In vitro resistance selections for Plasmodium falciparum dihydroorotate dehydrogenase inhibitors give mutants with multiple point mutations in the drug-binding site and altered growth.

Malaria is a preventable and treatable disease; yet half of the world's population lives at risk of infection, and an estimated 660,000 people die of malaria-related causes every year. Rising drug resistance threatens to make malaria untreatable, necessitating both the discovery of new antimalarial agents and the development of strategies to identify and suppress the emergence and spread of drug resistance. We focused on in-development dihydroorotate dehydrogenase (DHODH) inhibitors. Characterizing resistance pathways for antimalarial agents not yet in clinical use will increase our understanding of the potential for resistance. We identified resistance mechanisms of Plasmodium falciparum (Pf) DHODH inhibitors via in vitro resistance selections. We found 11 point mutations in the PfDHODH target. Target gene amplification and unknown mechanisms also contributed to resistance, albeit to a lesser extent. These mutant parasites were often hypersensitive to other PfDHODH inhibitors, which immediately suggested a novel combination therapy approach to preventing resistance. Indeed, a combination of wild-type and mutant-type selective inhibitors led to resistance far less often than either drug alone. The effects of point mutations in PfDHODH were corroborated with purified recombinant wild-type and mutant-type PfDHODH proteins, which showed the same trends in drug response as the cognate cell lines. Comparative growth assays demonstrated that two mutant parasites grew less robustly than their wild-type parent, and the purified protein of those mutants showed a decrease in catalytic efficiency, thereby suggesting a reason for the diminished growth rate. Co-crystallography of PfDHODH with three inhibitors suggested that hydrophobic interactions are important for drug binding and selectivity.

VKORC1 pharmacogenetics and pharmacoproteomics in patients on warfarin anticoagulant therapy: transthyretin precursor as a potential biomarker.

BACKGROUND: Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke. METHODS AND FINDING: We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation. CONCLUSION: This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism.

Palm fatty acid biodiesel: process optimization and study of reaction kinetics.

The relatively high cost of refined oils render the resulting fuels unable to compete with petroleum derived fuel. In this study, biodiesel is prepared from palm fatty acid (PFA) which is a by-product of palm oil refinery. The process conditions were optimized for production of palm fatty acid methyl esters. A maximum conversion of 94.4% was obtained using two step trans-esterification with 1:10 molar ratio of oil to methanol at 65°C. Sulfuric acid and Sodium hydroxide were used as acid and base catalyst respectively. The composition of fatty acid methyl esters (FAME) obtained was similar to that of palm oil. The biodiesel produced met the established specifications of biodiesel of American Society for Testing and Materials (ASTM). The kinetics of the trans-esterification reaction was also studied and the data reveals that the reaction is of first order in fatty acid and methanol (MeOH) and over all the reaction is of second order.

A prospective comparison of the platelet sequestration ability of three autotransfusion devices.

Although current autotransfusion devices have platelet sequestration capabilities, each has a unique technology to achieve the final platelet product. The purpose of this study was to assess the quality and quantity of platelets sequestered by three different autotransfusion devices. The three commercially available autotransfusion devices evaluated were Fresenius C.A.T.S (closed spiral chamber), Cobe BRAT 2 (Baylor bowl), and Haemonetic Cell Saver 5 (Latham bowl). Platelet sequestration was preformed in the automatic mode following the manufacturer's recommended sequestration protocols. The total number of platelets sequestered, percent recovery, and percent platelet function were assessed. Each device behaved similarly. There was a 2- to 3-fold increase in platelet count compared with baseline with only approximately 50-60% recovery, whereas there was approximately a 10% decrease in platelet function after processing compared with baseline. No statistical difference was noted in platelet function between the respective machines. However, there was a significant loss of platelet function observed with the actual process regardless of autotransfusion device used.

Quality of red blood cells using the Dideco Electa autotransfusion device.

The purpose of this study was to evaluate the quality of washed, concentrated red blood cells (RBCs) produced by the new Electa autotransfusion device from Cobe Cardiovascular (Dideco). Blood was collected intraoperatively in 16 patients undergoing cardiac surgery for whom routine cell savage was being used and then washed using the Electa. According to the manufacture's protocol. 125-mL bowls were used in the standard wash program. Reservoir and washed RBCs were analyzed for platelets (PLTs), leukocytes (WBCs), potassium (K+), and plasma-free hemoglobin (PFH) removal, as well as, hematocrit (Hct) and RBC recovery. The Electa cell saver produced a product with an average Hct of 58+/-5% and a RBC recovery rate of 87+/-10%. Its removal of waste products resulted in the washout of 54+/-18% WBCs, 87+/-6% PLTs, 91+/-4% K+, and 77+/-17% PFH. The Electa produces a good-quality washed RBC product that is comparable with other autotransfusion devices on the market.

Quality of red blood cells using autotransfusion devices: a comparative analysis.

Cell salvage devices are routinely used to process and wash red blood cells (RBCs) shed during surgical interventions. Although the principle theory of cell saving is the same, the actual process to achieve this is very different from one device to another. The purpose of this study was to compare the quality of washed, concentrated RBC produced by five very different cell-saving devices, specifically the Cobe BRAT 2, Medtronic Sequestra 1000, Haemonetics Cell Saver 5, Medtronic Autolog, and the Fresenius CATS. Reservoir and washed red blood cells were analyzed for hematocrit (Hct), platelets (PLT), leukocytes (WBC), potassium (K+), heparin, plasma-free hemoglobin (PFH), RBC mass recovery and recovery rate. The Haemonetics and BRAT 2 had the highest RBC recovery. All devices adequately removed heparin and potassium. The Medtronic Autolog had the highest removal of platelets and PFH; whereas, the BRAT had the lowest. Although the Autolog had the highest leukocyte removal, leukocytes were not adequately washed out by any of the autotransfusion devices. In conclusion, although all cell-saving devices use the same theory of centrifugation, the actual quality of the washed RBC product differs widely from one device to another.