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Imatinib (Gleevec) is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO) PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance.

Experimental design is a significant tool for optimization and validation for the development of HPLC methods to determine API in both human serum and pharmaceutical formulations. In this study, RP-HPLC method is developed and validated for the simultaneous determination of moxifloxacin and NSAIDs. In this experiment, Purospher STAR C18 column with optimum assay conditions (10:90, v/v, water: methanol, pH 2.75) used as mobile phase having flow rate of 1.5mL min-1 and screened at 240 nm. The experimental results exhibit reliability through accuracy (98-102%), precision (0.011-1.85%) and linearity (R2>0.999) in range of 0.15-40ÎgmL-1. The LOD and LOQ limits for moxifloxacin and NSAIDs are found to be 0.015 and 0.046 ÎgmL-1 respectively. The significant outcomes conclude that the developed method for assay is effectively suitable to human serum and pharmaceutical formulations and there is no interference from excipients of tablets and serum. The proposed method is useful for drug-interaction and investigation of moxifloxacin with NSAIDs. [ABSTRACT FROM AUTHOR]

Seven new hydrazinyl thiazole derivatives of piperidin-4-one (PE3-PE9) have been synthesized by cyclization of intermediate thiosemicarbazone derivative (PE2). Parent molecule (PE1) was synthesized by one pot total synthesis using Mannich condensation reaction. Percent yield of most of the compounds found in between 70-85%. Compounds were identified by spectroscopic analysis. In vivo analgesic activity was examined using tail flick method. one-way ANOVA was used to compare the mean latency time of synthesized derivatives with control and standard. Analgesic activity was discussed in terms of structural differences between compounds. Among all the derivatives thiosemicarbazone derivative showed good analgesic activity (195.24%). Methoxy (-OCH3) and bromo (-Br) containing thiazole derivative also showed good pain reducing property (167.62%, 203%) at a dose of 30mg/kg. [ABSTRACT FROM AUTHOR]

Benzimidazole and its derivatives found variety of biological activities, for the searching of its potent antiinflammatory analogues, we synthesized four novel 4-(2-keto-1-benzimidazollinyl) piperidine derivatives (Q1 to Q4) by refluxing piperidine with substituted imidazole and subjected to in-vitro anti-inflammatory (ROS, NO) and antibacterial activities, structures were elucidated using spectroscopic techniques. Results revealed that compound Q1 showed most effective anti-inflammatory activity with IC50 7.6±1.3 μg/ml compared with standard Ibuprofen having IC50 11.2±1.9μg/mL. Compound Q3 showed good activity for Nitrite accumulation by stimulating macrophages test similar to standard NG Methyl L-arginine acetate with IC50 value 24.2±0.8μg/mL. The antibacterial activity of these compounds were evaluated against selected Gram+ve E. faecalis, C. diphtheriae, S. aureus and Gram -ve organism E. coli, Enterobacter aerogenes and P. aeruginosa. Synthesized compounds showed low to moderate level of antibacterial activity Q1 showed the highest antibacterial activity against Enterococcus faecalis and Escherichia coli with zone of inhibition 18mm and Q3 showed highest activity against Corynebacterium diptheriae (ZOI:18mm). Structure-activity relationship (SAR) study revealed that among all the synthesized compounds unsubstituted naphthalene (Q1) and phenyl (Q3) ring containing derivatives were most potent. [ABSTRACT FROM AUTHOR]

In this present study seven novel thiazole derivatives (PM3-PM9) were synthesized by cyclization of key intermediate thiosemicarbazone (PM2), derived from 4-piperidone (PM1). The parent 4-piperidone was synthesized by Mannich condensation reaction with good yield (89%). All the derivatives were characterized by UV, IR, 1HNMR and mass spectral analysis. All the synthesized products were screened for their in vivo analgesic activities. Most of the tested compounds exhibited potential to reduce pain and some of them showed good analgesic properties. Thiosemicarbazone derivative showed most significant activity (p-value 0.01). All the thiazole derivatives exhibited dose dependent mild to good analgesic activities. Among thiazole derivatives, chloro and nitro substituted compounds (PM3, PM4, and PM5) showed highest analgesic activities. [ABSTRACT FROM AUTHOR]

