Simultaneous Quantification of Lamivudine, Zidovudine and Related Impurities in Fixed Oral Dosage Combination Using RP-HPLC with DAD detection

A simple and fast isocratic Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed and validated for the simultaneous determination of Lamivudine, zidovudine and their related impurities in tablets. The method consists of a mobile phase combination of Acetonitrile (HPLC grade) and Buffer (0.0680 g of Potassium Dihydrogen Orthophosphate, 0.3 ml of Triethylamine, pH adjusted to 8.0 with Orthophosphoric acid to a final volume preparation of 100 ml) in the ratio 10:90. Phenomenex Luna 5-µm C18 (2)-250 x 4.6-mm, 5-µm) was used as the stationary phase. The column oven was set to a temperature of 30±1oC. Quantification was achieved with a DAD detector set at 270 nm. Resolution was achieved at a short run time of 25 minutes. Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine and Zidovudine related impurity B eluted at 3.749±0.004, 4.862±0.013, 15.332±0.064, 21.201±0.076 and 23.682±0.117 respectively. Relative retention times (RRT) for lamivudine unknown related impurities with respect to Zidovudine were 0.15, 0.17, 0.30 and 0.59. RRT for Zidovudine unknown related impurities with respect to Zidovudine were 0.39 and 0.63.  The method was found to be specific, robust, accurate and precise for the estimation of Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine and Zidovudine related impurity B in fixed oral dosage tablets over the concentration ranges of 0.0204 mg/mL-0.0088 mg/mL, 0.0962 mg/mL-0.7699 mg/mL, 0.1929 mg/mL-1.5410 mg/mL and 0.0088 mg/mL-0.024 mg/mL respectively. The Correlation Coefficient (r2) for Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine and Zidovudine related impurity B were greater than 0.998. The LOD were found to be between 1.9x10-4 mg/mL to 2.69 x10-4 mg/mL.  The proposed method is precise, specific, accurate and robust for the simultaneous estimation of Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine, Zidovudine related impurity B and other related impurities in dosage forms. Keywords: Zidovudine related impurity C, Lamivudine, salicylic acid, Zidovudine, Zidovudine related impurity B, Lamivudine related impurities, Zidovudine related impurities RP-HPLC, Validation.

Relative Response Factor for Lamivudine and Zidovudine Related substances by RP-HPLC with DAD detection

A study was conducted to establish the Relative Response Factors for Zidovudine related impurity C, Lamivudine salicylic acid and Zidovudine related impurity B.  A simple and fast isocratic Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method was used for the simultaneous determination of the related impurities. The method consists of a mobile phase combination of Acetonitrile (HPLC grade) and Buffer (0.0680 g of Potassium Dihydrogen Orthophosphate, 0.3 ml of Triethylamine, pH adjusted to 8.0 with Orthophosphoric acid to a final volume preparation of 100 ml) in the ratio 10:90 using Phenomenex Luna 5-µm C18 (2)-250 x 4.6-mm, 5-µm) as a stationary phase, flow rate of 1.0 mL/min with detection at 270 nm. The RRF for Zidovudine related impurity C, Lamivudine salicylic acid and Zidovudine related impurity B were 2.07, 0.13 and 1.28 respectively.   Results obtained for quantification of the related substance in lamivudine and zidovudine single dose oral solid dosage form using the RRF and know standards shows no significant difference at 95 % confidence interval. The RRF can therefore be used for the quantification of know related impurities in lamivudine zidovudine oral dosage form using the stated chromatographic conditions

Characterization of renal cell carcinoma-associated constitutional chromosome abnormalities by genome sequencing.

