Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

OBJECTIVE: BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. METHODOLOGY: Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling  studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. RESULTS: A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. CONCLUSION: We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second  generation and third generation TKIs.

High frequency and poor prognosis of late childhood BCR-ABL-positive and MLL-AF4-positive ALL define the need for advanced molecular diagnostics and improved therapeutic strategies in pediatric B-ALL in Pakistan.

BACKGROUND: Fusion oncogenes (FOs) resulting from chromosomal abnormalities have an important role in leukemogenesis in pediatric B cell acute lymphoblastic leukemia (ALL). The most common FOs are BCR-ABL, MLL-AF4, ETV6-RUNX1, and TCF3-PBX1, all of which have important prognostic and drug selection implications. Moreover, frequencies of FOs have ethnic variations. We studied Pakistani frequencies of FOs, clinical pattern, and outcome in pediatric B-ALL. METHODS: FOs were studied in 188 patients at diagnosis using reverse transcriptase-polymerase chain reaction (RT-PCR) and interphase fluorescent in situ hybridization (FISH). Data were analyzed using SPSS version 17 (SPSS Inc., Chicago, IL, USA). RESULTS: FOs were detected in 87.2 % of patients. Mean overall survival was 70.9 weeks, 3-year survival was 31.9 %, and 3-year relapse-free survival was 18.1 %. Four patients died of drug toxicities. ETV6-RUNX1 (19.14 %) had better survival (110.9 weeks; p = 0.03); TCF3-PBX1 (2.1 %) was associated with inferior outcome and higher central nervous system (CNS) relapse risk; MLL-AF4 (18.1 %) was more common in the 8- to 15-year age group (24/34; p = 0.001) and was associated with organomegaly, low platelet count, and poor survival; and BCR-ABL (47.9 %) was associated with older age (7-15 years, 52/90), lower remission rates, shorter survival (43.73 ± 4.24 weeks) and higher white blood cell count. Overall, MLL-AF4 and BCR-ABL were detected in 66 % of B-ALL, presented in later childhood, and were associated with poor prognosis and inferior survival. CONCLUSIONS: This study reports the highest ethnic frequency of BCR-ABL FO in pediatric ALL, and is consistent with previous reports from our region. Poor prognosis BCR-ABL and MLL-AF4 was detected in two-thirds of pediatric B-ALL and is likely to be the reason for the already reported poor survival of childhood ALL in South-East Asia. Furthermore, MLL-AF4, usually most common in infants, presented in later childhood in most of the ALL patients, which was one of the unique findings in our study. The results presented here highlight the need for mandatory inclusion of molecular testing for pediatric ALL patients in clinical decision making, together with the incorporation of tyrosine kinase inhibitors, as well as hematopoietic stem cell transplantation facilities, to improve treatment outcome for patients in developing countries.

Interaction of anticancer drug methotrexate with DNA analyzed by electrochemical and spectroscopic methods.

Electrochemical DNA biosensor was used to study the interaction of methotrexate (MTX) with DNA immobilized on the bare surface of glassy carbon electrode (GCE). The binding mechanism of MTX with DNA was elucidated by using constant current potentiometric technique further supported by UV-Visible and FT-IR studies. The decrease in guanine peak area was used as an analytical signal for the interaction of drug with DNA in acetate buffer solution at pH 4.2 (20% ethanol). The binding constant (K) value calculated for MTX was 3.821×10(5)M(-1). UV-Visible studies indicated hyperchromic and hypsochromic shifts in the maximum absorption bands of MTX after interaction with DNA. FT-IR investigations of MTX-DNA interaction revealed significant changes in the characteristic IR absorption bands of all the bases and phosphate groups of DNA. Furthermore, the shift of characteristics bands of C=O, N-H, C-H and O-H groups of MTX endow evidence for the interaction of MTX with DNA supporting the intercalative binding between them.

Removal and recovery of zirconium from its aqueous solution by Candida tropicalis.

Removal and recovery of zirconium from dilute aqueous solutions by Candida tropicalis used as biosorbent, was studied by performing biosorption-desorption tests. This biosorbent was selected after screening a range of microbial species. The process was found to be highly dependent on initial pH and concentration of metal solution. At optimized experimental parameters, the maximum zirconium biosorption capacity of C. tropicalis was 179 mg Zr g(-1) dry weight of biosorbent. The adsorption distribution coefficient value of 3968 ml g(-1) was obtained for zirconium biosorption by C. tropicalis. Different theoretical thermodynamic models governing the adsorption behavior of zirconium were also tested. Zirconium biosorption was found to closely follow the Langmuir model. At low biomass concentrations it was found to follow pseudo-first-order kinetics. However when higher biomass concentrations were used kinetics was changed to pseudo-second-order. The zirconium bound to the biomass was stripped out (60.2% at S/L of 1.0 g of zirconium loaded biomass/l of eluent) using sodium bicarbonate and the biomass could be used for multiple sorption-desorption cycles.

