Proceedings of the 2015 International Conference on Green Computing and Internet of Things, ICGCIoT 2015 | Year: 2015
Speaker recognition has made great progress under the laboratory environment, but in real life the performance of speaker recognition system is affected by various factors including environmental noise. This paper studies the performance of speaker recognition system in noisy environment and presents Speaker recognition system using Mel-Frequency Cepstral Coefficients (MFCC) technique based on different classifiers likes Euclidean distance, Back-Propagation Neural Network (BPNN), Self Organizing Map (SOM). This paper presents comparative plots of different classifier. Speaker recognition system based on SOM Neural Network classifier is provide better recognition rate compare to BPNN and Euclidean Distance based systems. © 2015 IEEE.
Proceedings of the 2015 International Conference on Green Computing and Internet of Things, ICGCIoT 2015 | Year: 2015
Speaker recognition has made great progress under the laboratory environment, but in real life the performance of speaker recognition system is affected by various factors including environmental noise. This paper studies the performance of speaker recognition system in noisy environment and presents Speaker recognition system using modified Mel-Frequency Cepstral Coefficients (MFCC) technique based on different classifiers likes Euclidean distance, Back-Propagation Neural Network (BPNN), Self Organizing Map (SOM). Modified Mel-Frequency Cepstral Coefficients (MFCC) technique includes Blackman windowing instead of hamming window. This paper presents comparative plots of different classifiers based on modified Mel-Frequency Cepstral Coefficients (MFCC) technique. Speaker recognition system based on SOM Neural Network classifier provides better recognition rate compare to BPNN and Euclidean Distance based systems. © 2015 IEEE.
Subhash P.,JITS Karimnagar |
2015 International Conference on Advances in Computing, Communications and Informatics, ICACCI 2015 | Year: 2015
Authenticated mesh peering exchange (AMPE) is one of the core functionalities of wireless mesh network(WMN) that facilitates mesh routers to discover their peers (neighbours), securely. Even though the AMPE protocol prevents unauthorized neighbours from becoming part of the network, it fails to prevent relay attacks, where an attacker can simply relay frames used to establish peer-links. The motivation of an attacker is to convince two far-away nodes as neighbours, and make them commit to a non-existent link that acts as a wormhole later. In this paper, we address this problem of relay attacks and propose a secure neighbour discovery mechanism that detects non-existent network links. It relies on a ranking mechanism to compute relative distance between neighbours, and employs connectivity information to validate those links. © 2015 IEEE.
News Article | October 25, 2016
Strains of P. falciparum (Dd2, 3D7, D6, K1, NF54, V1/3, HB3, 7G8, FCB and TM90C2B) were obtained from the Malaria Research and Reference Reagent Resource Center (MR4). PfscDHODH, the transgenic P. falciparum line expressing S. cerevisiae DHODH19, was a gift from A. B. Vaidya. P. falciparum isolates were maintained with O-positive human blood in an atmosphere of 93% N , 4% CO , 3% O at 37 °C in complete culturing medium (10.4 g l−1 RPMI 1640, 5.94 g l−1 HEPES, 5 g l−1 albumax II, 50 mg l−1 hypoxanthine, 2.1 g l−1 sodium bicarbonate, 10% human serum and 43 mg l−1 gentamicin). Parasites were cultured in medium until parasitaemia reached 3–8%. Parasitaemia was determined by checking at least 500 red blood cells from a Giemsa-stained blood smear. For the compound screening, a parasite dilution at 2.0% parasitaemia and 2.0% haematocrit was created with medium. 25 μl of medium was dispensed into 384-well black, clear-bottom plates and 100 nl of each compound in DMSO was transferred into assay plates along with the control compound (mefloquine). Next, 25 μl of the parasite suspension in medium was dispensed into the assay plates giving a final parasitaemia of 1% and a final haematocrit of 1%. The assay plates were incubated for 72 h at 37 °C. 10 μl of detection reagent consisting of 10× SYBR Green I (Invitrogen; supplied in 10,000× concentration) in lysis buffer (20 mM Tris-HCl, 5 mM EDTA, 0.16% (w/v) Saponin, 1.6% (v/v) Triton X-100) was dispensed into the assay plates. For optimal staining, the assay plates were left at room temperature for 24 h in the dark. The assay plates were read with 505 dichroic mirrors with 485 nm excitation and 530 nm emission settings in an Envision (PerkinElmer). High-throughput screening hits were hierarchically clustered by structural similarity using average linkage on pairwise Jaccard distances43 between ECFP4 fingerprints44. Pipeline Pilot45 was used for fingerprint and distance calculation; clustering and heat-map generation was done in R (ref. 46). HepG2 cells (ATCC) were maintained in DMEM, 10% (v/v) FBS (Sigma), and 1% (v/v) antibiotic–antimycotic in a standard tissue culture incubator (37 °C, 5% CO ). P. berghei (ANKA GFP–luc) infected A. stephensi mosquitoes were obtained from the New York