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Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through Braf(V600E) or Kras(G12D) knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5(+ve) stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5(+ve) cells also results in cSCC development. These findings indicate that LGR5(+ve) stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis.
Members of the transforming growth factor beta (TGF-beta) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-beta superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4(-/-) primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.
Transforming growth factor beta (TGF-beta) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-beta action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-beta type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-beta type II receptor (TGFbetaRII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-beta, and (2) ALK5 acts also in conjunction with type II receptors other than TGFbetaRII.
Diamond Blackfan anaemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA affects about seven per million live births and presents during the first year of life. However, as mutated genes have been discovered in DBA, non-classical cases with less distinct phenotypes are being described in adults as well as children. In caring for these patients it is often difficult to have a clear understanding of the treatment options and their outcomes because of the lack of complete information on the natural history of the disease. The purpose of this document is to review the criteria for diagnosis, evaluate the available treatment options, including corticosteroid and transfusion therapies and stem cell transplantation, and propose a plan for optimizing patient care. Congenital anomalies, mode of inheritance, cancer predisposition, and pregnancy in DBA are also reviewed. Evidence-based conclusions will be made when possible; however, as in many rare diseases, the data are often anecdotal and the recommendations are based upon the best judgment of experienced clinicians. The recommendations regarding the diagnosis and management described in this report are the result of deliberations and discussions at an international consensus conference.
Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.
Prospective isolation is critical for understanding the cellular and molecular aspects of stem cell heterogeneity. Here, we identify the cell surface antigen CD9 as a positive marker that provides a simple alternative for hematopoietic stem cell isolation at high purity. Crucially, CD9 affords the capture of all hematopoietic stem cells in murine bone marrow in the absence of contaminating populations that lack authentic stem cell function. Using CD9 as a tool to subdivide hematopoietic stem-cell-containing populations, we provide evidence for heterogeneity at the cellular, functional, and molecular levels.
Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA.
Life-long production of blood from hematopoietic stem cells (HSCs) is a process of strict modulation. Intrinsic and extrinsic signals govern fate options like self-renewal - a cardinal feature of HSCs. Bone morphogenetic proteins (BMP) have an established role in embryonic hematopoiesis, but less is known about its functions in adulthood. Previously, SMAD-mediated BMP signaling has been proven dispensable for HSCs. However, the BMP Type II receptor (BMPR-II) is highly expressed in HSCs, leaving the possibility that BMPs function via alternative pathways. Here, we establish that BMP signaling is required for self-renewal of adult HSCs. Through conditional knockout we show that BMPR-II deficient HSCs have impaired self-renewal and regenerative capacity. BMPR-II deficient cells have reduced p38 activation, implying that non-SMAD pathways operate downstream of BMPs in HSCs. Indeed, a majority of primitive hematopoietic cells do not engage in SMAD-mediated responses downstream of BMPs in vivo. Furthermore, deficiency of BMPR-II results in increased expression of TJP1, a known regulator of self-renewal in other stem cells, and knockdown of TJP1 in primitive hematopoietic cells partly rescues the BMPR-II null phenotype. This suggests TJP1 may be a universal stem cell regulator. In conclusion, BMP signaling, in part mediated through TJP1, is required endogenously by adult HSCs to maintain self-renewal capacity and proper resilience of the hematopoietic system during regeneration.
The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.
Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by a specific deficiency in erythroid progenitors. Since some patients with DBA develop a reduction in thrombocytes and granulocytes with age, we asked whether multipotent hematopoietic progenitors from DBA patients had normal proliferative capacity in liquid expansion cultures. CD34(+) cells derived from DBA patients showed deficient proliferation in liquid culture containing IL-3, IL-6, and SCF. Single CD34(+) CD38(-) cells from DBA patients exhibited deficient proliferation recruitment in a limiting dilution assay containing IL-3, IL-6, SCF, Tpo, FL, and G-CSF or containing IL-3, IL-6, and SCF. Our findings suggest that the underlying hematopoietic defect in DBA may not be limited to the erythroid lineage. Since a fraction of DBA patients have a deficiency in ribosomal protein S19 (RPS19), we constructed lentiviral vectors containing the RPS19 gene for overexpression in hematopoietic progenitors from RPS19-deficient DBA patients. Enforced expression of the RPS19 transgene improved the proliferation of CD34(+) cells from DBA patients with RPS19 mutation. Similarly, enforced expression of RPS19 improved erythroid development of RPS19-deficient hematopoietic progenitors as determined by colony assays and erythroid differentiation cultures. These findings suggest that gene therapy for RPS19-deficient DBA is feasible.
For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood-brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.
Binding of steroid hormones to their cognate receptors regulates the growth of most prostate and breast cancers. We hypothesized that CYP11A inhibition might halt the synthesis of all steroid hormones, because CYP11A is the only enzyme that catalyses the first step of steroid hormone biosynthesis. We speculated that a CYP11A inhibitor could be administered safely provided that the steroids essential for life are replaced. Virtual screening and systematic structure-activity relationship optimization were used to develop ODM-208, the first-in-class, selective, nonsteroidal, oral CYP11A1 inhibitor. Safety of ODM-208 was assessed in rats and Beagle dogs, and efficacy in a VCaP castration-resistant prostate cancer (CRPC) xenograft mouse model, in mice and dogs, and in six patients with metastatic CRPC. Blood steroid hormone concentrations were measured using liquid chromatography-mass spectrometry. ODM-208 binds to CYP11A1 and inhibited its enzymatic activity. ODM-208 administration led to rapid, complete, durable, and reversible inhibition of the steroid hormone biosynthesis in an adrenocortical carcinoma cell model in vitro, in adult noncastrated male mice and dogs, and in patients with CRPC. All measured serum steroid hormone concentrations reached undetectable levels within a few weeks from the start of ODM-208 administration. ODM-208 was well tolerated with steroid hormone replacement. The toxicity findings were considered related to CYP11A1 inhibition and were reversed after stopping of the compound administration. Steroid hormone biosynthesis can be effectively inhibited with a small-molecule inhibitor of CYP11A1. The findings suggest that administration of ODM-208 is feasible with concomitant corticosteroid replacement therapy.
