Bladder cancer determination via two urinary metabolites: a biomarker pattern approach

The purpose of this study was to use metabonomic profiling to identify a potential specific biomarker pattern in urine as a noninvasive bladder cancer (BC) detection strategy. A liquid chromatography-mass spectrometry based method, which utilized both reversed phase liquid chromatography and hydrophilic interaction chromatography separations, was performed, followed by multivariate data analysis to discriminate the global urine profiles of 27 BC patients and 32 healthy controls. Data from both columns were combined, and this combination proved to be effective and reliable for partial least squares-discriminant analysis. Following a critical selection criterion, several metabolites showing significant differences in expression levels were detected. Receiver operating characteristic analysis was used for the evaluation of potential biomarkers. Carnitine C9:1 and component I, were combined as a biomarker pattern, with a sensitivity and specificity up to 92.6% and 96.9%, respectively, for all patients and 90.5% and 96.9%, respectively for low-grade BC patients. Metabolic pathways of component I and carnitine C9:1 are discussed. These results indicate that metabonomics is a practicable tool for BC diagnosis given its high efficacy and economization. The combined biomarker pattern showed better performance than single metabolite in discriminating bladder cancer patients, especially low-grade BC patients, from healthy controls.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

Mechanism of the mesenchymal-epithelial transition and its relationship with metastatic tumor formation

Cancer metastasis consists of a sequential series of events, and the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are recognized as critical events for metastasis of carcinomas. A current area of focus is the histopathological similarity between primary and metastatic tumors, and MET at sites of metastases has been postulated to be part of the process of metastatic tumor formation. Here, we summarize accumulating evidence from experimental studies that directly supports the role of MET in cancer metastasis, and we analyze the main mechanisms that regulate MET or reverse EMT in carcinomas. Given the critical role of MET in metastatic tumor formation, the potential to effectively target the MET process at sites of metastasis offers new hope for inhibiting metastatic tumor formation.     

MtDNA haplogroups M7 and B in southwestern Han Chinese at risk for acute mountain sickness

We conducted a case-control study to investigate the association of mitochondrial DNA (mtDNA) haplogroups with acute mountain sickness (AMS) in Han Chinese from southwestern (SW) China. Pearson's chi-square test or Fisher's exact test revealed significant reduction of mtDNA haplogroups D and M9, while a significant increase of haplogroup M7 in AMS subjects compared with non-AMS subjects. The multivariate logistic regression analysis after adjustment for body mass index (BMI), a risk factor of AMS in the present study, showed that both D and M9 were associated with significantly decreased risk of AMS, while M7 was associated with a significantly increased risk of AMS (OR=0.605, p=0.000; OR=0.037, p=0.001, and OR=2.419, p=0.001, respectively). In addition, further analysis stratified by the AMS severities indicated that haplogroup B was correlated with a 2.41-folds increased risk of developing severe AMS (95%C.I=1.288-4.514, p=0.006). Our findings provide evidence that, in SW Han Chinese, mtDNA haplogroups D and M9 are related to individual tolerance to AMS, while haplogroups M7 and B are risk factors for AMS.     

A missense mutation in PPARD causes a major QTL effect on ear size in pigs

Chinese Erhualian is the most prolific pig breed in the world. The breed exhibits exceptionally large and floppy ears. To identify genes underlying this typical feature, we previously performed a genome scan in a large scale White Duroc × Erhualian cross and mapped a major QTL for ear size to a 2-cM region on chromosome 7. We herein performed an identical-by-descent analysis that defined the QTL within a 750-kb region. Historically, the large-ear feature has been selected for the ancient sacrificial culture in Erhualian pigs. By using a selective sweep analysis, we then refined the critical region to a 630-kb interval containing 9 annotated genes. Four of the 9 genes are expressed in ear tissues of piglets. Of the 4 genes, PPARD stood out as the strongest candidate gene for its established role in skin homeostasis, cartilage development, and fat metabolism. No differential expression of PPARD was found in ear tissues at different growth stages between large-eared Erhualian and small-eared Duroc pigs. We further screened coding sequence variants in the PPARD gene and identified only one missense mutation (G32E) in a conserved functionally important domain. The protein-altering mutation showed perfect concordance (100%) with the QTL genotypes of all 19 founder animals segregating in the White Duroc × Erhualian cross and occurred at high frequencies exclusively in Chinese large-eared breeds. Moreover, the mutation is of functional significance; it mediates down-regulation of β-catenin and its target gene expression that is crucial for fat deposition in skin. Furthermore, the mutation was significantly associated with ear size across the experimental cross and diverse outbred populations. A worldwide survey of haplotype diversity revealed that the mutation event is of Chinese origin, likely after domestication. Taken together, we provide evidence that PPARD G32E is the variation underlying this major QTL.     