University Langone Medical Center Insectary. For assays, ∼17,500 HepG2 cells per well were added to a 384-well microtitre plate in duplicate. After 18–24 h at 37 °C the media was exchanged and compounds were added. After 1 h, parasites obtained from freshly dissected mosquitoes were added to the plates (4,000 parasites per well), the plates were spun for 10 min at 1,000 r.p.m. and then incubated at 37 °C. The final assay volume was 30 μl. After a 48-h incubation at 37 °C, Bright-Glo (Promega) was added to the parasite plate to measure relative luminescence. The relative signal intensity of each plate was evaluated with an EnVision (PerkinElmer) system. Micropatterned co-culture (MPCC) is an in vitro co-culture system of primary human hepatocytes organized into colonies and surrounded by supportive stromal cells. Hepatocytes in this format maintain a functional phenotype for up to 4–6 weeks without proliferation, as assessed by major liver-specific functions and gene expression47, 48, 49. In brief, 96-well plates were coated homogenously with rat-tail type I collagen (50 μg ml−1) and subjected to soft-lithographic techniques to pattern the collagen into 500-μm-island microdomains that mediate selective hepatocyte adhesion. To create MPCCs, cryopreserved primary human hepatocytes (BioreclamationIVT) were pelleted by centrifugation at 100g for 6 min at 4 °C, assessed for viability using Trypan blue exclusion (typically 70–90%), and seeded on micropatterned collagen plates (each well contained ~10,000 hepatocytes organized into colonies of 500 μM) in serum-free DMEM with 1% penicillin–streptomycin. The cells were washed with serum-free DMEM with 1% penicillin–streptomycin 2–3 h later and replaced with human hepatocyte culture medium48. 3T3-J2 mouse embryonic fibroblasts were seeded (7,000 cells per well) 24 h after hepatocyte seeding. 3T3-J2 fibroblasts were courtesy of H. Green50. MPCCs were infected with 75,000 sporozoites (NF54) (Johns Hopkins University) 1 day after hepatocytes were seeded48, 49. After incubation at 37 °C and 5% CO for 3 h, wells were washed once with PBS, and the respective compounds were added. Cultures were dosed daily. Samples were fixed on day 3.5 after infection. For immunofluorescence staining, MPCCs were fixed with −20 °C methanol for 10 min at 4 °C, washed twice with PBS, blocked with 2% BSA in PBS, and incubated with mouse anti-P. falciparum Hsp70 antibodies (clone 4C9, 2 μg ml−1) for 1 h at room temperature. Samples were washed with PBS then incubated with Alexa 488-conjugated secondary goat anti-mouse for 1 h at room temperature. Samples were washed with PBS, counterstained with the DNA dye Hoechst 33258 (Invitrogen; 1:1,000), and mounted on glass slides with fluoromount G (Southern Biotech). Images were captured on a Nikon Eclipse Ti fluorescence microscope. Diameters of developing liver stage parasites were measured and used to calculate the corresponding area. All rhesus macaques (Macaca mulatta) used in this study were bred in captivity for research purposes, and were housed at the Biomedical Primate Research Centre (BPRC; AAALAC-certified institute) facilities under compliance with the Dutch law on animal experiments, European directive 86/609/EEC and with the ‘Standard for Humane Care and Use of Laboratory Animals by Foreign Institutions’ identification number A5539-01, provided by the Department of Health and Human Services of the US National Institutes of Health. The local independent ethical committee first approved all protocols. Non-randomized rhesus macaques (male or female; 5−14 years old; one animal per month) were infected with 1 × 106 P. cynomolgi (M strain) blood-stage parasites, and bled at peak parasitaemia. Approximately 300 female A. stephensi mosquitoes (Sind-Kasur strain, Nijmegen University Medical Centre St Radboud) were fed with this blood as described previously51. Rhesus monkey hepatocytes were isolated from liver lobes as described by previously52. Sporozoite infections were performed within 3 days of hepatocyte isolation. Sporozoite inoculation of primary rhesus monkey hepatocytes was performed as described previously53, 54. On day 6, intracellular P. cynomolgi malaria parasites were fixed, stained with purified rabbit antiserum reactive against P. cynomolgi Hsp70.1 (ref. 53), and visualized with FITC-labelled goat anti-rabbit IgG antibodies. Quantification of small ‘hypnozoite’ exoerythrocytic forms (1 nucleus, a small round shape, a maximal diameter of 7 μm) or large ‘developing parasite’ exoerythrocytic forms (more than 1 nucleus, larger than 7 μm and round or irregular shape) was determined for each well using a high-content imaging system (Operetta, PerkinElmer). P. falciparum 3D7 stage IV–V gametocytes were isolated by discontinuous Percoll gradient centrifugation of parasite cultures treated with 50 mM N-acetyl-d-glucosamine for 3 days to kill asexual parasites. Gametocytes (1.0 × 105) were seeded in 96-well plates and incubated with compounds for 72 h. In vitro anti-gametocyte activity was measured using CellTiter-Glo (Promega). A detailed description of the method is published elsewhere55. In brief, NF54pfs16-LUC-GFP highly synchronous gametocytes were induced from a single intra-erythrocytic asexual replication cycle. On day 0 of gametocyte development, spontaneously generated gametocytes were removed by VarioMACS magnetic column (MAC) technology. Early stage I gametocytes were collected on day 2 of development and late-stage gametocytes (stage IV) on day 8 using MAC columns. Percentage parasitaemia and haematocrit was adjusted to 10 and 0.1, respectively. 