Efficacy of modified distillation sludge of rose (Rosa centifolia) petals for lead(II) and zinc(II) removal from aqueous solutions.

Removal of lead(II) and zinc(II) from aqueous solutions was studied using chemically modified distillation sludge of rose (Rosa centifolia) petals by pretreatment with NaOH, Ca(OH)(2), Al(OH)(3), C(6)H(6), C(6)H(5)CHO and HgCl(2). The adsorption capacity of biomass was found to be significantly improved. NaOH pretreated biomass showed remarkable increase in sorption capacity. Maximum adsorption of both metal ions was observed at pH 5. When Freundlich and Langmuir isotherms were tested, the latter had a better fit with the experimental data. The overall adsorption process was best described by pseudo second order kinetics. The thermodynamic assessment of the metal ion-Rosa centifolia biomass system indicated the feasibility and spontaneous nature of the process and DeltaG degrees was evaluated as ranging from -26.9501 to -31.652 KJmol(-1) and -24.1905 to -29.8923KJmol(-1) for lead(II) and zinc(II) sorption, respectively, in the concentration range 10-640mgL(-1). Distribution coefficient (D) showed that the concentration of metal ions at the sorbent-water interface is higher than the concentration in the continuous aqueous phase. Maximum adsorption capacity of biomass tends to be in the order Pb(II) (87.74mgg(-1))>Zn(II) (73.8mgg(-1)) by NaOH pretreated biomass.

Removal and recovery of uranium from aqueous solutions by Trichoderma harzianum.

Removal and recovery of uranium from dilute aqueous solutions by indigenously isolated viable and non-viable fungus (Trichoderma harzianum) and algae (RD256, RD257) was studied by performing biosorption-desorption tests. Fungal strain was found comparatively better candidate for uranium biosorption than algae. The process was highly pH dependent. At optimized experimental parameters, the maximum uranium biosorption capacity of T. harzianum was 612 mg U g(-1) whereas maximum values of uranium biosorption capacity exhibited by algal strains (RD256 and RD257) were 354 and 408 mg U g(-1) and much higher in comparison with commercially available resins (Dowex-SBR-P and IRA-400). Uranium biosorption by algae followed Langmuir model while fungus exhibited a more complex multilayer phenomenon of biosorption and followed pseudo-second-order kinetics. Mass balance studies revealed that uranium recovery was 99.9%, for T. harzianum, and 97.1 and 95.3% for RD256 and RD257, respectively, by 0.1M Hydrochloric acid which regenerated the uranium-free cell biomass facilitating the sorption-desorption cycles for better economic feasibility.

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

Electrochemical DNA biosensor for the study of ciprofloxacin-DNA interaction.

The interaction of ciprofloxacin with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of ciprofloxacin was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current at +0.9 V was used as an indicator for the interaction mechanism in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 1.33+/-0.02 x 10(4) and 1.32+/-0.08 x 10(4) M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak currents was observed in the range of 40-80 microM ciprofloxacin, with a detection limit of 24 microM with r=0.995 and 9 microM with r=0.999 by using constant current potentiometry and differential pulse voltammetry, respectively. Moreover, the influence of sodium and calcium ions was also studied to elucidate the mechanism of ciprofloxacin-DNA interaction at different solution conditions, and this proved to be helpful in understanding the ciprofloxacin-DNA interaction.

Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.

Intein-mediated protein trans-splicing and transgene containment in plastids.

Transgenes in plastids are contained by stringent maternal inheritance in most cultivated plant species and their expression yields high levels of protein with bona fide structure. Nevertheless, transfer of plastid genes to the nucleus has been reported, with implications for transgene containment. The significance of these transfers will depend on the likelihood that they will become functional nuclear genes. Recently a novel approach, intein-mediated protein trans-splicing, has been demonstrated promising to yield transgenic plants with greatly reduced risk of genetic outcrossing.

Phage phiC31 integrase: a new tool in plastid genome engineering.

Plastid transformation offers the unique advantages of high-level transgene expression and increased transgene containment compared with conventional transgenic technologies. The process relies on the homologous recombination machinery of the plastid incorporating foreign DNA into the plastome, which restricts the method to species where this type of incorporation works well. However, Pal Maliga and colleagues have recently reported a novel approach for integrating foreign DNA into the plastid genome that works independently of homologous recombination.

Phylogenetic analysis of different isolates of Sulfobacillus spp. isolated from uranium-rich environments and recovery of genes using integron-specific primers.

The isolation and phylogenetic characterization of acidophilic moderate thermophilic bacteria from different locations of uranium mines and a uranium processing mill in Pakistan is reported. The dominant culturable bacteria found were related to Sulfobacillus thermosulfidooxidans in all the samples analyzed. Different strains displayed different levels of identity (95-97%) to 16S rDNA of known strains of this species, indicating group heterogeneity. Genomic DNA from five isolates was subjected to amplification using integron-specific primers HS286 and HS287. Recovery of different integron-linked genes from one of the isolates indicated the usefulness of this approach for gene mining in place of traditional gene recovery methodologies.