N-Acyl and N-sulfonyloxazolidine-2,4-diones are pseudo-irreversible inhibitors of serine proteases

The synthesis, inhibitory activity and mode of action of oxazolidine-2,4-diones against porcine pancreatic elastase, here used as a model for human neutrophil elastase, are reported. The nature of N-substitution at the oxazolidine-2,4-dione scaffold has large effect on the inhibitory potency against elastase. N-Acyl and N-sulfonyloxazolidine-2,4-diones emerged as potent pseudo-irreversible inhibitors, displaying high second-order rate constants for PPE inactivation. The title compounds were also shown to be potent inhibitors of human neutrophil elastase (HNE) and proteinase-3, and weak inhibitors of human cathepsin G. The results herein presented show that the oxazolidine-2,4-diones represent a new promising class of serine protease inhibitors.     

Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: preliminary study

The production of recombinant proteins in the milk of non-transgenic goats can be achieved by transducing the mammary gland with recombinant adenoviral vectors. However, this process involves several regulatory issues. The current study evaluates the biosafety of this production system. We present a preliminary biosafety profile based on detection of adenoviral particles in different body fluids and the antibody response after adenoviral transduction of the goat mammary gland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenoviral particles were detected in the milk until day 4 after transduction, they were absent in serum, saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus inactivation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant proteins using adenoviral vectors.     

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.     

Engineered Mammalian Vector to Express EGFP-Tagged Proteins as Biomarkers

Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvβ3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvβ3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.     

Milk yield persistency in Brazilian Gyr cattle based on a random regression model

With the objective of evaluating measures of milk yield persistency, 27,000 test-day milk yield records from 3362 first lactations of Brazilian Gyr cows that calved between 1990 and 2007 were analyzed with a random regression model. Random, additive genetic and permanent environmental effects were modeled using Legendre polynomials of order 4 and 5, respectively. Residual variance was modeled using five classes. The average lactation curve was modeled using a fourth-order Legendre polynomial. Heritability estimates for measures of persistency ranged from 0.10 to 0.25. Genetic correlations between measures of persistency and 305-day milk yield (Y305) ranged from -0.52 to 0.03. At high selection intensities for persistency measures and Y305, few animals were selected in common. As the selection intensity for the two traits decreased, a higher percentage of animals were selected in common. The average predicted breeding values for Y305 according to year of birth of the cows had a substantial annual genetic gain. In contrast, no improvement in the average persistency breeding value was observed. We conclude that selection for total milk yield during lactation does not identify bulls or cows that are genetically superior in terms of milk yield persistency. A measure of persistency represented by the sum of deviations of estimated breeding value for days 31 to 280 in relation to estimated breeding value for day 30 should be preferred in genetic evaluations of this trait in the Gyr breed, since this measure showed a medium heritability and a genetic correlation with 305-day milk yield close to zero. In addition, this measure is more adequate at the time of peak lactation, which occurs between days 25 and 30 after calving in this breed.     

Molecular characterization of Corynebacterium pseudotuberculosis isolated from goats using ERIC-PCR

Corynebacterium pseudotuberculosis, the infectious agent of caseous lymphadenitis (CLA), is responsible for substantial economic losses in goat and sheep production. Molecular characterization of C. pseudotuberculosis isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR has shown promising results in genotyping strains isolated from sheep with CLA. We evaluated the genetic diversity of C. pseudotuberculosis isolates collected from the Sert?o region of the Pernambuco (PE) State, Brazil, and investigated the potential of ERIC-PCR as a tool for the molecular typing of strains of C. pseudotuberculosis isolated from goats. Thirty-two C. pseudotuberculosis strains isolated from goats in the municipalities of Floresta and Ibimirim, PE, C. pseudotuberculosis type strain ATCC 19410, the 1002 vaccine strain, and a field isolate of Rhodococcus equi were fingerprinted using the primers ERIC-1R and ERIC-2 and the primer pair ERIC- 1R+ERIC-2. Using 100% similarity as the cutoff, 8, 10, and 7 genotypes were obtained with ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively. The Hunter-Gaston discriminatory index calculated for the ERIC-1-PCR was 0.75. The index for the ERIC-2-PCR was 0.88, and the index for the ERIC-1+2-PCR was 0.79. Among goat isolates of C. pseudotuberculosis, three, two and four genotypes (found by ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively) had been previously described among sheep isolates from Minas Gerais State, Brazil. These results showed that ERIC-PCR has good discriminatory power and typeability, making it a useful tool for discrimination among C. pseudotuberculosis isolates from goats.     

Simultaneous determination of sulfamethoxazole and trimethoprim in biological fluids for high-throughput analysis: comparison of HPLC with ultraviolet and tandem mass spectrometric detection

The comparison of two methods based on online solid phase extraction-liquid chromatography with UV (SPE-LC-UV) or mass spectrometry detection (SPE-LC-MS/MS) for the simultaneous quantification of sulfamethoxazole (SMZ) and trimethoprim (TMP) is presented. The methods were validated and proved to be accurate. The analysis of standard samples for SMZ at concentrations of 0.5, 1.5, 25 and 50microg/mL demonstrated a relative standard deviation of less than 6% for both methods (n=18), while TMP samples at concentrations of 0.05, 0.15, 1.5 and 5.0microg/mL were analyzed with R.S.D. of less than 4% (n=18). The method with mass spectrometric detection was approximately six times more sensitive than the method with ultraviolet detection. The total run time for the SPE-LC-MS/MS was 2.5min per sample as opposed to 18.0min for the SPE-LC-UV method. The method with MS detection in comparison with UV detection proved to be more rugged and was successfully applied to pharmacokinetics studies.     

Protein identification pipeline for the homology-driven proteomics

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ Orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download.