Proteomics of Trypanosoma evansi Infection in Rodents

Background: Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS).Methodology/Principal Findings: Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Conclusions/Significance: Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.

Characterization of viral proteins ofOryctes baculovirus and comparison between two geographical isolates

BacilliformOryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infectedOryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reportedOryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins stained positive for glycosylation. Comparison between the viral proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

An intelligent approach using support vector machines for monitoring and identification of faults on transmission systems

Power system disturbances are often caused by faults on transmission lines. When faults occur in a power system, the protective relays detect the fault and initiate tripping of appropriate circuit breakers, which isolate the affected part from the rest of the power system. Generally Extra High Voltage (EHV) transmission substations in power systems are connected with multiple transmission lines to neighboring substations. In some cases mal-operation of relays can happen under varying operating conditions, because of inappropriate coordination of relay settings. Due to these actions the power system margins for contingencies are decreasing. Hence, power system protective relaying reliability becomes increasingly important. In this paper an approach is presented using Support Vector Machine (SVM) as an intelligent tool for identifying the faulted line that is emanating from a substation and finding the distance from the substation. Results on 24-bus equivalent EHV system, part of Indian southern grid, are presented for illustration purpose. This approach is particularly important to avoid mal-operation of relays following a disturbance in the neighboring line connected to the same substation and assuring secure operation of the power systems.

Epidemiology of candidemia in Finland

Candida species are an important cause of nosocomial bloodstream infections in hospitalized patients worldwide, with associated high mortality, excess length of stay and costs. Main contributors to candidemias is profound immunosuppression due to serious underlying condition or intensive treatments leading to an increasing number of susceptible patients. The rank order of causative Candida species varies over time and in different geographic locations. The aim of this study was to obtain information on epidemiology of candidemia in Finland, to identify trends in incidence, causative species, and patient populations at risk. In order to reveal possible outbreaks and assess the value of one molecular typing method, restriction enzyme analysis (REA), in epidemiological study, we analyzed C. albicans bloodstream isolates in Uusimaa region in Southern Finland during eight years. The data from the National Infectious Disease Register were used to assess the incidence and epidemiological features of candidemia cases. In Helsinki University Central Hospital (HUCH) all patients with blood culture yielding any Candida spp. were identified from laboratory log-books and from Finnish Hospital Infection Program. All the patients with a stored blood culture isolate of C. albicans were identified through microbiology laboratory logbooks, and stored isolates were genotyped with REA in the National Institute for Health and Welfare (former KTL). The incidence of candidemia in Finland is globally relatively low, but increased between between 1990s and 2000s. The incidence was highest in males >65 years of age, but incidence rates for patients <1-15 years were lower during 2000s than during 1990s. In HUCH the incidence of candidemia remained low and constant during our 18 years of observation, but a significant shift in patient-populations at risk was observed, associated with patients treated in intensive care units, such as premature neonates and surgical patients. The predominating causative species in Finland and in HUCH is C. albicans, but the proportion of C. glabrata increased considerably. The crude one-month case fatality was constantly high between 28-33%. REA differentiated efficiently between C. albicans blood culture isolates and no clusters were observed in the hospitals involved, despite of abundant transfer of patients among them. Candida spp. are an important cause of nosocomial blood stream infections in Finland, and continued surveillance is necessary to determine the overall trends and patient groups at risk, and reduce the impact of these infections in the future. Molecular methods provide an efficient tool for investigation of suspected outbreak and should be available in the future in Finland, also.

Diverse pathways for salicin utilization in Shigella sonnei and Escherichia coli carrying an impaired bgl operon

Utilization of the aryl-beta-glucosides salicin or arbutin in most wild-type strains of E. coli is achieved by a single-step mutational activation of the bgl operon. Shigella sonnei, a branch of the diverse E. coli strain tree, requires two sequential mutational steps for achieving salicin utilization as the bglB gene, encoding the phospho-beta-glucosidase B, harbors an inactivating insertion. We show that in a natural isolate of S. sonnei, transcriptional activation of the gene SSO1595, encoding a phospho-beta-glucosidase, enables salicin utilization with the permease function being provided by the activated bgl operon. SSO1595 is absent in most commensal strains of E. coli, but is present in extra-intestinal pathogens as bgcA, a component of the bgc operon that enables beta-glucoside utilization at low temperature. Salicin utilization in an E. coli bglB laboratory strain also requires a two-step activation process leading to expression of BglF, the PTS-associated permease encoded by the bgl operon and AscB, the phospho-beta-glucosidase B encoded by the silent asc operon. BglF function is needed since AscF is unable to transport beta-glucosides as it lacks the IIA domain involved in phopho-relay. Activation of the asc operon in the Sal(+) mutant is by a promoter-up mutation and the activated operon is subject to induction. The pathway to achieve salicin utilization is therefore diverse in these two evolutionarily related organisms; however, both show cooperation between two silent genetic systems to achieve a new metabolic capability under selection.

