabira Orange, we determined their kinetics in vivo and demonstrated that CD150+CD9+Sca1- stress-BFU-Es provide a massive but transient radioprotective erythroid wave, followed by multi-lineage reconstitution from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Whole genome transcriptional analysis revealed that stress-BFU-Es express gene signatures more associated with erythropoiesis and proliferation compared to steady-state BFU-Es, and are BMP-responsive. Evaluation of chromatin accessibility through ATAC sequencing reveals enhanced and differential accessibility to binding sites of the chromatin-looping transcription factor CTCF in stress-BFU-Es compared to steady-state BFU-Es. Our findings offer molecular insight to the unique capacity of stress-BFU-Es to rapidly form erythroid cells in response to anemia and constitute an important step towards identifying novel erythropoiesis stimulating agents.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 ( rps29 ) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34 + hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34 + cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

α 1 -microglobulin (A1M) is a small protein present in vertebrates including humans. It has several physiologically relevant properties, including binding of heme and radicals as well as enzymatic reduction, that are used in the protection of cells and tissue. Research has revealed that A1M can ameliorate heme and ROS-induced injuries in cell cultures, organs, explants and animal models. Recently, it was shown that A1M could reduce hemolysis in vitro, observed with several different types of insults and sources of RBCs. In addition, in a recently published study, it was observed that mice lacking A1M (A1M-KO) developed a macrocytic anemia phenotype. Altogether, this suggests that A1M may have a role in RBC development, stability and turnover. This opens up the possibility of utilizing A1M for therapeutic purposes in pathological conditions involving erythropoietic and hemolytic abnormalities. Here, we provide an overview of A1M and its potential therapeutic effect in the context of the following erythropoietic and hemolytic conditions: Diamond-Blackfan anemia (DBA), 5q-minus myelodysplastic syndrome (5q-MDS), blood transfusions (including storage), intraventricular hemorrhage (IVH), preeclampsia (PE) and atherosclerosis., Competing Interests: B.Å., M.G. and S.R.H. are co-founders and minority shareholders of Guard Therapeutics International AB (former A1M Pharma AB), which holds patent rights on medical uses of A1M.

Cyclin-dependent kinase 8 (CDK8) is a mediator complex-associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8-and its paralog CDK19-have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow-derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF-κB) ligand (RANKL)-induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate-resistant acid phosphatase (TRAP) activity and C-terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF-κB, nuclear factor of activated T-cells 1 (NFATc1), dendritic cell-specific transmembrane protein (DC-STAMP), TRAP, and cathepsin K in RANKL-stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration., (© 2019 Wiley Periodicals, Inc.)

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.

Purpose: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg., Experimental Design: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys., Results: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys., Conclusions: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML., (©2018 American Association for Cancer Research.)

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Imaging subsurface rock formations or geological objects like oil and gas reservoirs, mineral deposits, cavities or even magmatic plumbing systems under active volcanoes has been for many years a major quest of geoscientists. Since these subsurface objects cannot be observed directly, different indirect methods have been developed. These methods are all based on variations of certain physical properties of the subsurface materials that can be detected from the ground surface or from boreholes. To determine the density distribution, a new imaging technique using cosmic-ray muon detectors deployed in a borehole has been developed and a first prototype of a borehole muon detector successfully tested. In addition to providing a static image of the subsurface density in three dimensions (or three-dimensional tomography), borehole muography can also inform on the variations of density with time, which recently became of major importance with the injection of large volumes of fluids, mainly water and CO 2 , in porous subsurface reservoirs (e.g. aquifer storage and recovery, wastewater disposal, enhanced oil recovery and carbon sequestration). This raises several concerns about the risk of leakage and the mechanical integrity of the reservoirs. Determining the field scale induced displacement of fluids by geophysical methods like muography is thus a priority.This article is part of the Theo Murphy meeting issue 'Cosmic-ray muography'., (© 2018 The Author(s).)

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Diamond-Blackfan anemia is a congenital erythroid hypoplasia and is associated with physical malformations and a predisposition to cancer. Twenty-five percent of patients with Diamond-Blackfan anemia have mutations in a gene encoding ribosomal protein S19 (RPS19). Through overexpression of RPS19 using a lentiviral vector with the spleen focus-forming virus promoter, we demonstrated that the Diamond-Blackfan anemia phenotype can be successfully treated in Rps19-deficient mice. In our present study, we assessed the efficacy of a clinically relevant promoter, the human elongation factor 1α short promoter, with or without the locus control region of the β-globin gene for treatment of RPS19-deficient Diamond-Blackfan anemia. The findings demonstrate that these vectors rescue the proliferation defect and improve erythroid development of transduced RPS19-deficient bone marrow cells. Remarkably, bone marrow failure and severe anemia in Rps19-deficient mice was cured with enforced expression of RPS19 driven by the elongation factor 1α short promoter. We also demonstrate that RPS19-deficient bone marrow cells can be transduced and these cells have the capacity to repopulate bone marrow in long-term reconstituted mice. Our results collectively demonstrate the feasibility to cure RPS19-deficient Diamond-Blackfan anemia using lentiviral vectors with cellular promoters that possess a reduced risk of insertional mutagenesis., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)