45 μl of parasite sample were added to PerkinElmer Cell carrier poly-d-lysine imaging plates containing 5 μl of test compound at 16 doses, including control wells containing 4% DMSO and 50 μM puromycin (0.4% and 5 μM final concentrations, respectively), the plates sealed with a membrane (Breatheasy or 4ti-05 15/ST) and incubated for 72 h in standard incubation conditions of 5% CO , 5% O , 90% N and 60% humidity at 37 °C. After incubation, 5 μl of 0.07 μg ml−1 MitoTracker Red CM-H2XRos (MTR) (Invitrogen) in PBS was added to each well, and plates were resealed with membranes and incubated overnight under standard conditions. The following day, the plates were brought to room temperature for at least one hour before being measured on the Opera QEHS Instrument. Image analysis was performed using an Acapella (PerkinElmer)-based algorithm that identifies gametocytes of the expected morphological shape with respect to degree of elongation and specifically those parasites that are determined as viable by the MitoTracker Red CM-H2XRos fluorescence size and intensity. IC values were determined using GraphPad Prism 4, using a 4-parameter log dose, nonlinear regression analysis, with sigmoidal dose–response (variable slope) curve fit. P. falciparum transmission-blocking activity of BRD7929 was assessed in a standard membrane feeding assay as previously described56. In brief, P. falciparumNF54 hsp70-GFP-luc reporter parasites were cultured up to stage V gametocytes (day 14). Test compounds were serially diluted in DMSO and subsequently in RPMI medium to reach a final DMSO concentration of 0.1%. Diluted compound was either pre-incubated with stage V gametocytes for 24 h (indirect mode) or directly added to the blood meal (direct mode). Gametocytes were adjusted to 50% haematocrit, 50% human serum and fed to A. stephensi mosquitoes. All compound dilutions were tested in duplicate in independent feeders. After 8 days, mosquitoes were collected and the relative decrease in oocysts density in the midgut was determined by measurement of luminescence signals in 24 individual mosquitoes from each cage. For each vehicle (control) cage, an additional 10 mosquitoes were dissected and examined by microscopy to determine the baseline oocyst intensity. In vitro resistance selections were performed as previously described15. In brief, approximately 1 × 109 P. falciparum Dd2 parasites were treated with 60 nM (EC ) or 150 nM (10 × EC ) of BRD1095 in each of four independent flasks for 3–4 days. After the compounds were removed, the cultures were maintained in compound-free complete RPMI growth medium with regular media exchange until healthy parasites reappeared. Once parasitaemia reached 2–4%, compound pressure was repeated and these steps were executed for about 2 months until the initial EC shift was observed. Three out of four independent selections pressured at 60 nM developed a phenotypic EC shift. None of the selections pressured at 150 nM resulted in resistant parasites. After an initial shift in the dose–response phenotype was observed, selection at an increased concentration was repeated in the same manner until at least a threefold shift in EC was observed. Selected parasites were then cloned by limiting dilution. BRD73842-resistant selections were conducted in a similar manner except that parasites were initially treated at 0.5 μM (10× EC ) for 4 days or 150 nM (EC ) for 2 days in each of two independent flasks. The Y1356N mutant was derived from a flask pressured at 0.5 μM and the L1418F mutant was developed from one of the flasks exposed to the 150 nM. DNA libraries were prepared for sequencing using the Illumina Nextera XT kit (Illumina), and quality-checked before sequencing on a Tapestation. Libraries were clustered and run as 100-bp paired-end reads on an Illumina HiSeq 2000 in RapidRun mode, according to the manufacturer’s instructions. Samples were analysed by aligning to the P. falciparum 3D7 reference genome (PlasmoDB v. 11.1). To identify SNVs and CNVs, a sequencing pipeline developed for P. falciparum (Plasmodium Type Uncovering Software, Platypus) was used as previously described, with the exception of an increase in the base quality filter from 196.5 to 1,000 (ref. 57). Substrate-dependent inhibition of recombinant P. falciparum DHODH protein was assessed in an in vitro assay in 384-well clear plates (Corning 3640) as described previously58. A 20-point dilution series of inhibitor concentrations were assayed against 2–10 nM protein with 500 μM l-dihydroorotate substrate (excess), 18 μM dodecylubiquinone electron acceptor (~K ), and 100 μM 2,6-dichloroindophenol indicator dye in assay buffer (100 mM HEPES pH 8.0, 150 mM NaCl, 5% glycerol, 0.5% Triton X-100). Assays were incubated at 25 °C for 20 min and