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.

Berry phenolics: isolation, analysis, identification, and antioxidant properties

The main objectives in this thesis were to isolate and identify the phenolic compounds in wild (Sorbus aucuparia) and cultivated rowanberries, European cranberries (Vaccinium microcarpon), lingonberries (Vaccinium vitis-idaea), and cloudberries (Rubus chamaemorus), as well as to investigate the antioxidant activity of phenolics occurring in berries in food oxidation models. In addition, the storage stability of cloudberry ellagitannin isolate was studied. In wild and cultivated rowanberries, the main phenolic compounds were chlorogenic acids and neochlorogenic acids with increasing anthocyanin content depending on the crossing partners. The proanthocyanidin contents of cranberries and lingonberries were investigated, revealing that the lingonberry contained more rare A-type dimers than the European cranberry. The liquid chromatography mass spectrometry (LC-MS) analysis of cloudberry ellagitannins showed that trimeric lambertianin C and sanguiin H-10 were the main ellagitannins. The berries, rich in different types of phenolic compounds including hydroxycinnamic acids, proanthocyanidins, and ellagitannins, showed antioxidant activity toward lipid oxidation in liposome and emulsion oxidation models. All the different rowanberry cultivars prevented lipid oxidation in the same way, in spite of the differences in their phenolic composition. In terms of liposomes, rowanberries were slightly more effective antioxidants than cranberry and lingonberry phenolics. Greater differences were found when comparing proanthocyanidin fractions. Proanthocyanidin dimers and trimers of both cranberries and lingonberries were most potent in inhibiting lipid oxidation. Antioxidant activities and antiradical capacities were also studied with hydroxycinnamic acid glycosides. The sinapic acid derivatives of the hydroxycinnamic acid glycosides were the most effective at preventing lipid oxidation in emulsions and liposomes and scavenging radicals in DPPH assay. In liposomes and emulsions, the formation of the secondary oxidation product, hexanal, was inhibited more than that of the primary oxidation product, conjugated diene hydroperoxides, by hydroxycinnamic acid derivatives. This indicates that they are principally chain-breaking antioxidants rather than metal chelators, although they possess chelating activity as well. The storage stability test of cloudberry ellagitannins was performed by storing ellagitannin isolate and ellagitannins encapsulated with maltodextrin at different relative vapor pressures. The storage stability was enhanced by the encapsulation when higher molecular weight maltodextrin was used. The best preservation was achieved when the capsules were stored at 0 or 33% relative vapor pressures. In addition, the antioxidant activities of encapsulated cloudberry extracts were followed during the storage period. Different storage conditions did not alter the antioxidant activity, even though changes in the ellagitannin contents were seen. The current results may be of use in improving the oxidative stability of food products by using berries as natural antioxidants.

Development of a probe-based blotting technique for the detection of Tobacco streak virus

Digoxigenin (DIG)-labeled DNA probe was developed for a sensitive and rapid detection of the Tobacco streak virus (TSV) isolates in India by dot-blot and tissue print hybridization techniques. DIG-labeled DNA probe complementary to the coat protein (CP) region of TSV sunflower isolate was designed and used to detect the TSV presence at field levels. Dot-blot hybridization was used to check a large number of TSV isolates with a single probe. In addition, a sensitivity of the technique was examined with the different sample extraction methods. Another technique, the tissue blot hybridization offered a simple, reliable procedure and did not require a sample processing. Thus, both non-radioactively labeled probe techniques could facilitate the sample screening during TSV outbreaks and offer an advantage in quarantine services.

Diastereomeric Drug Glucuronides: Enzymatic Glucuronidation and Analysis by Capillary Electrophoresis and Liquid Chromatography-Mass Spectrometry

Foreign compounds, such as drugs are metabolised in the body in numerous reactions. Metabolic reactions are divided into phase I (functionalisation) and phase II (conjugation) reactions. Uridine diphosphoglucuronosyltransferase enzymes (UGTs) are important catalysts of phase II metabolic system. They catalyse the transfer of glucuronic acid to small lipophilic molecules and convert them to hydrophilic and polar glucuronides that are readily excreted from the body. Liver is the main site of drug metabolism. Many drugs are racemic mixtures of two enantiomers. Glucuronidation of a racemic compound yields a pair of diastereomeric glucuronides. Stereoisomers are interesting substrates in glucuronidation studies since some UGTs display stereoselectivity. Diastereomeric glucuronides of O-desmethyltramadol (M1) and entacapone were selected as model compounds in this work. The investigations of the thesis deal with enzymatic glucuronidation and the development of analytical methods for drug metabolites, particularly diastereomeric glucuronides. The glucuronides were analysed from complex biological matrices, such as urine or from in vitro incubation matrices. Various pretreatment techniques were needed to purify, concentrate and isolate the analytes of interest. Analyses were carried out by liquid chromatography (LC) with ultraviolet (UV) or mass spectrometric (MS) detection or with capillary electromigration techniques. Commercial glucuronide standards were not available for the studies. Enzyme-assisted synthesis with rat liver microsomes was therefore used to produce M1 glucuronides as reference compounds. The glucuronides were isolated by LC/UV and ultra performance liquid chromatography (UPLC)/MS, while tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed in structural characterisation. The glucuronides were identified as phenolic O-glucuronides of M1. To identify the active UGT enzymes in (±)-M1 glucuronidation recombinant human UGTs and human tissue microsomes were incubated with (±)-M1. The study revealed that several UGTs can catalyse (±)-M1 glucuronidation. Glucuronidation in human liver microsomes like in rat liver microsomes is stereoselective. The results of the studies showed that UGT2B7, most probably, is the main UGT responsible for (±)-M1 glucuronidation in human liver. Large variation in stereoselectivity of UGTs toward (±)-M1 enantiomers was observed. Formation of M1 glucuronides was monitored with a fast and selective UPLC/MS method. Capillary electromigration techniques are known for their high resolution power. A method that relied on capillary electrophoresis (CE) with UV detection was developed for the separation of tramadol and its free and glucuronidated metabolites. The suitability of the method to identify tramadol metabolites in an authentic urine samples was tested. Unaltered tramadol and four of its main metabolites were detected in the electropherogram. A micellar electrokinetic chromatography (MEKC) /UV method was developed for the separation of the glucuronides of entacapone in human urine. The validated method was tested in the analysis of urine samples of patients. The glucuronides of entacapone could be quantified after oral entacapone dosing.

Characterization of the genome of Oryctes baculovirus, a viral biocide of the insect pest Oryctes rhinoceros

Oryctes baculovirus is a viral biocide exploited for the control of the insect pest Oryctes rhinoceros. We have recently established a physical map of the genome of the Indian isolate of Oryctes baculovirus (OBV-KI). Here we examine the genomic relatedness between OBV-KI and OBV-PV505, the type isolate (originally from the Philippines), by DNA reassociation kinetics and by the use of restriction endonucleases. On the basis of differences in restriction-enzyme profiles between the two genomes, and previously reported differences in protein profiles and antigenic makeup, we propose the taxonomic status of a variant of Oryctes baculovirus for the Indian isolate.

Serotypic and genotypic characterization of human serotype 10 rotaviruses from asymptomatic neonates

Human rotaviruses were isolated from asymptomatic neonates at various hospitals and clinics in the city of Bangalore, India, and were found to be subgroup I specific and possess long RNA patterns (M. Sukumaran, K. Gowda, P. P. Maiya, T. P. Srinivas, M. S. Kumar, S. Aijaz, R. R. Reddy, L. Padilla, H. B. Greenberg, and C. D. Rao, Arch. Virol. 126:239-251, 1992). Three of these strains were adapted to tissue culture and found by serotype analysis and neutralization assays to be of serotype 10, a serotype commonly found in cattle but infrequently found in humans and not previously identified in neonates. By RNA-RNA hybridization, a high level of relatedness to a serotype 10 bovine rotavirus strain and a low-to-medium level of relatedness to a human rotavirus strain were observed. Since this human isolate shares a genogroup with bovine rotavirus, it is likely that it originated by interspecies transmission. A human rotavirus strain isolated from asymptomatic neonates and similar to bovine rotavirus might represent a good vaccine candidate.