In search of potent molecules having antioxidant activity the present work was designed to synthesize 2, 6- diphenylpiperidine-4-one compounds (1a and 1b) and their imine derivatives (2a, 2b, 3a, and 3b). Compounds 1a and 1b were synthesized by Mannich condensation reaction. The method was found to be simple, convenient with high yield and products were easily separated. Compounds 1a and 1b serves as an intermediate for the preparation of highly functionalized novel imine derivatives. Oxime (2a, 2b) and carbothioamide (3a, 3b) derivatives of 1a and 1b compounds were produced by condensation reaction with hydroxyl amine hydrochloride and thiosemicarbazide respectively. These compounds were characterized by IR, EI-mass and 1HNMR spectroscopy. The antioxidant activity of compounds was analyzed by 1, 1- dipheny1-2-picrylhydrazyl (DPPH) assay method. It was found that substituted aryl derivatives containing phenol and methoxy groups (1b, 2b and 3b) showed better antioxidant activity (IC50 values rang from 1.84- 4.53μg/ml) than unsubstituted aryl derivative (1a, 2a and 3a) (IC50 rang from 6.46-11.13μg/ml). Compound 1b exhibited excellent antioxidant activity (IC50 1.84±0.15μg/ml) comparable to standard ascorbic acid (IC501.65± 0.16μg/ml). [ABSTRACT FROM AUTHOR]

The title compound, C31H46NO7+·Cl-, was synthesized by a one-pot Mannich condensation reaction. In the molecule, the piperidinone ring adopts a chair conformation, and the trimethoxy-substituted benzene rings and octyl chain are arranged equatorially. In the crystal, centrosymmetric dimers are linked into layers parallel to (011) by N-H...Cl and C-H...Cl hydrogen bonds. A Hirshfeld surface analysis indicates that the most important contributions for the crystal packing are O...H (20.5%) interactions followed by C...H (7.8%), Cl...H (5.5%), C...C (1.2%), C...O (0.5%) and Cl...O (0.4%) interactions. [ABSTRACT FROM AUTHOR]

Mutations in rpsL gene associated with resistance to streptomycin were screened in multiple drug resistant and randomly collected isolates of M. tuberculosis from Pakistan. Mutations were detected by PCR amplification of rpsL gene followed by dot-blot hybridization with allele-specific radioactively-labeled probes and by PCR-RFLP. Out of 45 isolates, 31 were resistant to streptomycin, while 14 were sensitive by conventional drug sensitivity assay. The rpsL43 mutation was found in 7 out of 31 isolates tested (22.58%) while rpsL88 was found in 4 out of 31(12.90%) isolates. The rpsL43 was also found in 27% isolates collected randomly, while rpsL88 was found in 5.4% isolates. All susceptible isolates had no mutation when probed with rpsL43 or rpsL88. The presence of rpsL43 mutation in randomly collected isolates indicates a high level of resistance to streptomycin. [ABSTRACT FROM AUTHOR]

Background: Microdeletions of the azoospermia factor locus of the long arm of Y chromosome are an etiological factor of severe oligozoospermia or azoospermia. Objective: The aim of this study was to investigate the prevalence of Y- chromosome microdeletions in AZF region and their role in infertility in Pakistani population. Materials and Methods: The type of deletions in AZF locus were detected in infertile men (n=113) and the association of Y chromosome microdeletions with male infertility was assessed by including men (50) with normal karyotype and having children. Y chromosome microdeletions were detected by multiplex PCR using 10 sequence tagged sites namely sY81, sY130, sY141, sY142, sY155, sY157, sY160, sY182, sY231, and sY202 that covered all three regions of AZF. Results: Individuals with severe oligozoospermia showed 2.86% deletion frequency in AZFc region as compared to azoospermic males (5.5%). Conclusion: The results of our study showed that deletions in Y chromosome are not playing major part in male infertility. Moreover, multiplex-PCR strategy might preferably be employed for the detection of Y chromosome microdeletions allied to male infertility. [ABSTRACT FROM AUTHOR]

The insertion element IS6110 has been used extensively for the detection and strain differentiation of M. tuberculosis. The presence and copy number of IS6110 varies geographically. We have investigated the presence and copy number of this insertion sequence in M. tuberculosis isolates collected throughout Pakistan. All 92 confirmed cultures of M. tuberculosis from Pakistan were found to contain this insertion sequence when checked by PCR using primers based on IS6110 sequence. The copy number determined by the standard IS6110 restriction fragment length polymorphism varied from 2?18. Among 46 cultures used for copy number estimation 32 strains (69.5%) contained more than 10 copies of IS6110. The percentage of isolates having up to 5 copies of IS6110 was 6.25, while 21.7% isolates had copy number between 5 and 11. Our results confirmed the presence of IS6110 in all isolates of M. tuberculosis and showed that IS6110 is a useful marker for the detection and DNA fingerprinting of M. tuberculosis strains originating from Pakistan. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the frequency and clinical significance of gr/gr and b2/b3 deletions in Pakistani population. Methodology: In this study, we analyzed Y chromosome deletions using markers sY1191 and sY1291 in patients with azoospermia (n=26), severe oligozoospermia (n=29), moderate oligozoospermia (n=24) and normozoospermia (n=9) using multiplex PCR. Control group consisted of fertile men with normal spermatogenesis (n=30) having children. Results: The overall deletion frequency of gr/gr was 6.8 % (6 /88) while that of b2/b3 was 5.6 % (5/88). gr/gr deletions were detected in 4 men with severe oligozoospermia, 1 with azoospermia and 1 normospermic (but infertile) man. b2/b3 deletion was found in 3 severe oligozoospermic and 2 azoospermic men. None of the control group had either gr/gr or b2/b3 deletion. Conclusion: Our results indicate that both gr/gr and b2/b3 deletions are a risk factor for spermatogenic failure in the population under study. [ABSTRACT FROM AUTHOR]

The article focuses on a study which described the establishment of a very sensitive polymerase chain reaction (PCR) to detect mutations in an imatinib-resistant chronic myeloid leukemia (CML). C944T and T1052C mutations were detected which led to complete partial imatinib resistance. The study served as the first report of multiple point mutations conferring primary imatinib resistance in the same patient.

The emergence of multidrug resistant strains of Mycobacterium tuberculosis is a threat to tuberculosis control programs. The most effective strategy to control the spread of these strains is to use an accurate and rapid drug susceptibility test. Resistance to rifampicin, one of the most potent first line anti-TB drugs, is found to be aβociated with mutations in "hot-spot" region of RNA polymerase gene (rpoB) are geographically distributed. M. tuberculosis isolates from various regions of Pakistan were collected and mutations were detected by reverse hybridization using in-house line probe aβay. The rpoB gene from 45 isolates was cloned and sequenced to validate the results obtained by line probe aβay. The most common mutations were found to be in codon 531 (56.45%) followed by codon 526 (9.68%), 516 (4.84%) and 518 (3.22%). Certain silent mutations and several novel mutations outside the "hot-spot" region were also found. © 2014 Friends Science Publishers [ABSTRACT FROM AUTHOR]

Infra-species taxonomy is a prerequisite to compare features such as virulence in different pathogen lineages. Mycobacterium tuberculosis complex taxonomy has rapidly evolved in the last 20 years through intensive clinical isolation, advances in sequencing and in the description of fast-evolving loci (CRISPR and MIRU-VNTR). On-line tools to describe new isolates have been set up based on known diversity either on CRISPRs (also known as spoligotypes) or on MIRU-VNTR profiles. The underlying taxonomies are largely concordant but use different names and offer different depths. The objectives of this study were 1) to explicit the consensus that exists between the alternative taxonomies, and 2) to provide an on-line tool to ease classification of new isolates. Genotyping (24-VNTR, 43-spacers spoligotypes, IS6110-RFLP) was undertaken for 3,454 clinical isolates from the Netherlands (2004-2008). The resulting database was enlarged with African isolates to include most human tuberculosis diversity. Assignations were obtained using TB-Lineage, MIRU-VNTRPlus, SITVITWEB and an algorithm from Borile et al. By identifying the recurrent concordances between the alternative taxonomies, we proposed a consensus including 22 sublineages. Original and consensus assignations of the all isolates from the database were subsequently implemented into an ensemble learning approach based on Machine Learning tool Weka to derive a classification scheme. All assignations were reproduced with very good sensibilities and specificities. When applied to independent datasets, it was able to suggest new sublineages such as pseudo-Beijing. This Lineage Prediction tool, efficient on 15-MIRU, 24-VNTR and spoligotype data is available on the web interface "TBminer." Another section of this website helps summarizing key molecular epidemiological data, easing tuberculosis surveillance. Altogether, we successfully used Machine Learning on a large dataset to set up and make available the first consensual taxonomy for human Mycobacterium tuberculosis complex. Additional developments using SNPs will help stabilizing it.

Background: Microdeletions of the azoospermia factor locus of the long arm of Y chromosome are an etiological factor of severe oligozoospermia or azoospermia., Objective: The aim of this study was to investigate the prevalence of Y-chromosome microdeletions in AZF region and their role in infertility in Pakistani population., Materials and Methods: The type of deletions in AZF locus were detected in infertile men (n=113) and the association of Y chromosome microdeletions with male infertility was assessed by including men (50) with normal karyotype and having children. Y chromosome microdeletions were detected by multiplex PCR using 10 sequence tagged sites namely sY81, sY130, sY141, sY142, sY155, sY157, sY160, sY182, sY231, and sY202 that covered all three regions of AZF., Results: Individuals with severe oligozoospermia showed 2.86% deletion frequency in AZFc region as compared to azoospermic males (5.5%)., Conclusion: The results of our study showed that deletions in Y chromosome are not playing major part in male infertility. Moreover, multiplex-PCR strategy might preferably be employed for the detection of Y chromosome microdeletions allied to male infertility.