Sample records for brucei rhodesiense diagnostic

  1. A search for Trypanosoma brucei rhodesiense diagnostic antigens by proteomic screening and targeted cloning.

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    Theresa Manful

    Full Text Available BACKGROUND: The only available diagnostic method for East African trypanosomiasis is light microscopy of blood samples. A simple immunodiagnostic would greatly aid trypanosomiasis control. METHODOLOGY AND PRINCIPAL FINDINGS: To find trypanosome proteins that are specifically recognised by sera from human sleeping sickness patients, we have screened the Trypanosoma brucei brucei proteome by Western blotting. Using cytosolic, cytoskeletal and glycosomal fractions, we found that the vast majority of abundant trypanosome proteins is not specifically recognised by patient sera. We identified phosphoglycerate kinase (PGKC, heat shock protein (HSP70, and histones H2B and H3 as possible candidate diagnostic antigens. These proteins, plus paraflagellar rod protein 1, rhodesain (a cysteine protease, and an extracellular fragment of the Trypanosoma brucei nucleoside transporter TbNT10, were expressed in E. coli and tested for reactivity with patient and control sera. Only TbHSP70 was preferentially recognized by patient sera, but the sensitivity and specificity were insufficient for use of TbHSP70 alone as a diagnostic. Immunoprecipitation using a native protein extract revealed no specifically reacting proteins. CONCLUSIONS: No abundant T. brucei soluble, glycosomal or cytoskeletal protein is likely to be useful in diagnosis. To find useful diagnostic antigens it will therefore be necessary to use more sophisticated proteomic methods, or to test a very large panel of candidate proteins.

  2. Localization of serum resistance-associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei. (United States)

    Bart, Jean-Mathieu; Cordon-Obras, Carlos; Vidal, Isabel; Reed, Jennifer; Perez-Pastrana, Esperanza; Cuevas, Laureano; Field, Mark C; Carrington, Mark; Navarro, Miguel


    African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.

  3. Population genetics of Trypanosoma brucei rhodesiense: clonality and diversity within and between foci.

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    Craig W Duffy


    Full Text Available African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda and Southern (Malawi Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.

  4. Immunospecific immunoglobulins and IL-10 as markers for Trypanosoma brucei rhodesiense late stage disease in experimentally infected vervet monkeys

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    Ngotho, Maina; Kagira, J.M.; Jensen, Henrik Michael Elvang


    OBJECTIVE: To determine the usefulness of IL-10 and immunoglobulin M (IgM) as biomarkers for staging HAT in vervet monkeys, a useful pathogenesis model for humans. METHODS: Vervet monkeys were infected with Trypanosoma brucei rhodesiense and subsequently given sub-curative and curative treatment 28...

  5. Loop-mediated isothermal amplification (LAMP method for rapid detection of Trypanosoma brucei rhodesiense.

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    Zablon Kithinji Njiru

    Full Text Available Loop-mediated isothermal amplification (LAMP of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62 degrees C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml and 0.1 pg (1 trypanosome/ml using heat-treated buffy coat, while the detection limit for conventional SRA PCR was approximately 1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.

  6. Trypanosoma brucei rhodesiense infection in a German traveller returning from the Masai Mara area, Kenya, January 2012. (United States)

    Wolf, T; Wichelhaus, T; Gottig, S; Kleine, C; Brodt, H R; Just-Nuebling, G


    In January 2012, a case of Human African Trypanosomiasis (HAT) has been identified in Germany in a traveller returning from the Masai Mara area in Kenya. The 62-year-old man had travelled to the Masai Mara game park from 18 to 19 January 2012 and developed fever on 28 January. The infection with Trypanosoma brucei rhodesiense was confirmed by laboratory testing three days hereafter.

  7. Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populations and their cross-resistance. (United States)

    Bernhard, Sonja C; Nerima, Barbara; Mäser, Pascal; Brun, Reto


    Resistance to melarsoprol and pentamidine was induced in bloodstream-form Trypanosoma brucei rhodesiense STIB 900 in vitro, and drug sensitivity was determined for melarsoprol, pentamidine and furamidine. The resistant populations were also inoculated into immunosuppressed mice to verify infectivity and to monitor whether rodent passage selects for clones with altered drug sensitivity. After proliferation in the mouse, trypanosomes were isolated and their IC(50) values to the three drugs were determined. To assess the stability of drug-induced resistance, drug pressure was ceased for 2 months and the drug sensitivity was determined again. Resistance was stable, with a few exceptions that are discussed. Drug IC(50)s indicated cross-resistance among all drugs, but to varying extents: resistance of the melarsoprol-selected and pentamidine-selected trypanosomes to pentamidine was the same, but the pentamidine-selected trypanosome population showed lower resistance to melarsoprol than the melarsoprol-selected trypanosomes. Interestingly, both resistant populations revealed the same intermediate cross-resistance to furamidine. Resistant trypanosome populations were characterised by molecular means, referring to the status of the TbAT1 gene. The melarsoprol-selected population apparently had lost TbAT1, whereas in the pentamidine-selected trypanosome population it was still present.

  8. Complete coding sequence, sequence analysis and transmembrane topology modelling of Trypanosoma brucei rhodesiense putative oligosaccharyl transferase (TbOST II). (United States)

    Baticados, Waren N; Inoue, Noboru; Sugimoto, Chihiro; Nagasawa, Hideyuki; Baticados, Abigail M


    The partial nucleotide sequence of putative Trypanosoma brucei rhodesiense oligosaccharyl transferase gene was previously reported. Here, we describe the determination of its full-length nucleotide sequence by Inverse PCR (IPCR), subsequent biological sequence analysis and transmembrane topology modelling. The full-length DNA sequence has an Open Reading Frame (ORF) of 2406 bp and encodes a polypeptide of 801 amino acid residues. Protein and DNA sequence analyses revealed that homologues within the genome of other kinetoplastid and various origins exist. Protein topology analysis predicted that Trypanosoma brucei rhodesiense putative oligosaccharyl transferase clone II (TbOST II) is a transmembrane protein with transmembrane helices in probably an N(cytosol)-C(cytosol) orientation. Data from the GenBank database assembly and sequence analyses in general clearly state that TbOST II is the STT3 subunit of OST in T.b. rhodesiense that necessitates further characterisation and functional studies with RNAi. TbOST II sequence had been deposited in the GenBank (accession number GU245937).

  9. Coenzyme Q10 prevented full blown splenomegaly and decreased melarsoprol-induced reactive encephalopathy in mice infected with Trypanosoma brucei rhodesiense

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    James Nyabuga Nyariki; John Kibuthu Thuita; Grace Kemunto Nyambati; Alfred Orina Isaac


    Objective: To establish the modulatory effects of coenzyme Q10 on experimental trypanosome infections in mice and evaluate the risk of occurrence and severity of melarsoprol-induced post treatment reactive encephalopathy (PTRE). Methods: Female Swiss white mice were orally administered with 200 mg/kg of coenzyme Q10 after which they were intraperitoneally inoculated with Trypanasoma brucei rhodesiense (T. b. rhodesiense). The resultant infection was allowed to develop and simulate all phases of human African trypanosomiasis and PTRE. Parasitaemia development, packed cell volume, haematological and pathological changes were determined. Results:A histological study in the brain tissue of T. b. rhodesiense infected mice demonstrated neuroinflammatory pathology which was highly amplified in the PTRE-induced groups. A prominent reduction in the severity of the neuroinflammatory response was detected when coenzyme-Q10 was administered. Furthermore, the mean tissue weight of spleen to body ratio in coenzyme Q10 supplemented group was significantly (P Conclusions: The capacity of coenzyme Q10 to alter the pathogenesis of T. b. rhodesiense infection in mice and following treatment with melarsoprol, may find application by rendering humans and animals less susceptible to deleterious effects of trypanosome infection such as splenomegaly and melarsoprol-induced PTRE and neurotoxicity.