Constitutional translocations, typically involving chromosome 3, have been recognized as a rare cause of inherited predisposition to renal cell carcinoma (RCC) for four decades. However, knowledge of the molecular basis of this association is limited. We have characterized the breakpoints by genome sequencing (GS) of constitutional chromosome abnormalities in five individuals who presented with RCC. In one individual with constitutional t(10;17)(q11.21;p11.2), the translocation breakpoint disrupted two genes: the known renal tumor suppressor gene (TSG) FLCN (and clinical features of Birt-Hogg-Dubé syndrome were detected) and RASGEF1A. In four cases, the rearrangement breakpoints did not disrupt known inherited RCC genes. In the second case without chromosome 3 involvement, the translocation breakpoint in an individual with a constitutional t(2;17)(q21.1;q11.2) mapped 12 Kb upstream of NLK. Interestingly, NLK has been reported to interact indirectly with FBXW7 and a previously reported RCC-associated translocation breakpoint disrupted FBXW7. In two cases of constitutional chromosome 3 translocations, no candidate TSGs were identified in the vicinity of the breakpoints. However, in an individual with a constitutional chromosome 3 inversion, the 3p breakpoint disrupted the FHIT TSG (which has been reported previously to be disrupted in two apparently unrelated families with an RCC-associated t(3;8)(p14.2;q24.1). These findings (a) expand the range of constitutional chromosome rearrangements that may be associated with predisposition to RCC, (b) confirm that chromosome rearrangements not involving chromosome 3 can predispose to RCC, (c) suggest that a variety of molecular mechanisms are involved the pathogenesis of translocation-associated RCC, and (d) demonstrate the utility of GS for investigating such cases

Improved annotation of 3' untranslated regions and complex loci by combination of strand-specific direct RNA sequencing, RNA-seq and ESTs

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct annotation is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3-prime untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3-prime polyadenylation sites to within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3-prime UTR re-annotation (including extension of one 3-prime UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental dataComment: 44 pages, 9 figure

Features of mammalian microRNA promoters emerge from polymerase II chromatin immunoprecipitation data

Background: MicroRNAs (miRNAs) are short, non-coding RNA regulators of protein coding genes. miRNAs play a very important role in diverse biological processes and various diseases. Many algorithms are able to predict miRNA genes and their targets, but their transcription regulation is still under investigation. It is generally believed that intragenic miRNAs (located in introns or exons of protein coding genes) are co-transcribed with their host genes and most intergenic miRNAs transcribed from their own RNA polymerase II (Pol II) promoter. However, the length of the primary transcripts and promoter organization is currently unknown. Methodology: We performed Pol II chromatin immunoprecipitation (ChIP)-chip using a custom array surrounding regions of known miRNA genes. To identify the true core transcription start sites of the miRNA genes we developed a new tool (CPPP). We showed that miRNA genes can be transcribed from promoters located several kilobases away and that their promoters share the same general features as those of protein coding genes. Finally, we found evidence that as many as 26% of the intragenic miRNAs may be transcribed from their own unique promoters. Conclusion: miRNA promoters have similar features to those of protein coding genes, but miRNA transcript organization is more complex. © 2009 Corcoran et al

Cost of Mating and Insemination Capacity of a Genetically Modified Mosquito Aedes aegypti OX513A Compared to Its Wild Type Counterpart

The idea of implementing genetics-based insect control strategies modelled on the traditional SIT is becoming increasingly popular. In this paper we compare a genetically modified line of Aedes aegypti carrying a tetracycline repressible, lethal positive feedback system (OX513A) with its wild type counterpart with respect to their insemination capacities and the cost of courtship and mating. Genetically modified males inseminated just over half as many females as the wild type males during their lifetime. Providing days of rest from mating had no significant effect on the total number of females inseminated by males of either line, but it did increase their longevity. Producing sperm had a low cost in terms of energy investment; the cost of transferring this sperm to a receptive female was much higher. Continued mating attempts with refractory females suggest that males could not identify refractory females before investing substantial energy in courtship. Although over a lifetime OX513A males inseminated fewer females, the number of females inseminated over the first three days, was similar between males of the two lines, suggesting that the identified cost of RIDL may have little impact on the outcome of SIT-based control programmes with frequent releases of the genetically modified males

Nuclear Receptor SHP Activates miR-206 Expression via a Cascade Dual Inhibitory Mechanism

MicroRNAs play a critical role in many essential cellular functions in the mammalian species. However, limited information is available regarding the regulation of miRNAs gene transcription. Microarray profiling and real-time PCR analysis revealed a marked down-regulation of miR-206 in nuclear receptor SHP−/− mice. To understand the regulatory function of SHP with regard to miR-206 gene expression, we determined the putative transcriptional initiation site of miR-206 and also its full length primary transcript using a database mining approach and RACE. We identified the transcription factor AP1 binding sites on the miR-206 promoter and further showed that AP1 (c-Jun and c-Fos) induced miR-206 promoter transactivity and expression which was repressed by YY1. ChIP analysis confirmed the physical association of AP1 (c-Jun) and YY1 with the endogenous miR-206 promoter. In addition, we also identified nuclear receptor ERRγ (NR3B3) binding site on the YY1 promoter and showed that YY1 promoter was transactivated by ERRγ, which was inhibited by SHP (NROB2). ChIP analysis confirmed the ERRγ binding to the YY1 promoter. Forced expression of SHP and AP1 induced miR-206 expression while overexpression of ERRγ and YY1 reduced its expression. The effects of AP1, ERRγ, and YY1 on miR-206 expression were reversed by siRNA knockdown of each gene, respectively. Thus, we propose a novel cascade “dual inhibitory” mechanism governing miR-206 gene transcription by SHP: SHP inhibition of ERRγ led to decreased YY1 expression and the de-repression of YY1 on AP1 activity, ultimately leading to the activation of miR-206. This is the first report to elucidate a cascade regulatory mechanism governing miRNAs gene transcription

Genetic determinants of co-accessible chromatin regions in activated T cells across humans.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression

Long-term all-sites cancer mortality time trends in Ohio, USA, 1970–2001: differences by race, gender and age

BACKGROUND: There were significant changes in cancer mortality in the USA over the last several decades, in the whole country and in particular states. However, no in depth analysis has been published so far, dealing with changes in mortality time trends in the state of Ohio. Since the state of Ohio belongs to the states of relatively high level of all-sites mortality in both males and females, it is of interest to analyze recent changes in mortality rates, as well as to compare them with the situation in the rest of the USA. The main aim of this study was to analyze, describe and interpret all-sites cancer mortality time trends in the population of the State of Ohio. METHODS: Cancer mortality data by age, sex, race and year for the period 1970–2001 were obtained from the Surveillance Research Program of the National Cancer Institute SEER*Stat software. A joinpoint regression methodology was used to provide estimated annual percentage changes (EAPCs) and to detect points in time where significant changes in the trends occurred. RESULTS: In both, males and females mortality rates were higher in blacks compared with whites. The difference was bigger in males (39.9%) than in women (23.3%). Mortality rates in Ohio are generally higher than average USA rates – an overall difference was 7.5% in men in 1997–2001, and 6.1% in women. All-sites mortality trends in Ohio and in the whole USA are similar. However, in general, mortality rates in Ohio remained elevated compared with the USA rates throughout the entire analyzed period. The exceptions are the rates in young and middle-aged African Americans. CONCLUSION: Although direction of time trends in Ohio are similar in Ohio and the whole US, Ohio still have cancer mortality rates higher than the US average. In addition, there is a significant discrepancy between white and black population of Ohio in all-sites mortality level, with disadvantage for Blacks. To diminish disparities in cancer mortality between African Americans and white inhabitants of Ohio efforts should be focused on increasing knowledge of black people regarding healthy lifestyle and behavioral risk factors, but also on diminishing socioeconomic differences, and last but not least, on better access to medical care

Interventions for preventing oral mucositis for patients with cancer receiving treatment

Interventions for preventing oral mucositis for patients with cancer receiving treatmentTreatment for cancer (including bone marrow transplant) can cause oral mucositis (severe ulcers in the mouth). This painful condition can cause difficulties in eating, drinking and swallowing, and may also be associated with infections which may require the patient to stay longer in hospital. Different strategies are used to try and prevent this condition, and the review of trials found that some of these are effective. Two interventions, cryotherapy (ice chips) and keratinocyte growth factor (palifermin®) showed some benefit in preventing mucositis. Sucralfate is effective in reducing the severity of mucositis, and a further seven interventions, aloe vera, amifostine, intravenous glutamine, granulocyte‐colony stimulating factor (G‐CSF), honey, laser and antibiotic lozenges containing polymixin/tobramycin/amphotericin (PTA) showed weaker evidence of benefit. These were evaluated in patients with different types of cancer, undergoing different types of cancer treatment. Benefits may be restricted to the disease and treatment combinations evaluated