then assessed via OD . Data were normalized to 1% DMSO and excess inhibitor (25 μM DSM265; ref. 7). Substrate-dependent inhibition of recombinant human DHODH protein was assessed in an in vitro assay in 384-well clear plates (Corning 3640) as described previously59. A 20-point dilution series of inhibitor concentrations was assayed against 13 nM protein with 1 mM l-dihydroorotate substrate (excess), 100 μM dodecylubiquinone electron acceptor, and 60 μM 2,6-dichloroindophenol indicator dye in assay buffer (50 mM Tris HCl pH 8.0, 150 mM KCl, 0.1% Triton X-100). Assays were incubated at 25 °C for 20 min and then assessed via OD . Data were normalized to 1% DMSO and no enzyme. The synthetic gene for full-length P. vivax PI4K (PVX_098050) was synthesized from GeneArt (ThermoScientific), and was expressed and purified as previously described20. Aliquots of P. vivax PI4Kβ were flash-frozen in liquid nitrogen and stored at −80°C. Full-length human PI4KB (uniprot gene Q9UBF8-2) was expressed and purified as previously described60. 100 nM extruded lipid vesicles were made to mimic Golgi organelle vesicles (20% phosphatidylinositol, 10% phosphatidylserine, 45% phosphatidylcholine and 25% phosphatidylethanolamine) in lipid buffer (20 mM HEPES pH 7.5 (room temperature), 100 mM KCl, 0.5 mM EDTA). Lipid kinase assays were carried out using the Transcreener ADP2 FI Assay (BellBrook Labs) following the published protocol as previously described61. 4-μl reactions ran at 21 °C for 30 min in a buffer containing 30 mM HEPES pH 7.5, 100 mM NaCl, 50 mM KCl, 5 mM MgCl , 0.25 mM EDTA, 0.4% (v/v) Triton X-100, 1 mM TCEP, 0.5 mg ml−1 Golgi-mimic vesicles and 10 μM ATP. P. vivax PI4Kβ was used at 7.5 nM and human PI4KB was used at 200 nM. Fluorescence intensity was measured using a Spectramax M5 plate reader with excitation at 590 nm and emission at 620 nm (20-nm bandwidth). IC values were calculated from triplicate inhibitor curves using GraphPad Prism software. The model was built using the SWISS-MODEL online resource62, 63, 64 and Prime65 (Schrödinger Release 2015-2: Prime, version 4.0, Schrödinger), with human PheRS (PDB accession 3L4G) as a template for P. falciparum PheRS (PlasmoDB Gene ID: PF3D7_0109800). The template was chosen based on highest sequence identity and similarity identified via PSI-BLAST. Target-template alignment was made using ProMod-II and validated with Prime STA. Coordinates from residues that were conserved between the target and the template were copied from the template to the model, and remaining sites were remodelled using segments from known structures. The side chains were then rebuilt, and the model was finally refined using a force field. Protein sequences of both α- (PF3D7_0109800) and β- (PF3D7_1104000) subunits of cytoplasmic P. falciparum PheRS were obtained from PlasmoDB ( http://plasmodb.org/plasmo/). Full length α- and β-subunit gene sequences optimized for expression in E. coli were cloned into pETM11 (Kanamycin resistance) and pETM20 (ampicillin resistance) expression vectors using Nco1 and Kpn1 sites and co-transformed into E. coli B834 cells. Protein expression was induced by addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and cells were grown until an OD of 0.6–0.8 was reached at 37 °C. They were then allowed to grow at 18 °C for 20 h after induction. Cells were separated by centrifugation at 5,000g for 20 min and the bacterial pellets were suspended in a buffer consisting of 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 4 mM β-mercaptoethanol, 15% (v/v) glycerol, 0.1 mg ml−1 lysozyme and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by sonication and cleared by centrifugation at 20,000g for 1 h. The supernatant was applied on to prepacked NiNTA column (GE Healthcare), and bound proteins were eluted by gradient-mixing with elution buffer (50 mM Tris–HCl (pH 7.5), 80 mM NaCl, 4 mM β-mercaptoethanol, 15% (v/v) glycerol, 1 M imidazole). Pure fractions were pooled and loaded on to heparin column for further purification. Again, bound proteins were eluted using gradient of heparin elution buffer 50 mM Tris–HCl (pH 7.5), 1 M NaCl, 4 mM β-mercaptoethanol, 15% (v/v) glycerol). Pure fractions were again pooled and dialysed overnight into a buffer containing 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 4 mM β-mercaptoethanol, 1 mM DTT and 0.5 mM EDTA. TEV protease (1:50 ratio of protease:protein) was added to the protein sample and incubated at 20 °C for 24 h to remove the polyhistidine tag. Protein was further purified via gel-filtration chromatography on a GE HiLoad 60/600 Superdex column in 50 mM Tris–HCl (pH 7.5), 200 mM NaCl, 4 mM β-mercaptoethanol, 1 mM MgCl . The eluted protein (a heterodimer of P. falciparum cPheRS) were collected, assessed for purity via SDS–PAGE and stored at −80 °C. Nuclear encoded tRNAPhe from P. falciparum was synthesized using an in vitro transcription method as described earlier22, 66. Aminoacylation and enzyme inhibition assays for P. falciparum cytosolic PheRS were performed as described earlier22, 67. Enzymatic assays were performed in buffer containing 30 mM HEPES (pH 7.5), 150 mM NaCl, 30 mM KCl, 50 mM MgCl , 1 mM DTT, 100 μM ATP, 100 μM l-phenylalanine, 15 μM P. falciparum tRNAPhe, 2 U ml−1 E. coli inorganic pyrophosphatase (NEB) and 500 nM recombinant P. falciparum PheRS at 3 °C. Reactions at different time points were stopped by the addition of 40 mM EDTA and subsequent transfer to ice. Recombinant maltose binding protein was used as negative control. The cPheRS inhibition assays were performed using inhibitor concentrations of 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 5 μM and 10 μM for strong binders and 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 500 μM for weaker binders in the assay buffer. Enzymatic and inhibition experiments were performed twice in triplicate. Mammalian cells (HepG2, A549, and HEK293) were obtained from the ATCC and cultured routinely in DMEM with 10% FBS and 1% (v/v) antibiotic–antimycotic. For cytotoxicity assays, 1 × 106 cells were seeded into 384-well plates 1 day before compound treatment. Cells were treated with ascending doses of compound for 72 h, and viability was measured using Cell-Titer Glo (Promega). All cell lines were tested for Mycoplasma contamination using Universal mycoplasma Detection Kit (ATCC). In vitro characterization assays (protein binding, microsomal stability, hepatocyte stability, cytochrome P450 (CYP) inhibition, and aqueous solubility) were performed according to industry-standard techniques. Ion channel inhibition studies were performed using the Q-Patch system using standard techniques. All animal experiments were conducted in compliance with institutional policies and appropriate regulations and were approved by the institutional animal care and use committees for each of the study sites (the Broad Institute, 0016-09-14; Harvard School of Public Health, 03228; Eisai, 13-05, 13-07, 14-C-0027). No method of randomization or blinding was used in this study. CD-1 mice (n = 4 per experimental group; female; 6–7-week-old; 20–24 g, Charles River) were intravenously inoculated with approximately 1 × 105 P. berghei (ANKA GFP-luc) blood-stage parasites 24 h before treatment and compounds were administered orally (at 0 h). Parasitaemia was monitored by the in vivo imaging system (IVIS SpectrumCT, Perkinelmer) to acquire the bioluminescence signal (150 mg kg-1 of luciferin was intraperitoneally injected approximately 10 min before imaging). In addition, blood smear samples were obtained from each mouse periodically, stained with Giemsa, and viewed under a microscope for visual detection of blood parasitaemia. Animals with parasitaemia exceeding 25% were humanely euthanized. CD-1 mice (n = 4 per experimental group; female; 6–7-week-old; 20–24 g, Charles River) were inoculated intravenously with approximately 1 × 105 P. berghei (ANKA GFP-luc) sporozoites freshly dissected from A. stephensi mosquitoes. Immediately after infection, the mice were treated with single oral doses of BRD7929; infection was monitored as described for the P. berghei erythrocytic-stage assay. For time-course experiments, the time of compound treatment (single oral dose of 10 mg kg−1) was varied from 5 days before infection to 2 days after infection. CD-1 (n = 3 per experimental group; female; 6–7-week-old; 21–24 g, Charles River) mice were infected with P. berghei (ANKA GFP-luc) for 96 h before treatment with vehicle or BRD7929 (day 0). On day 2, female A. stephensi mosquitoes were allowed to feed on the mice for 20 min. After 1 week (day 9), the midguts of the mosquitoes were dissected out and oocysts were enumerated microscopically (12.5× magnification). In vivo adapted P. falciparum (3D7HLH/BRD) were selected as described previously68. In brief, NSG mice (n = 2 per experimental group; female; 4–5-week-old; 19–21 g; The Jackson Laboratory) were intraperitoneally injected with 1 ml of human erythrocytes (O-positive, 50% haematocrit, 50% RPMI 1640 with 5% albumax) daily to generate mice with humanized circulating erythrocytes (huRBC NSG). Approximately 2 × 107 blood-stage P. falciparum 3D7HLH/BRD (ref. 69) were intravenously infected to huRBC NSG mice and >1% parasitaemia was achieved 5 weeks after infection. After three in vivo passages, the parasites were frozen and used experimentally. Approximately 48 h after infection with 1 × 107 blood-stage of P. falciparum 3D7HLH/BRD, the mean parasitaemia was approximately 0.4%. huRBC NSG mice were orally treated with a single dose of compound and parasitaemia was monitored for 30 days by IVIS to acquire the bioluminescence signal (150 mg kg-1 of luciferin was intraperitoneally injected approximately 10 min before imaging). huRBC NSG mice (n = 2 per experimental group; female; 4–5-week-old; 18–20 g; Jackson Laboratory) were infected with blood-stage P. falciparum 3D7HLH/BRD for 2 weeks to allow the development of mature gametocytes. Subsequently, the mice were treated with a single oral dose of BRD7929. Blood samples were collected for 11 days. For molecular detection of parasite stages, 40 μl of blood was obtained from control and treated mice. In brief, total RNA was isolated from blood samples using RNeasy Plus Kit with genomic DNA eliminator columns (Qiagen). First-strand cDNA synthesis was performed from extracted RNA using SuperScript III First-Strand Synthesis System (Life Technologies). Parasite stages were quantified using a stage-specific qRT–PCR assay as described previously33, 69. Primers were designed to measure transcript levels of PF3D7_0501300 (ring stage parasites), PF3D7_1477700 (immature gametocytes) and PF3D7_1031000 (mature gametocytes). Primers for PF3D7_1120200 (P. falciparum UCE) transcript were used as a constitutively expressed parasite marker. The assay was performed using cDNA in a total reaction volume of 20 μl, containing primers for each gene at a final concentration of 250 nM. Amplification was performed on a Viia7 qRT–PCR machine (Life Technologies) using SYBR Green Master Mix (Applied Biosystems) with the following reaction conditions: 1 cycle × 10 min at 95 °C and 40 cycles × 1 s at 95 °C and 20 s at 60 °C. Each cDNA sample was run in triplicate and the mean C value was used for the analysis. C values obtained above the cut-off (negative control) for each marker were considered negative for the presence of specific transcripts. Blood samples from each mouse before parasite inoculation were also tested for ‘background noise’ using the same primer sets. No amplification was detected from any samples. FRG knockout on C57BL/6 (human repopulated, >70%) mice (huHep FRG knockout; n = 2 per experimental group; female; 5.5–6-month-old; 19–21 g; Yecuris) were inoculated intravenously with approximately 1 × 105 P. falciparum (NF54HT-GFP-luc) sporozoites and BRD7929 was administered as a single 10 mg kg−1 oral dose one day after inoculation31. Infection was monitored daily by IVIS. Daily engraftment of human erythrocytes (0.4 ml, O-positive, 50% haematocrit, 50% RPMI 1640 with 5% albumax) was initiated 5 days after inoculation. For qPCR analysis, blood samples (40 μl) were collected 7 days after inoculation. For molecular detection of the blood-stage parasite, 40 μl of blood was obtained from control and treated mice. In brief, total RNA was isolated from blood samples using RNeasy Plus Kit with genomic DNA eliminator columns (Qiagen). First-strand cDNA synthesis was performed from extracted RNA using SuperScript III First-Strand Synthesis System (Life Technologies). The presence of the blood-stage parasites was quantified using a highly stage-specific qRT–PCR assay as described previously33, 70. Primers were designed to measure transcript levels of PF3D7_1120200 (P. falciparum UCE). The assay was performed using cDNA in a 20 μl total reaction volume containing primers for each gene at a final concentration of 250 nM. Amplification was performed on a Viia7 qRT–PCR machine (Life Technologies) using SYBR Green Master Mix (Applied Biosystems) and the reaction conditions are as follows: 1 cycle × 10 min at 95 °C and 40 cycles × 1 s at 95 °C and 20 s at 60 °C. Each cDNA sample was run in triplicate and the mean C value was used for the analysis. C values obtained above the cut-off (negative control) for each marker were considered negative for presence of specific transcripts. Blood samples from each mouse were also tested for background noise using the same primer sets before parasite inoculation. No amplification was detected from any samples. In vitro cultures of P. falciparum Dd2, with the initial inocula ranging from 105 to 109 parasites, were maintained in complete medium supplemented with 20 nM of BRD7929 (EC against Dd2). Media was replaced with fresh compound added daily and cultures monitored for 60 days to identify propensity for recrudescent parasitaemia as described34. Atovaquone was used as a control (EC = 2 nM). Solubility was determined in PBS pH 7.4 with 1% DMSO. Each compound was prepared in triplicate at 100 μM in both 100% DMSO and PBS with 1% DMSO. Compounds were allowed to equilibrate at room temperature with a 750 r.p.m. vortex shake for 18 h. After equilibration, samples were analysed by UPLC–MS (Waters) with compounds detected by single-ion reaction detection on a single quadrupole mass spectrometer. The DMSO samples were used to create a two-point calibration curve to which the response in PBS was fit. Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology) for both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 μl) with PBS pH 7.4 added to the other side (350 μl). Compounds were incubated at 37 °C for 5 h with 350 r.p.m. orbital shaking. After incubation, samples were analysed by UPLC–MS (Waters) with compounds detected by SIR detection on a single quadrupole mass spectrometer. The required potency to inhibit the hERG channel in expressed cell lines were evaluated using an automated patch-clamp system (QPatch-HTX). Pharmacokinetics of BRD3444 and BRD1095 were performed by Shanghai ChemPartner Co. Ltd., following single intravenous and oral administrations to female CD-1 mice. BRD3444 and BRD1095 were formulated in 70% PEG400 and 30% aqueous glucose (5% in H O) for intravenous and oral dosing. Test compounds were dosed as a bolus solution intravenously at 0.6 mg kg−1 (dosing solution; 70% PEG400 and 30% aqueous glucose, 5% in H O) or dosed orally by gavage as a solution at 1 mg kg−1 (dosing solution; 70% PEG400 and 30% aqueous glucose, 5% in H O) to female CD-1 mice (n = 9 per dose route). Pharmacokinetic