Thermolabile ribonucleases from antarctic psychrotrophic bacteria: detection of the enzyme in various bacteria and purification from Pseudomonas fluorescens

Thirteen terrestrial psychrotrophic bacteria from Antarctica were screened for the presence of a thermolabile ribonuclease (RNAase-HL). The enzyme was detected in three isolates of Pseudomonas fluorescens and one isolate of Pseudomonas syringae. It was purified from one P. Fluorescens isolate and the molecular mass of the enzyme as determined by SDS-PAGE was 16 kDa. RNAase-HL exhibited optimum activity around 40 degrees C at pH 7.4. It could hydrolyse Escherichia coli RNA and the synthetic substrates poly(A), poly(C), poly(U) and poly(A-U). Unlike the crude RNAase from mesophilic P. Fluorescens and pure bovine pancreatic RNAase A which were active even at 65 degrees C, RNAase-HL was totally and irreversibly inactivated at 65 degrees C.

Differential influence of ions on the copy number of plasmids in Thiobacillus ferrooxidans

Thiobacillus ferrooxidans MAL4-1, an isolate from Malanjkhand copper mines, India, was adapted to grow in the presence of high concentration (30 gL(-1)) of Cu2+, resulting in a 15-fold increase in its tolerance to Cu2+. While wild-type T. ferrooxidans MAL4-1 contained multiple plasmids, cultures adapted to Cu2+ concentrations of 20 gL(-1) or more showed a drastic reduction in the copy number of the plasmids. The reduction for three of the plasmids was estimated to be over 50-fold. Examination of the plasmid profiles of the strains adapted to high concentration of SO42- anion (as Na2SO4 or ZnSO4) indicated that the reduction in plasmid copy number is not owing to SO42- anion, but is specific for Cu2+. The effect of mercury on the plasmids was similar to that of copper. Deadaptation of the Cu2+- Or Hg2+-adapted T. ferrooxidans resulted in restoration of the plasmids to the original level within the first passage. The fact that the plasmid copy number, in general, is drastically reduced in Cu2+-adapted T. ferrooxidans suggests that resistance to copper is chromosome mediated. This is the first report of a selective negative influence of copper ions on the copy number of plasmids in T. ferrooxidans.

Fuzzy logic intelligent system for gas turbine module and system fault isolation

A fuzzy logic intelligent system is developed for gas-turbine fault isolation. The gas path measurements used for fault isolation are exhaust gas temperature, low and high rotor speed, and fuel flow. These four measurements are also called the cockpit parameters and are typically found in almost all older and newer jet engines. The fuzzy logic system uses rules developed from a model of performance influence coefficients to isolate engine faults while accounting for uncertainty in gas path measurements. It automates the reasoning process of an experienced powerplant engineer. Tests with simulated data show that the fuzzy system isolates faults with an accuracy of 89% with only the four cockpit measurements. However, if additional pressure and temperature probes between the compressors and before the burner, which are often found in newer jet engines, are considered, the fault isolation accuracy rises to as high as 98%. In addition, the additional sensors are useful in keeping the fault isolation system robust as quality of the measured data deteriorates.

Generalized model for infrared perception from an engine exhaust

A comprehensive scheme has been developed for the prediction of radiation from engine exhaust and its incidence on an arbitrarily located sensor. Existing codes have been modified for the simulation of flows inside nozzles and jets. A novel view factor computation scheme has been applied for the determination of the radiosities of the discrete panels of a diffuse and gray nozzle surface. The narrowband model has been used to model the radiation from the gas inside the nozzle and the nonhomogeneous jet. The gas radiation from the nozzle inclusive of nozzle surface radiosities have been used as boundary conditions on the jet radiation. Geometric modeling techniques have been developed to identify and isolate nozzle surface panels and gas columns of the nozzle and jet to determine the radiation signals incident on the sensor. The scheme has been validated for intensity and heat flux predictions, and some useful results of practical importance have been generated to establish its viability for infrared signature analysis of jets.