ETV6-RUNX1 is associated with childhood acute B-lymphoblastic leukemia (cALL) functioning as a first-hit mutation that initiates a clinically silent pre-leukemia in utero. Because lineage commitment hierarchies differ between embryo and adult, and the impact of oncogenes is cell-context dependent, we hypothesized that the childhood affiliation of ETV6-RUNX1 cALL reflects its origins in a progenitor unique to embryonic life. We characterize the first emerging B cells in first-trimester human embryos, identifying a developmentally restricted CD19-IL-7R+ progenitor compartment, which transitions from a myeloid to lymphoid program during ontogeny. This developmental series is recapitulated in differentiating human pluripotent stem cells (hPSCs), thereby providing a model for the initiation of cALL. Genome-engineered hPSCs expressing ETV6-RUNX1 from the endogenous ETV6 locus show expansion of the CD19-IL-7R+ compartment, show a partial block in B lineage commitment, and produce proB cells with aberrant myeloid gene expression signatures and potential: features (collectively) consistent with a pre-leukemic state.
The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.
Retroviral overexpression of the transcription factor HOXB4 results in a rapid increase in proliferation of murine hematopoietic stem cells both in vivo and in vitro. Therefore, we asked whether transient overexpression of HOXB4 would increase proliferation of human primitive hematopoietic progenitors. Transient overexpression of HOXB4 was generated in umbilical cord blood (CB) CD34(+) cells by a recombinant adenovirus (AdHOXB4) expressing HOXB4 together with the enhanced green fluorescent protein (GFP). Transduced, GFP(+) cells were cultured in serum-free medium containing cytokines that primarily support the growth of primitive hematopoietic progenitors. In contrast to previous findings using retroviral overexpression of HOXB4, we did not observe any increase in proliferation of primitive progenitors or increased colony formation of clonogenic progenitors, including progenitor progeny from long-term culture-initiating cells following adenoviral vector overexpression of HOXB4 in CB CD34(+) cells. However, enforced expression of HOXB4 by the adenoviral vector significantly increased myeloid differentiation of primitive hematopoietic progenitors. Since retroviral vectors generate low and continuous levels of transgene expression in contrast to the high, transient levels generated by the adenoviral vector, our findings suggest that the high levels of HOXB4 expression generated by AdHOXB4 in human CB CD34(+) cells direct the cells toward a myeloid differentiation program rather than increased proliferation.
Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1.
Enterotoxigenic Escherichia coli (ETEC) cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP) on the regulation of production and secretion of heat labile (LT) enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.
Hematopoietic stem cells (HSCs) are maintained in hypoxic niches in endosteal regions of bones. Here we demonstrate that Cripto and its receptor GRP78 are important regulators of HSCs in the niche. Flow cytometry analyses revealed two distinct subpopulations of CD34(-)KSL cells based on the expression of GRP78, and these populations showed different reconstitution potential in transplantation assays. GRP78(+)HSCs mainly reside in the endosteal area, are more hypoxic, and exhibit a lower mitochondrial potential, and their HSC capacity was maintained in vitro by Cripto through induction of higher glycolytic activity. Additionally, HIF-1α KO mice have decreased numbers of GRP78(+)HSCs and reduced expression of Cripto in the endosteal niche. Furthermore, blocking GRP78 induced a movement of HSCs from the endosteal to the central marrow area. These data suggest that Cripto/GRP78 signaling is an important pathway that regulates HSC quiescence and maintains HSCs in hypoxia as an intermediary of HIF-1α.
Gaucher disease type 1 (GD1) is an inherited lysosomal disorder with multisystemic effects in patients. Hallmark symptoms include hepatosplenomegaly, cytopenias, and bone disease with varying degrees of severity. Mutations in a single gene, glucosidase beta acid 1 (GBA1), are the underlying cause for the disorder, resulting in insufficient activity of the enzyme glucocerebrosidase, which in turn leads to a progressive accumulation of the lipid component glucocerebroside. In this study, we treat mice with signs consistent with GD1, with hematopoietic stem/progenitor cells transduced with a lentiviral vector containing an RNA transcript that, after reverse transcription, results in codon-optimized cDNA that, upon its integration into the genome encodes for functional human glucocerebrosidase. Five months after gene transfer, a highly significant reduction in glucocerebroside accumulation with subsequent reversal of hepatosplenomegaly, restoration of blood parameters, and a tendency of increased bone mass and density was evident in vector-treated mice compared to non-treated controls. Furthermore, histopathology revealed a prominent reduction of Gaucher cell infiltration after gene therapy. The vector displayed an oligoclonal distribution pattern but with no sign of vector-induced clonal dominance and a typical lentiviral vector integration profile. Cumulatively, our findings support the initiation of the first clinical trial for GD1 using the lentiviral vector described here.
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.
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