  10. Analysis of risk factors for T. brucei rhodesiense sleeping sickness within villages in south-east Uganda

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    Odiit Martin


    Full Text Available Abstract Background Sleeping sickness (HAT caused by T.b. rhodesiense is a major veterinary and human public health problem in Uganda. Previous studies have investigated spatial risk factors for T.b. rhodesiense at large geographic scales, but none have properly investigated such risk factors at small scales, i.e. within affected villages. In the present work, we use a case-control methodology to analyse both behavioural and spatial risk factors for HAT in an endemic area. Methods The present study investigates behavioural and occupational risk factors for infection with HAT within villages using a questionnaire-based case-control study conducted in 17 villages endemic for HAT in SE Uganda, and spatial risk factors in 4 high risk villages. For the spatial analysis, the location of homesteads with one or more cases of HAT up to three years prior to the beginning of the study was compared to all non-case homesteads. Analysing spatial associations with respect to irregularly shaped geographical objects required the development of a new approach to geographical analysis in combination with a logistic regression model. Results The study was able to identify, among other behavioural risk factors, having a family member with a history of HAT (p = 0.001 as well as proximity of a homestead to a nearby wetland area (p Conclusion Spatial risk factors for HAT are maintained across geographical scales; this consistency is useful in the design of decision support tools for intervention and prevention of the disease. Familial aggregation of cases was confirmed for T. b. rhodesiense HAT in the study and probably results from shared behavioural and spatial risk factors amongmembers of a household.

  11. No gold standard estimation of the sensitivity and specificity of two molecular diagnostic protocols for Trypanosoma brucei spp. in Western Kenya.

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    Barend Mark de Clare Bronsvoort

    Full Text Available African animal trypanosomiasis is caused by a range of tsetse transmitted protozoan parasites includingTrypanosoma vivax, Trypanosoma congolense and Trypansoma brucei. In Western Kenya and other parts of East Africa two subspecies of T. brucei, T.b. brucei and the zoonoticT.b. rhodesiense, co-circulate in livestock. A range of polymerase chain reactions (PCR have been developed as important molecular diagnostic tools for epidemiological investigations of T. brucei s.l. in the animal reservoir and of its zoonotic potential. Quantification of the relative performance of different diagnostic PCRs is essential to ensure comparability of studies. This paper describes an evaluation of two diagnostic test systems for T. brucei using a T. brucei s.l. specific PCR [1] and a single nested PCR targeting the Internal Transcribed Spacer (ITS regions of trypanosome ribosomal DNA [2]. A Bayesian formulation of the Hui-Walter latent class model was employed to estimate their test performance in the absence of a gold standard test for detecting T.brucei s.l. infections in ear-vein blood samples from cattle, pig, sheep and goat populations in Western Kenya, stored on Whatman FTA cards. The results indicate that the system employing the T. brucei s.l. specific PCR (Se1=0.760 had a higher sensitivity than the ITS-PCR (Se2=0.640; both have high specificity (Sp1=0.998; Sp2=0.997. The true prevalences for livestock populations were estimated (pcattle=0.091, ppigs=0.066, pgoats=0.005, psheep=0.006, taking into account the uncertainties in the specificity and sensitivity of the two test systems. Implications of test performance include the required survey sample size; due to its higher sensitivity and specificity, the T. brucei s.l. specific PCR requires a consistently smaller sample size than the ITS-PCR for the detection of T. brucei s.l. However the ITS-PCR is able to simultaneously screen samples for other pathogenic trypanosomes and may thus be, overall, a better

  12. Clinical presentation of T.b. rhodesiense sleeping sickness in second stage patients from Tanzania and Uganda.

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    Irene Kuepfer

    Full Text Available BACKGROUND: A wide spectrum of disease severity has been described for Human African Trypanosomiasis (HAT due to Trypanosoma brucei rhodesiense (T.b. rhodesiense, ranging from chronic disease patterns in southern countries of East Africa to an increase in virulence towards the north. However, only limited data on the clinical presentation of T.b. rhodesiense HAT is available. From 2006-2009 we conducted the first clinical trial program (Impamel III in T.b. rhodesiense endemic areas of Tanzania and Uganda in accordance with international standards (ICH-GCP. The primary and secondary outcome measures were safety and efficacy of an abridged melarsoprol schedule for treatment of second stage disease. Based on diagnostic findings and clinical examinations at baseline we describe the clinical presentation of T.b. rhodesiense HAT in second stage patients from two distinct geographical settings in East Africa. METHODOLOGY/PRINCIPAL FINDINGS: 138 second stage patients from Tanzania and Uganda were enrolled. Blood samples were collected for diagnosis and molecular identification of the infective trypanosomes, and T.b. rhodesiense infection was confirmed in all trial subjects. Significant differences in diagnostic parameters and clinical signs and symptoms were observed: the median white blood cell (WBC count in the cerebrospinal fluid (CSF was significantly higher in Tanzania (134 cells/mm(3 than in Uganda (20 cells/mm(3; p<0.0001. Unspecific signs of infection were more commonly seen in Uganda, whereas neurological signs and symptoms specific for HAT dominated the clinical presentation of the disease in Tanzania. Co-infections with malaria and HIV did not influence the clinical presentation nor treatment outcomes in the Tanzanian study population. CONCLUSIONS/SIGNIFICANCE: We describe a different clinical presentation of second stage T.b. rhodesiense HAT in two distinct geographical settings in East Africa. In the ongoing absence of sensitive diagnostic

  13. Wild chimpanzees are infected by Trypanosoma brucei

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    Milan Jirků


    Finally, we demonstrated that the mandrill serum was able to efficiently lyse T. b. brucei and T. b. rhodesiense, and to some extent T. b. gambiense, while the chimpanzee serum failed to lyse any of these subspecies.

  14. Estimating the burden of rhodesiense sleeping sickness during an outbreak in Serere, eastern Uganda

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    Coleman Paul G


    Full Text Available Abstract Background Zoonotic sleeping sickness, or HAT (Human African Trypanosomiasis, caused by infection with Trypanosoma brucei rhodesiense, is an under-reported and neglected tropical disease. Previous assessments of the disease burden expressed as Disability-Adjusted Life Years (DALYs for this infection have not distinguished T.b. rhodesiense from infection with the related, but clinically distinct Trypanosoma brucei gambiense form. T.b. rhodesiense occurs focally, and it is important to assess the burden at the scale at which resource-allocation decisions are made. Methods The burden of T.b. rhodesiense was estimated during an outbreak of HAT in Serere, Uganda. We identified the unique characteristics affecting the burden of rhodesiense HAT such as age, severity, level of under-reporting and duration of hospitalisation, and use field data and empirical estimates of these to model the burden imposed by this and other important diseases in this study population. While we modelled DALYs using standard methods, we also modelled uncertainty of our parameter estimates through a simulation approach. We distinguish between early and late stage HAT morbidity, and used disability weightings appropriate for the T.b. rhodesiense form of HAT. We also use a model of under-reporting of HAT to estimate the contribution of un-reported mortality to the overall disease burden in this community, and estimate the cost-effectiveness of hospital-based HAT control. Results Under-reporting accounts for 93% of the DALY estimate of rhodesiense HAT. The ratio of reported malaria cases to reported HAT cases in the same health unit was 133:1, however, the ratio of DALYs was 3:1. The age productive function curve had a close correspondence with the HAT case distribution, and HAT cases occupied more patient admission time in Serere during 1999 than all other infectious diseases other than malaria. The DALY estimate for HAT in Serere shows that the burden is much greater

  15. Adaptation of Trypanosoma rhodesiense to hypohaptoglobinaemic serum requires transcription of the APOL1 resistance gene in a RNA polymerase I locus. (United States)

    Lecordier, Laurence; Uzureau, Pierrick; Tebabi, Patricia; Brauner, Jonathan; Benghiat, Fleur Samantha; Vanhollebeke, Benoit; Pays, Etienne


    Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.

  16. Human African trypanosomiasis in a traveler: diagnostic pitfalls. (United States)

    Meltzer, Eyal; Leshem, Eyal; Steinlauf, Shmuel; Michaeli, Shulamit; Sidi, Yechezkel; Schwartz, Eli


    Abstract. An Israeli traveler returning from Tanzania presented with a relapsing febrile illness. A diagnosis of Trypanosoma brucei rhodesiense infection was established by blood smear after nearly a month. Blood polymerase chain reaction failed to provide an early diagnosis of human African trypanososmiasis. Recognition of suggestive signs should prompt physicians to perform repeated tests before ruling out human African trypanososmiasis.

  17. Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy. (United States)

    Martin, M B; Grimley, J S; Lewis, J C; Heath, H T; Bailey, B N; Kendrick, H; Yardley, V; Caldera, A; Lira, R; Urbina, J A; Moreno, S N; Docampo, R; Croft, S L; Oldfield, E


    We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.

  18. A luciferase based viability assay for ATP detection in 384-well format for high throughput whole cell screening of Trypanosoma brucei brucei bloodstream form strain 427

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    Avery Vicky M


    Full Text Available Abstract Background Human African Trypanosomiasis (HAT is caused by two trypanosome species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Current drugs available for the treatment of HAT have significant issues related to toxicity, administration regimes with limited effectiveness across species and disease stages, thus there is a considerable need to find alternative drugs. A well recognised approach to identify new drug candidates is high throughput screening (HTS of large compound library collections. Results We describe here the development of a luciferase based viability assay in 384-well plate format suitable for HTS of T.b.brucei. The parameters that were explored to determine the final HTS assay conditions are described in detail and include DMSO tolerability, Z', diluents and cell inoculum density. Reference compound activities were determined for diminazene, staurosporine and pentamidine and compared to previously published IC50 data obtained. The assay has a comparable sensitivity to reference drugs and is more cost effective than the 96-well format currently reported for T.b.brucei. Conclusion Due to the reproducibility and sensitivity of this assay it is recommended for potential HTS application. As it is commercially available this assay can also be utilised in many laboratories for both large and small scale screening.

  19. Classical clinical signs in rats experimemtally infected with Trypanosoma brucei

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    Nwoha Rosemary Ijeoma Ogechi; Omamegbe Joseph Omolathebu


    Objective:To investigate clinical signs in Trypanosoma brucei infection in albino rats. Methods:Fourteen rats grouped into 2 with 7 rats in each group were used to determine classical clinical manifestation of Trypanosoma brucei infection in rats. Group A rats were uninfected control and Group B rats were infected with Trypanosoma brucei. Results:Parasitaemia was recorded in Group B by (3.86±0.34) d and the peak of parasitaemia was observed at Day 5 post infection. Classical signs observed included squint eyes, raised whiskers, lethargy, no weight loss, pyrexia, isolation from the other rats, and starry hair coat. Conclusions:These signs could be diagnostic or aid in diagnosis of Trypanosoma brucei infection in rats.

  20. Taxonomy Icon Data: Trypanosoma brucei [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Trypanosoma brucei Trypanosoma brucei Trypanosoma_brucei_L.png Trypanosoma_brucei_NL.png Trypanosoma_bruce...i_S.png Trypanosoma_brucei_NS.png ...

  1. Discovery of Inhibitors of Trypanosoma brucei by Phenotypic Screening of a Focused Protein Kinase Library. (United States)

    Woodland, Andrew; Thompson, Stephen; Cleghorn, Laura A T; Norcross, Neil; De Rycker, Manu; Grimaldi, Raffaella; Hallyburton, Irene; Rao, Bhavya; Norval, Suzanne; Stojanovski, Laste; Brun, Reto; Kaiser, Marcel; Frearson, Julie A; Gray, David W; Wyatt, Paul G; Read, Kevin D; Gilbert, Ian H


    A screen of a focused kinase inhibitor library against Trypanosoma brucei rhodesiense led to the identification of seven series, totaling 121 compounds, which showed >50 % inhibition at 5 μm. Screening of these hits in a T. b. brucei proliferation assay highlighted three compounds with a 1H-imidazo[4,5-b]pyrazin-2(3H)-one scaffold that showed sub-micromolar activity and excellent selectivity against the MRC5 cell line. Subsequent rounds of optimisation led to the identification of compounds that exhibited good in vitro drug metabolism and pharmacokinetics (DMPK) properties, although in general this series suffered from poor solubility. A scaffold-hopping exercise led to the identification of a 1H-pyrazolo[3,4-b]pyridine scaffold, which retained potency. A number of examples were assessed in a T. b. brucei growth assay, which could differentiate static and cidal action. Compounds from the 1H-imidazo[4,5-b]pyrazin-2(3H)-one series were found to be either static or growth-slowing and not cidal. Compounds with the 1H-pyrazolo[3,4-b]pyridine scaffold were found to be cidal and showed an unusual biphasic nature in this assay, suggesting they act by at least two mechanisms.

