Identification of TEL-AML1 (ETV6-RUNX1) associated DNA and its impact on mRNA and protein output using ChIP, mRNA expression arrays and SILAC

The contribution of the most common reciprocal translocation in childhood B-cell precursor leukemia t(12;21)(p13;q22) to leukemia development is still under debate. Direct as well as secondary indirect effects of the TEL-AML1 fusion protein are commonly recorded by using cell lines and patient samples, often bearing the TEL-AML1 fusion protein for decades. To identify direct targets of the fusion protein a short-term induction of TEL-AML1 is needed. We here describe in detail the experimental procedure, quality controls and contents of the ChIP, mRNA expression and SILAC datasets associated with the study published by Linka and colleagues in the Blood Cancer Journal [1] utilizing a short term induction of TEL-AML1 in an inducible precursor B-cell line model

Pharmacokinetics and pharmacodynamics of sildenafil in a patient treated with human immunodeficiency virus protease inhibitors

We describe a 44-year-old male patient with human immunodeficiency virus (HIV) infection and pulmonary arterial hypertension who was treated with several protease inhibitors and with sildenafil. In order to guide treatment with sildenafil, the pharmacokinetics and dynamics of sildenafil were monitored at various time points. In comparison with healthy subjects, the maximal concentration in plasma (Cmax), area under the curve (AUC), and elimination half-life of sildenafil were approximately doubled in the patient. After increasing the sildenafil dose to ensure therapeutic drug levels over 24 hours, the pulmonary arterial hypertension and physical performance of the patient improved significantly. We conclude that the elimination of sildenafil is impaired in patients treated with protease inhibitors, but to a lesser extent than predicted from single-dose studies reported in the literature. Patients treated concomitantly with protease inhibitors and sildenafil need close monitoring of plasma levels, pharmacodynamics, and toxicity of sildenafil in order to be treated optimally

Peroxisomal transporters associated with β-oxidation

Peroxisomes perform a range of different functions, including β-oxidation of fatty acids and synthesis and degradation of bioactive lipid-derived molecules. A key feature of peroxisomes is their role in metabolic pathways which are shared between several subcellular compartments, including mitochondria, chloroplasts and cytosol. Transport across the peroxisomal membrane is therefore essential for the coordination of metabolism. Although transport proteins are very likely required for import of substrates and cofactors, export of intermediates and products and the operation of redox shuttles, relatively few peroxisomal transporters have been identified to date. We have identified and characterised two peroxisomal transport systems which are required for β-oxidation in the model plant, Arabidopsis thaliana. Peroxisomal Nucleotide Carrier 1 and 2 were identified by homology with the yeast peroxisomal adenine nucleotide carrier and were shown by complementation and in vitro uptake assays to catalyse the counter exchange of ATP with AMP [1]. Inducible RNAi lines demonstrated that import of ATP into peroxisomes is essential for activation of fatty acids during seedling establishment and plays a role in other β-oxidation reactions such as auxin metabolism. Arabidopsis also contains a single peroxisomal ABC transporter, COMATOSE (CTS), which has been identified in at least four independent forward genetic screens. Analysis of cts null mutants has demonstrated that CTS plays key roles in a number of developmental and physiological processes, including germination, seedling establishment, fertility and root growth [2]. We demonstrate that the different roles of CTS in planta are separable by mutagenesis [3] and can be related to different biochemical roles, specifically the ability to metabolise distinct substrates such as fatty acids and hormone precursors via β-oxidation. Taken together, these findings strongly suggest that CTS is a broad specificity transporter which mediates uptake of substrates for β-oxidation into the peroxisome. Here, we present biochemical characterisation of heterologously-expressed CTS, provide evidence for its role as a transporter of fatty acyl-CoAs and compare its activity to that of yeast and mammalian homologues

Hypomorphic Mutations in the Central Fanconi Anemia Gene FANCD2 Sustain a Significant Group of FA-D2 Patients with Severe Phenotype. Running title : FA-D2 phenotype and FANCD2 mutations

Premi a l'excel·lència investigadora. Àmbit de les Ciències de la Salut. 2008FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a central role in DNA double-strand type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 FA patients from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3 to 6% of FA patients registered in various datasets. Malformations are frequent in FA-D2 patients and hematological manifestations appear earlier and progress more rapidly when compared to patients from all other FA groups combined, as represented by the International Fanconi Anemia Registry, IFAR. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared alleles. There were no biallelic null mutations so that residual FANCD2 protein of both isotypes was observed in all patients' cell lines available. These analyses suggest that unlike in a knock-out mouse model, total absence of FANCD2 is not existing in FA-D2 patients due to constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations are involved, the result generally is a relatively severe form of FA

Overnight transduction with foamyviral vectors restores the long-term repopulating activity of Fancc−/− stem cells

Fanconi anemia (FA) is a complex genetic disorder characterized by congenital abnormalities, bone marrow failure, and myeloid malignancies. Identification of 13 FA genes has been instrumental to explore gene transfer technologies aimed at correction of autologous FA-deficient stem cells. To date, 3 human FA stem cell gene therapy trials with standard 4-day transduction protocols using gammaretroviral vectors failed to provide clinical benefit. In addition, 2- to 4 day ex vivo manipulation of bone marrow from mice containing a disruption of the homologue of human FANCC (Fancc) results in a time-dependent increase in apoptosis and a risk for malignant transformation of hematopoietic cells. Here, we show that a 14-hour transduction period allows a foamyviral vector construct expressing the human FANCC cDNA to efficiently transduce murine FA stem cells with 1 to 2 proviral integrations per genome. Functionally, the repopulating activity of Fancc−/− stem cells from reconstituted mice expressing the recombinant FANCC transgene was comparable with wild-type controls. Collectively, these data provide evidence that short-term transduction of c-kit+ cells with a foamyviral vector is sufficient for functional correction of a stem cell phenotype in a murine FA model. These data could have implications for future gene therapy trials for FA patients

Hypomorphic Mutations in the Gene Encoding a Key Fanconi Anemia Protein, FANCD2, Sustain a Significant Group of FA-D2 Patients with Severe Phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand–type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%–6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA–non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA