A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by diff

beta-thalassemia haplotypes in Romania in the context of genetic mixing in the Mediterranean area

The purpose of this meta-study was to investigate β-thalassemia (β-thal) mutations and their chromosomal background in order to highlight the origin and spread of thalassemia alleles in the European and Mediterranean areas. Screening of more than 100 new Romanian β-thal alleles was also conducted. The results suggest an ancient introduction of mutations at codon 39 (C > T) (HBB: c.118C > T) and IVS-I-6 (T > C) (HBB: c.92 + 6T > C) in Romania. A comparative study was performed based on restriction fragment length polymorphism (RFLP) haplotypes associated with β-thal mutations in Romania and in Mediterranean countries. Each common β-thal allele from different populations exhibits a high degree of haplotype similarity, a sign of a clear unicentric origin for the IVS-I-110 (G > A) (HBB: c.93-21G > A), IVS-I-6, IVS-II-745 (C > G) (HBB: c.316-106C > G) and codon 39 mutations (the 17a [+ - - - - + +], 13c [ - + + - - - +], 17c [ + - - - - - +] and 14a [- + + - + + + ] ancestral RFLP background, respectively), followed by recurrent recombination events. This study also showed that geographic distances played a major role in shaping the spread of the predominant β-thal alleles, whereas no genetic boundaries were detected between broad groups of populations living in the Middle East, Europe and North Africa. The analyses revealed some discrepancies concerning Morocco and Serbia, which suggest some peculiar genetic flows. Marked variations in β(A) were observed between Southeast Asia and the Mediterranean, whereas a relative genetic homogeneity was found around the Mediterranean Basin. This homogeneity is undoubtedly the result of the high level of specific historic human migrations that occurred in this area

Scanning of beta-globin gene for identification of beta-thalassemia mutation in Romanian population

beta-Thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemia pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/?l. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f)

Assessment of individual molecular response in chronic myeloid leukemia patients with atypical BCR-ABL1 fusion transcripts. Recommendations by the EUTOS cooperative network

Purpose: Approximately 1–2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients. Methods: BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR). Results: In total, 330 blood samples (2–34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios. Conclusions: Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy.</p

Vanadium(V) Complexes with Substituted 1,5-bis(2-hydroxybenzaldehyde)carbohydrazones and Their Use As Catalyst Precursors in Oxidation of Cyclohexane

Six dinuclear vanadium­(V) complexes have been synthesized: NH₄[(VO₂)₂(^HLH)] (NH₄[<b>1</b>]), NH₄[(VO₂)₂(^{<i>t</i>‑Bu}LH)] (NH₄[<b>2</b>]), NH₄[(VO₂)₂(^ClLH)] (NH₄[<b>3</b>]), [(VO₂)­(VO)­(^HLH)­(CH₃O)] (<b>4</b>), [(VO₂)­(VO)­(^{<i>t</i>‑Bu}LH)­(C₂H₅O)] (<b>5</b>), and [(VO₂)­(VO)­(^ClLH)­(CH₃O)­(CH₃OH/H₂O)] (<b>6</b>) (where ^HLH₄ = 1,5-bis­(2-hydroxybenzaldehyde)­carbohydrazone, ^{<i>t</i>‑Bu}LH₄ = 1,5-bis­(3,5-di-<i>tert</i>-butyl-2-hydroxybenzaldehyde)­carbohydrazone, and ^ClLH₄ = 1,5-bis­(3,5-dichloro-2-hydroxybenzaldehyde)­carbohydrazone). The structures of NH₄[<b>1</b>] and <b>4</b>–<b>6</b> have been determined by X-ray diffraction (XRD) analysis. In all complexes, the triply deprotonated ligand accommodates two V ions, using two different binding sites ONN and ONO separated by a diazine unit −N–N–. In two pockets of NH₄[<b>1</b>], two identical VO₂⁺ entities are present, whereas, in those of <b>4</b>–<b>6</b>, two different VO₂⁺ and VO³⁺ are bound. The highest oxidation state of V ions was corroborated by X-ray data, indicating the presence of alkoxido ligand bound to VO³⁺ in <b>4</b>–<b>6</b>, charge density measurements on <b>4</b>, magnetic susceptibility, NMR spectroscopy, spectroelectrochemistry, and density functional theory (DFT) calculations. All four complexes characterized by XRD form dimeric associates in the solid state, which, however, do not remain intact in solution. Compounds NH₄[<b>1</b>], NH₄[<b>2</b>], and <b>4</b>–<b>6</b> were applied as alternative selective homogeneous catalysts for the industrially significant oxidation of cyclohexane to cyclohexanol and cyclohexanone. The peroxidative (with <i>tert</i>-butyl hydroperoxide, TBHP) oxidation of cyclohexane was performed under solvent-free and additive-free conditions and under low-power microwave (MW) irradiation. Cyclohexanol and cyclohexanone were the only products obtained (high selectivity), after 1.5 h of MW irradiation. Theoretical calculations suggest a key mechanistic role played by the carbohydrazone ligand, which can undergo reduction, instead of the metal itself, to form an active reduced form of the catalyst

Vanadium(V) complexes with substituted 1,5-bis(2- hydroxybenzaldehyde)carbohydrazones and their use as catalyst precursors in oxidation of cyclohexane

Six dinuclear vanadium(V) complexes have been synthesized: NH4[(VO2)(2)((LH)-L-H)] (NH4[1]), NH4[(VO2)(2)((LH)-L-tBu)] (NH4[2]), NH4[(VO2)(2)((LH)-L-Cl)] (NH4[3]), [(VO2)(2)(VO) ((LH)-L-H) (CH3O)] (4), [(VO2) (VO) (t-BuLH) (C2H5O)] (5), and [ (VO2) (VO) (Cl-LH) (CH3O)(CH3OH/H2O)] (6) (where (LH4)-L-H = 1,5-bis(2-hydroxybenzaldehyde)carb ohydrazon e, t-BuLH4 = 1,5-bis(3,5-di-tert-butyl-2-hydroxybenzaldehyde) carbohydrazone, and (LH4)-L-cl = 1,5-bis(3,5-dichloro-2-hydroxybenzaldehyde)carbohydrazone). The structures of NH4[1] and 4-6 have been determined by X-ray diffraction (XRD) analysis. In all complexes, the triply deprotonated ligand accommodates two V ions, using two different binding sites ONN and ONO separated by a diazine unit -N-N-. In two pockets of NH4[1], two identical VO2+ entities are present, whereas, in those of 4-6, two different VO2+ and VO3+ are bound. The highest oxidation state of V ions was corroborated by X-ray data, indicating the presence of alkoxido ligand bound to VO3+ in 4-6, charge density measurements on 4, magnetic susceptibility, NMR spectroscopy, spectroelectrochemistry, and density functional theory (DFT) calculations. All four complexes characterized by XRD form dimeric associates in the solid state, which, however, do not remain intact in solution. Compounds NH4[1], NH4[2], and 4-6 were applied as alternative selective homogeneous catalysts for the industrially significant oxidation of cyclohexane to cyclohexanol and cyclohexanone. The peroxidative (with tert-butyl hydroperoxide, TBHP) oxidation of cyclohexane was performed under solvent -free and additive -free conditions and under low-power microwave (MW) irradiation. Cyclohexanol and cyclohexanone were the only products obtained (high selectivity), after 1.5 h of MW irradiation. Theoretical calculations suggest a key mechanistic role played by the carbohydrazone ligand, which can undergo reduction, instead of the metal itself, to form an active reduced form of the catalyst.PTDC/QEQERQ/1648/2014PTDC/QEQ-QIN/3967/2014info:eu-repo/semantics/publishedVersio

Vanadium(V) Complexes with Substituted 1,5-bis(2-hydroxybenzaldehyde)carbohydrazones and Their Use As Catalyst Precursors in Oxidation of Cyclohexane

Six dinuclear vanadium­(V) complexes have been synthesized: NH₄[(VO₂)₂(^HLH)] (NH₄[<b>1</b>]), NH₄[(VO₂)₂(^{<i>t</i>‑Bu}LH)] (NH₄[<b>2</b>]), NH₄[(VO₂)₂(^ClLH)] (NH₄[<b>3</b>]), [(VO₂)­(VO)­(^HLH)­(CH₃O)] (<b>4</b>), [(VO₂)­(VO)­(^{<i>t</i>‑Bu}LH)­(C₂H₅O)] (<b>5</b>), and [(VO₂)­(VO)­(^ClLH)­(CH₃O)­(CH₃OH/H₂O)] (<b>6</b>) (where ^HLH₄ = 1,5-bis­(2-hydroxybenzaldehyde)­carbohydrazone, ^{<i>t</i>‑Bu}LH₄ = 1,5-bis­(3,5-di-<i>tert</i>-butyl-2-hydroxybenzaldehyde)­carbohydrazone, and ^ClLH₄ = 1,5-bis­(3,5-dichloro-2-hydroxybenzaldehyde)­carbohydrazone). The structures of NH₄[<b>1</b>] and <b>4</b>–<b>6</b> have been determined by X-ray diffraction (XRD) analysis. In all complexes, the triply deprotonated ligand accommodates two V ions, using two different binding sites ONN and ONO separated by a diazine unit −N–N–. In two pockets of NH₄[<b>1</b>], two identical VO₂⁺ entities are present, whereas, in those of <b>4</b>–<b>6</b>, two different VO₂⁺ and VO³⁺ are bound. The highest oxidation state of V ions was corroborated by X-ray data, indicating the presence of alkoxido ligand bound to VO³⁺ in <b>4</b>–<b>6</b>, charge density measurements on <b>4</b>, magnetic susceptibility, NMR spectroscopy, spectroelectrochemistry, and density functional theory (DFT) calculations. All four complexes characterized by XRD form dimeric associates in the solid state, which, however, do not remain intact in solution. Compounds NH₄[<b>1</b>], NH₄[<b>2</b>], and <b>4</b>–<b>6</b> were applied as alternative selective homogeneous catalysts for the industrially significant oxidation of cyclohexane to cyclohexanol and cyclohexanone. The peroxidative (with <i>tert</i>-butyl hydroperoxide, TBHP) oxidation of cyclohexane was performed under solvent-free and additive-free conditions and under low-power microwave (MW) irradiation. Cyclohexanol and cyclohexanone were the only products obtained (high selectivity), after 1.5 h of MW irradiation. Theoretical calculations suggest a key mechanistic role played by the carbohydrazone ligand, which can undergo reduction, instead of the metal itself, to form an active reduced form of the catalyst