parameters of BRD7929 and BRD3316 were determined in CD-1 mice. BRD7929 and BRD3316 were formulated in 10% ethanol, 4% Tween, 86% saline for both intravenous and oral dosing. Pharmacokinetic parameters were estimated by non-compartmental model using WinNonlin 6.2. Pharmacokinetic parameters for BRD7929 and BRD3316 were estimated by a non-compartmental model using proprietary Eisai software. Pharmacokinetic parameters of BRD7539 and BRD9185 were determined in CD-1 mice. Compounds were formulated in 70% PEG300 and 30% (5% glucose in H O) at 0.5 mg ml−1 for oral dosing, and 5% DMSO, 10% cremophor, and 85% H O at 0.25 mg ml−1 for intravenous formulation. Pharmacokinetic parameters were estimated by non-compartmental model using WinNonlin 6.2. Pharmacokinetics of BRD7539 and BRD9185 were performed by WuXi AppTec. The protocol was approved by Eisai IACUC, 13-07, 13, 05, and 14-c-0027. Compounds were evaluated in vitro to determine their metabolic stability in incubations containing liver microsomes or hepatocytes of mouse and human. In the presence of NADPH, liver microsomes (0.2 mg ml−1) from mouse (CD-1) and human were incubated with compounds (0.5 and 5 μM) for 0, 10 and 90 min. The depletion of compounds in the incubation mixtures, determined using liquid chromatography tandem mass spectromety LC–MS/MS, was used to estimate K and V values and determine half-lives for both mouse and human microsomes. Compounds were evaluated in vitro for the potential inhibition of human cytochrome P450 (CYP) isoforms using human liver microsomes. Two concentrations (1 and 10 μM) of compound were incubated with pooled liver microsomes (0.2 mg ml−1) and a cocktail mixture of probe substrates for selective CYP isoform. The selective activities tested were CYP1A2-mediated phenacetin O-demethylation, CYP2C8-mediated rosiglitazone para-hydroxylation, CYP2C9-mediated tolbutamide 4′-hydroxylation, CYP2C19-mediated (S)-mephenytoin 4′-hydroxylation, CYP2D6-mediated (±)-bufuralol 1′-hydroxylation and, CYP3A4/5-mediated midazolam 1′-hydroxylation. The positive controls tested were α-naphthoflavone for CYP1A2, montelukast for CYP2C8, sulfaphenazole for CYP2C9, tranylcypromine for CYP2C19, quinidine for CYP2D6, and ketoconazole for CYP3A4/5. The samples were analysed by LC–MS/MS. IC values were estimated using nonlinear regression. The time-dependent inactivation potential of compounds were assessed in human liver microsomes for CYP2C9, CYP2D6, and CYP3A4/5 by determining K and k values when appropriate. Two concentrations (6 and 30 μM) of compound were incubated in primary reaction mixtures containing phosphate buffer and 0.2 mg ml−1 human liver microsomes for 0, 5, and 30 min in a 37 °C water bath. The reactions were initiated by the addition of NADPH. Phosphate buffer was substituted for NADPH solution for control. At the respective times, 25 μl of primary incubation was diluted tenfold into pre-incubated secondary incubation mixture containing each CYP-selective probe substrate in order to assess residual activity. The second incubation time was 10 min. The probe substrates used for CYP1A, 2C9, CYP2C19, CYP2D6, and CYP3A4 were phenacetin (50 μM), tolbutamide (500 μM), (S)-mephenytoin (20 μM), bufuralol (50 μM), and midazolam (30 μM), respectively. The CYP time- dependent inhibitors used were furafyllin, tienilic acid, ticlopidine, paroxetin and troleandomycin for CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, at two concentrations. The samples were analysed by LC–MS/MS.
Hota P.K.,UCE |
Barisal A.K.,UCE |
Chakrabarti R.,Jadavpur University
International Journal of Electrical Power and Energy Systems | Year: 2010
This paper presents a newly developed optimization approach involving a modified bacterial foraging algorithm (MBFA) applied for the solution of the economic and emission load dispatch (EELD) problem. The approach utilizes the natural selection of global optimum bacterium having successful foraging strategies in the fitness function. The bacterial foraging algorithm (BFA) appears to be a robust and reliable optimization algorithm for the solution of the EELD problems. To obtain the best compromising solution a fuzzy decision making approach using MBFA is applied to the standard IEEE 30-bus six generator test system and a Taiwan power system of 40 generating units with valve point loading effects. The results confirm the potential and effectiveness of the proposed algorithm compared to various methods such as, linear programming (LP), multi-objective stochastic search technique (MOSST), differential evolution (DE), non-dominated sorting genetic algorithm (NSGA), niched pareto genetic algorithm (NPGA), strength pareto evolutionary algorithm (SPEA) and fuzzy clustering based particle swarm optimization (FCPSO) performed in different central load dispatch centers to solve EELD problems. The quality and usefulness of the proposed algorithm is demonstrated through its application to two standard test systems in comparison with the other existing techniques. The current proposal was found to be better than, or at least comparable to them considering the quality of the solutions obtained. © 2010 Elsevier Ltd. All rights reserved.
Suvarchala P.V.L.,Research Scholar |
Kumar S.S.,UCE |
Mohan B.C.,P.A. College
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2013
Iris recognition is the most reliable and dependable biometric system as the features of human eye are invariant and distinctive for every individual. Present iris recognition algorithms are tested using the bench mark databases which are assumed to be almost ideal except for eyelid and eyelash occlusions and rotational inconsistencies. It has been discussed elaborately by Daugman in  that, non-ideal imaging conditions affect the "authentics" distribution in the decision environment graph. Getting motivation from this observation, all possible non-ideal imaging conditions and Charge Coupled Device (CCD) noise are simulated and applied on the available databases. Legendre moments, introduced by Teague can achieve translation and scale invariance and also, close to zero value of redundancy measure, so that the moments correspond to distinct and autonomous features of the image. In the proposed method it is proved that, they can also work very well on noise affected features when trained and tested using SVMs. The performance of the Exact Legendre Moments (ELM) on UBIRIS and CASIA datasets proves to be very good with Correct Recognition Rate (CRR) = 99.6% under non-ideal imaging conditions and CCD noise. © Springer-Verlag 2013.
Srikala K.,JNTUH College of Engineering |
Global Conference on Communication Technologies, GCCT 2015 | Year: 2015
Computational grids have the potential for solving large-scale and scientific problems using geographically distributed and heterogeneous resources. In addition to the challenges of managing and scheduling resources reliable challenges arise because the grid infrastructure is unreliable. There are two major problems in Scheduling the Grid 1) Efficient Scheduling of jobs 2) Providing fault tolerance in a reliable manner. Most of the existing strategies do not provide fault tolerance for scheduling the workflows. There are some algorithms which provide fault tolerance but, they do a significant measure of redundant computation to provide fault tolerance. This paper addresses this issue and reduces the redundant work by using a group level table of data. This technique is suitable for workflow of jobs. © 2015 IEEE.
Patil G.M.,P.A. College |
Subba Rao K.,UCE |
Niranjan U.C.,MIT |
Journal of Mechanics in Medicine and Biology | Year: 2010
This paper presents a new approach in the field of electrocardiogram (ECG) feature extraction system based on the discrete wavelet transform (DWT) coefficients using Daubechies Wavelets. Real ECG signals recorded in lead II configuration are chosen for processing. The ECG signal was acquired by a battery operated, portable ECG data acquisition and signal processing module. In the second step the ECG signal was denoised using soft thresholding with Symlet4 wavelet. Further denoising was achieved by removing the corresponding wavelet coefficients at higher levels of decomposition. Later the ECG data files were converted to .txt files and subsequently to. mat files before being imported into the Matlab 7.4.0 environment for the computation of the decomposition coefficients. The QRS complexes were grouped as normal or myocardial ischaemic ones based on these decomposition coefficients. The algorithm developed by us was evaluated with control database comprising 120 records and validated using 60 records making up test database. By using the DWT coefficients, we have successfully achieved the myocardial ischaemia detection rates up to 97.5% with the technique developed by us for control data and up to 100% for validation test data. © 2010 World Scientific Publishing Company.
Radhika K.,CBIT |
Venugopal Reddy A.,UCE
IET Conference Publications | Year: 2012
Next generation wireless networks are evolving towards the integration of various wireless networks to provide high bandwidth and QoS support for real-time multimedia applications to mobile users in a seamless manner. Vertical Handoff Decision (VHD) problem is one of the crucial design issues that need to be resolved in order to provide seamless mobility in heterogeneous wireless environment. In this paper, we present two models to solve VHD problem based on Fuzzy MCDM and Game Theory approaches. Further we analyze the sensitivity of these models and propose an AHP based analytical model to evaluate their performance in terms of a set of decision criteria including number of parameters, user preference, scalability and elimination of unnecessary handoffs.
Mallaiah K.,UCE |
Ramachandram S.,UCE |
Proceedings - 4th IEEE International Conference on Computer and Communication Technology, ICCCT 2013 | Year: 2013
Encrypting Credit card numbers, Social Security Numbers in huge legacy databases has become a very complex task as encrypted data normally increases in size and changes its format, with traditional encryption algorithms. A number of cryptographic methods have been introduced to provide security to credit card numbers. But, a major problem in adopting these cryptographic algorithms is the requirement to change the existing schema and underlying applications to incorporate the encrypted data. Format Preserving Encryption is a solution to the above mentioned problem, wherein the encrypted data 'fits' into the existing schema. In this paper, we are analyzing the performance of Format Preserving Encryption (FIPS 74-8) over block ciphers such as AES, DES, 3DES, Blowfish on numeric data (credit cards numbers) with different keys. It has been observed that FPE (FIPS 74-8) over AES with 192 bits key gives better performance with 16198.5ns for an average encryption and decryption time of 1000 credit card numbers. Performances of Blowfish and AES with Key size 192 are neck to neck with respect to 1000 sixteen digits credit card numbers. We have also discussed overhead of FPE (FIPS-74-8). FIPS 74-8 was a NIST standard based on DES. Instead of using a block cipher DES, use of AES or Blowfish will give better performance and security for Format preserving of numeric data. © 2013 IEEE.