An improved approach for extraction and high-performance liquid chromatography analysis of paraquat in human plasma

A simple, sensitive, reliable and economical HPLC method for quantifying paraquat concentration in human plasma has been developed, using diethyl paraquat as an internal standard. The drugs were extracted from the sample and separated on Xtimate C18 column with a mobile phase of 15% acetonitrile in 0.1M orthophosphoric acid containing SDS (150 mg/l). The pH of the mobile phase was adjusted to 3 with triethylamine and the detection wavelength was 256 nm for both paraquat and the internal standard. The average extraction recoveries were 91.9%. Good linearity (R(2)=0.9984) was observed throughout the range of 0.02-10 μg/ml in 0.5 ml plasma. The overall accuracy of this method was 97.6-107.3% and the lower limit of detection was 0.01 μg/ml. The intra- and inter-day variations were lower than 3.65% and 2.64%, respectively. We used this method to examine the paraquat concentrations of 53 patients with acute paraquat intoxication of whom 26 (49.1%) survived. In conclusion, this method was suitable for quantification of paraquat plasma concentration in toxicological samples. It was helpful in both assessing the severity of intoxication and predicting the outcome of paraquat poisoning.     

Determination of unbound vismodegib (GDC-0449) concentration in human plasma using rapid equilibrium dialysis followed by solid phase extraction and high-performance liquid chromatography coupled to mass spectrometry

A rapid equilibrium dialysis (RED) assay followed by a solid phase extraction (SPE) high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of unbound vismodegib in human plasma was developed and validated. The equilibrium dialysis was carried out using 0.3 mL plasma samples in the single-use plate RED system at 37°C for 6h. The dialysis samples (0.1 mL) were extracted using a Strata-X-C 33u Polymeric Strong Cation SPE plate and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization (ESI) mass spectrometry. The standard curve, which ranged from 0.100 to 100 ng/mL for vismodegib, was fitted to a 1/x(2) weighted linear regression model. The lower limit of quantitation (LLOQ, 0.100 ng/mL) was sufficient to quantify unbound concentrations of vismodegib after dialysis. The intra-assay precision of the LC-MS/MS assay, based on the four analytical QC levels (LLOQ, low, medium and high), was within 7.7% CV and inter-assay precision was within 5.5% CV. The assay accuracy, expressed as %Bias, was within ±4.0% of the nominal concentration values. Extraction recovery of vismodegib was between 77.9 and 84.0%. The assay provides a means for accurate assessment of unbound vismodegib plasma concentrations in clinical studies.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Synthesis of 9‐(Heteroarylmethylidene)amino Derivatives of Homocamptothecin with Biological Activities

Six 9‐(heteroarylmethylidene)amino derivatives, 2a – 2f , of homocamptothecin were synthesized for the first time by total synthesis in 22 steps and biologically evaluated as inhibitors of topoisomerase I. Moreover, the antitumor activities of 2a – 2f against three human tumor cell lines, i.e., A‐549, MDA‐MB‐435, and HCT‐116, were determined and the results showed that compound 2c was the most active homocamptothecin derivative against the A‐549 (IC₅₀=0.046 μM ) and HTC‐116 tumor cells (IC₅₀=3.67 μM ), with a ca. 50 times higher activity than the reference drug topotecan (TPT) against the lung cancer cell line A‐549.