Erythroblast Enucleation

Even though the production of orthochromatic erythroblasts can be scaled up to fulfill clinical requirements, enucleation remains one of the critical rate-limiting steps in the production of transfusable red blood cells. Mammalian erythrocytes extrude their nucleus prior to entering circulation, likely to impart flexibility and improve the ability to traverse through capillaries that are half the size of erythrocytes. Recently, there have been many advances in our understanding of the mechanisms underlying mammalian erythrocyte enucleation. This review summarizes these advances, discusses the possible future directions in the field, and evaluates the prospects for improved ex vivo production of red blood cells

In vivo genetic manipulation of inner ear connexin expression by bovine adeno-Associated viral vectors

We have previously shown that in vitro transduction with bovine adeno-associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin-deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. The aims of this study were to manipulate inner ear connexin expression in vivo using BAAV vectors, and to identify the optimal route of vector delivery. Injection of a BAAV vector encoding a bacterial Cre recombinase via canalostomy in adult mice with floxed connexin 26 (Cx26) alleles promoted Cre/LoxP recombination, resulting in decreased Cx26 expression, decreased endocochlear potential, increased hearing thresholds, and extensive loss of outer hair cells. Injection of a BAAV vector encoding GFP-tagged Cx30 via canalostomy in P4 mice lacking connexin 30 (Cx30) promoted formation of Cx30 gap junctions at points of contacts between adjacent non-sensory cells of the cochlear sensory epithelium. Levels of exogenous Cx30 decayed over time, but were still detectable four weeks after canalostomy. Our results suggest that persistence of BAAV-mediated gene replacement in the cochlea is limited by the extensive remodeling of the organ of Corti throughout postnatal development and associated loss of non-sensory cells

Single dose oral dexamethasone versus multi-dose prednisolone in the treatment of acute exacerbations of asthma in children who attend the emergency department: study protocol for a randomized controlled trial.

BACKGROUND: Asthma is a major cause of pediatric morbidity and mortality. In acute exacerbations of asthma, corticosteroids reduce relapses, subsequent hospital admission and the need for ß2-agonist therapy. Prednisolone is relatively short-acting with a half-life of 12 to 36 hours, thereby requiring daily dosing. Prolonged treatment course, vomiting and a bitter taste may reduce patient compliance with prednisolone. Dexamethasone is a long-acting corticosteroid with a half-life of 36 to 72 hours. It is used frequently in children with croup and bacterial meningitis, and is well absorbed orally. The purpose of this trial is to examine whether a single dose of oral dexamethasone (0.3 mg/kg) is clinically non-inferior to prednisolone (1 mg/kg/day for three days) in the treatment of exacerbations of asthma in children who attend the Emergency Department. METHODS/DESIGN: This is a randomized, non-inferiority, open-label clinical trial. After informed consent with or without assent, patients will be randomized to either oral dexamethasone 0.3 mg/kg stat or prednisolone 1 mg/kg/day for three days. The primary outcome measure is the comparison between the Pediatric Respiratory Assessment Measure (PRAM) across both groups on Day 4. The PRAM score, a validated, responsive and reliable tool to determine asthma severity in children aged 2 to 16 years, will be performed by a clinician blinded to treatment allocation. Secondary outcomes include relapse, hospital admission and requirement for further steroid therapy. Data will be analyzed on an intention-to-treat and a per protocol basis. With a sample size of 232 subjects (105 in each group with an estimated 10% loss to follow-up), we will be able to reject the null hypothesis - that the population means of the experimental and control groups are equal with a probability (power) of 0.9. The Type I error probability associated with this test (of the null hypothesis) is 0.05. DISCUSSION: This clinical trial may provide evidence that a shorter steroid course using dexamethasone can be used in the treatment of acute pediatric asthma, thus eliminating the issue of compliance to treatment. REGISTRATION: ISRCTN26944158 and EudraCT Number 2010-022001-18

Small molecule inhibition of CBP/catenin interactions eliminates drug resistant clones in acute lymphoblastic leukemia

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia, however little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CBP) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300, leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using ChIP assay, we demonstrate occupancy by CBP of the survivin promoter, which is decreased by ICG-001 in primary ALL. CBP-mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL

Oncogenic deubiquitination controls tyrosine kinase signaling and therapy response in acute lymphoblastic leukemia

Dysregulation of kinase signaling pathways favors tumor cell survival and therapy resistance in cancer. Here, we reveal a posttranslational regulation of kinase signaling and nuclear receptor activity via deubiquitination in T cell acute lymphoblastic leukemia (T-ALL). We observed that the ubiquitin-specific protease 11 (USP11) is highly expressed and associates with poor prognosis in T-ALL. USP11 ablation inhibits leukemia progression in vivo, sparing normal hematopoiesis. USP11 forms a complex with USP7 to deubiquitinate the oncogenic lymphocyte cell-specific protein-tyrosine kinase (LCK) and enhance its activity. Impairment of LCK activity leads to increased glucocorticoid receptor (GR) expression and glucocorticoids sensitivity. Genetic knockout of USP7 improved the antileukemic efficacy of glucocorticoids in vivo. The transcriptional activation of GR target genes is orchestrated by the deubiquitinase activity and mediated via an increase in enhancer-promoter interaction intensity. Our data unveil how dysregulated deubiquitination controls leukemia survival and drug resistance, suggesting previously unidentified therapeutic combinations toward targeting leukemia

Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 lysine 27 trimethylation

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL)1 and polysomy 21 is the most frequent somatic aneuploidy amongst all B-ALLs2. Yet, the mechanistic links between chr.21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chr.21q22 confers murine progenitor B cell self-renewal in vitro, maturation defects in vivo, and B-ALL with either BCR-ABL or CRLF2 with activated JAK2. Chr.21q22 triplication suppresses H3K27me3 in progenitor B cells and B-ALLs, and “bivalent” genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Strikingly, human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Finally, overexpression of HMGN1, a nucleosome remodeling protein encoded on chr.21q223–5, suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo

Building the Future Therapies for Down Syndrome:The Third International Conference of the T21 Research Society

Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6-9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer's disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar-ma-cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21

hnRNP A1 and hnRNP F Modulate the Alternative Splicing of Exon 11 of the Insulin Receptor Gene

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5′ GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3′ end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5′ splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression

Targeting megakaryocytic induced fibrosis by AURKA inhibition in the myeloproliferative neoplasms

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF

BAAV Mediated GJB2 Gene Transfer Restores Gap Junction Coupling in Cochlear Organotypic Cultures from Deaf Cx26Sox10Cre Mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26Sox10Cre mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10–Cre line. Cx26Sox10Cre mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans