High sensitivity assays for docetaxel and paclitaxel in plasma using solid-phase extraction and high-performance liquid chromatography with UV detection

BACKGROUND: The taxanes paclitaxel and docetaxel have traditionally been used in high doses every third week in the treatment of cancer. Lately there has been a trend towards giving weekly low doses to improve the therapeutic index. This article describes the development of high performance liquid chromatographic (HPLC) methods suitable for monitoring taxane levels in patients, focusing on patients receiving low-dose therapy. METHODS: Paclitaxel and docetaxel were extracted from human plasma by solid phase extraction, and detected by absorbance at 227 nm after separation by reversed phase high performance liquid chromatography. The methods were validated and their performance were tested using samples from patients receiving paclitaxel or docetaxel. RESULTS: The limits of quantitation were 1 nM for docetaxel and 1.2 nM for paclitaxel. For both compounds linearity was confirmed from the limit of quantitation up to 1000 nM in plasma. The recoveries ranged between 92% and 118% for docetaxel and between 76% and 104% for paclitaxel. Accuracy and precision were within international acceptance criteria, that is within ± 15%, except at the limit of quantitation where values within ± 20% are acceptable. Low-dose patients included in an on going clinical trial had a median docetaxel concentration of 2.8 nM at 72 hours post infusion. Patients receiving 100 mg/m(2 )of paclitaxel had a mean paclitaxel concentration of 21 nM 48 hours after the end of infusion. CONCLUSION: We have developed an HPLC method using UV detection capable of quantifying 1 nM of docetaxel in plasma samples. The method should be useful for pharmacokinetic determinations at all relevant doses of docetaxel. Using a similar methodology paclitaxel can be quantified down to a concentration of 1.2 nM in plasma with acceptable accuracy and precision. We further demonstrate that the previously reported negative influence of Cremophor EL on assay performance may be overcome by degradation of the detergent by incubation with lipase

Antimalarial Therapy Selection for Quinolone Resistance among Escherichia coli in the Absence of Quinolone Exposure, in Tropical South America

BACKGROUND: Bacterial resistance to antibiotics is thought to develop only in the presence of antibiotic pressure. Here we show evidence to suggest that fluoroquinolone resistance in Escherichia coli has developed in the absence of fluoroquinolone use. METHODS: Over 4 years, outreach clinic attendees in one moderately remote and five very remote villages in rural Guyana were surveyed for the presence of rectal carriage of ciprofloxacin-resistant gram-negative bacilli (GNB). Drinking water was tested for the presence of resistant GNB by culture, and the presence of antibacterial agents and chloroquine by HPLC. The development of ciprofloxacin resistance in E. coli was examined after serial exposure to chloroquine. Patient and laboratory isolates of E. coli resistant to ciprofloxacin were assessed by PCR-sequencing for quinolone-resistance-determining-region (QRDR) mutations. RESULTS: In the very remote villages, 4.8% of patients carried ciprofloxacin-resistant E. coli with QRDR mutations despite no local availability of quinolones. However, there had been extensive local use of chloroquine, with higher prevalence of resistance seen in the villages shortly after a Plasmodium vivax epidemic (p<0.01). Antibacterial agents were not found in the drinking water, but chloroquine was demonstrated to be present. Chloroquine was found to inhibit the growth of E. coli in vitro. Replica plating demonstrated that 2-step QRDR mutations could be induced in E. coli in response to chloroquine. CONCLUSIONS: In these remote communities, the heavy use of chloroquine to treat malaria likely selected for ciprofloxacin resistance in E. coli. This may be an important public health problem in malarious areas

Analysis of physical pore space characteristics of two pyrolytic biochars and potential as microhabitat

Background and Aims Biochar amendment to soil is a promising practice of enhancing productivity of agricultural systems. The positive effects on crop are often attributed to a promotion of beneficial soil microorganisms while suppressing pathogens e.g. This study aims to determine the influence of biochar feedstock on (i) spontaneous and fungi inoculated microbial colonisation of biochar particles and (ii) physical pore space characteristics of native and fungi colonised biochar particles which impact microbial habitat quality. Methods Pyrolytic biochars from mixed woods and Miscanthus were investigated towards spontaneous colonisation by classical microbiological isolation, phylogenetic identification of bacterial and fungal strains, and microbial respiration analysis. Physical pore space characteristics of biochar particles were determined by X-ray μ-CT. Subsequent 3D image analysis included porosity, surface area, connectivities, and pore size distribution. Results Microorganisms isolated from Wood biochar were more abundant and proliferated faster than those from the Miscanthus biochar. All isolated bacteria belonged to gram-positive bacteria and were feedstock specific. Respiration analysis revealed higher microbial activity for Wood biochar after water and substrate amendment while basal respiration was on the same low level for both biochars. Differences in porosity and physical surface area were detected only in interaction with biochar-specific colonisation. Miscanthus biochar was shown to have higher connectivity values in surface, volume and transmission than Wood biochars as well as larger pores as observed by pore size distribution. Differences in physical properties between colonised and non-colonised particles were larger in Miscanthus biochar than in Wood biochar. Conclusions Vigorous colonisation was found on Wood biochar compared to Miscanthus biochar. This is contrasted by our findings from physical pore space analysis which suggests better habitat quality in Miscanthus biochar than in Wood biochar. We conclude that (i) the selected feedstocks display large differences in microbial habitat quality as well as physical pore space characteristics and (ii) physical description of biochars alone does not suffice for the reliable prediction of microbial habitat quality and recommend that physical and surface chemical data should be linked for this purpose

Comparison of Proteomic and Transcriptomic Profiles in the Bronchial Airway Epithelium of Current and Never Smokers

Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear.Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in <or=5% of airway samples from a previously published dataset.1D-PAGE coupled with LC-MS/MS effectively profiled the airway epithelium proteome and identified proteins expressed at different levels as a result of cigarette smoke exposure. While there was a strong correlation between protein and transcript detection within the same sample, we also identified proteins whose corresponding transcripts were not detected by microarray. This noninvasive approach to proteomic profiling of airway epithelium may provide additional insights into the field of injury induced by tobacco exposure

Shake a tail feather: the evolution of the theropod tail into a stiff aerodynamic surface

Theropod dinosaurs show striking morphological and functional tail variation; e.g., a long, robust, basal theropod tail used for counterbalance, or a short, modern avian tail used as an aerodynamic surface. We used a quantitative morphological and functional analysis to reconstruct intervertebral joint stiffness in the tail along the theropod lineage to extant birds. This provides new details of the tail's morphological transformation, and for the first time quantitatively evaluates its biomechanical consequences. We observe that both dorsoventral and lateral joint stiffness decreased along the non-avian theropod lineage (between nodes Theropoda and Paraves). Our results show how the tail structure of non-avian theropods was mechanically appropriate for holding itself up against gravity and maintaining passive balance. However, as dorsoventral and lateral joint stiffness decreased, the tail may have become more effective for dynamically maintaining balance. This supports our hypothesis of a reduction of dorsoventral and lateral joint stiffness in shorter tails. Along the avian theropod lineage (Avialae to crown group birds), dorsoventral and lateral joint stiffness increased overall, which appears to contradict our null expectation. We infer that this departure in joint stiffness is specific to the tail's aerodynamic role and the functional constraints imposed by it. Increased dorsoventral and lateral joint stiffness may have facilitated a gradually improved capacity to lift, depress, and swing the tail. The associated morphological changes should have resulted in a tail capable of producing larger muscular forces to utilise larger lift forces in flight. Improved joint mobility in neornithine birds potentially permitted an increase in the range of lift force vector orientations, which might have improved flight proficiency and manoeuvrability. The tail morphology of modern birds with tail fanning capabilities originated in early ornithuromorph birds. Hence, these capabilities should have been present in the early Cretaceous, with incipient tail-fanning capacity in the earliest pygostylian birds

A second generation human haplotype map of over 3.1 million SNPs

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r(2) of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r(2) of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62863/1/nature06258.pd