  2. Trypanosoma b. rhodesiense (WRATat Serodeme): Purification and Characterization of Surface Antigens for the Vaccine Development Program. (United States)


    149-151 (1975). 7. Snary, D. and L. Hudson: Trypanosoma cruzi cell surface proteins: Identifi- cation of one major glycoprotein. FEBS Lett. 100: 166...7 AD-A095 616 COLORADO STATE UNIV FORT COLLINS COLL OF VETERINARY --ETC F/B 6/5 TRYPANOSOMA B. RHODESIENSE (WRATAT SERODEME): PURIFICATION AND --ETC...U) APR 0 A R KEILMAN DAMD17-79-C-9117 UNCLASSIFIED NL ///I///I//IIf~f~fllf EEEEEL 1LE.. AD_ REPORT NUMBER 1 Trypanosoma b. rhodesiense (WRATat

  3. An Overview of Trypanosoma brucei Infections: An Intense Host–Parasite Interaction (United States)

    Ponte-Sucre, Alicia


    Trypanosoma brucei rhodesiense and T. brucei gambiense, the causative agents of Human African Trypanosomiasis, are transmitted by tsetse flies. Within the vector, the parasite undergoes through transformations that prepares it to infect the human host. Sequentially these developmental stages are the replicative procyclic (in which the parasite surface is covered by procyclins) and trypo-epimastigote forms, as well as the non-replicative, infective, metacyclic form that develops in the vector salivary glands. As a pre-adaptation to their life in humans, metacyclic parasites begin to express and be densely covered by the Variant Surface Glycoprotein (VSG). Once the metacyclic form invades the human host the parasite develops into the bloodstream form. Herein the VSG triggers a humoral immune response. To avoid this humoral response, and essential for survival while in the bloodstream, the parasite changes its cover periodically and sheds into the surroundings the expressed VSG, thus evading the consequences of the immune system activation. Additionally, tools comparable to quorum sensing are used by the parasite for the successful parasite transmission from human to insect. On the other hand, the human host promotes clearance of the parasite triggering innate and adaptive immune responses and stimulating cytokine and chemokine secretion. All in all, the host–parasite interaction is extremely active and leads to responses that need multiple control sites to develop appropriately. PMID:28082973

  4. Identification and characterization of hundreds of potent and selective inhibitors of Trypanosoma brucei growth from a kinase-targeted library screening campaign.

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    Rosario Diaz


    Full Text Available In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2 cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053. This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.

  5. Identification and characterization of hundreds of potent and selective inhibitors of Trypanosoma brucei growth from a kinase-targeted library screening campaign. (United States)

    Diaz, Rosario; Luengo-Arratta, Sandra A; Seixas, João D; Amata, Emanuele; Devine, William; Cordon-Obras, Carlos; Rojas-Barros, Domingo I; Jimenez, Elena; Ortega, Fatima; Crouch, Sabrinia; Colmenarejo, Gonzalo; Fiandor, Jose Maria; Martin, Jose Julio; Berlanga, Manuela; Gonzalez, Silvia; Manzano, Pilar; Navarro, Miguel; Pollastri, Michael P


    In the interest of identification of new kinase-targeting chemotypes for target and pathway analysis and drug discovery in Trypanosomal brucei, a high-throughput screen of 42,444 focused inhibitors from the GlaxoSmithKline screening collection was performed against parasite cell cultures and counter-screened against human hepatocarcinoma (HepG2) cells. In this way, we have identified 797 sub-micromolar inhibitors of T. brucei growth that are at least 100-fold selective over HepG2 cells. Importantly, 242 of these hit compounds acted rapidly in inhibiting cellular growth, 137 showed rapid cidality. A variety of in silico and in vitro physicochemical and drug metabolism properties were assessed, and human kinase selectivity data were obtained, and, based on these data, we prioritized three compounds for pharmacokinetic assessment and demonstrated parasitological cure of a murine bloodstream infection of T. brucei rhodesiense with one of these compounds (NEU-1053). This work represents a successful implementation of a unique industrial-academic collaboration model aimed at identification of high quality inhibitors that will provide the parasitology community with chemical matter that can be utilized to develop kinase-targeting tool compounds. Furthermore these results are expected to provide rich starting points for discovery of kinase-targeting tool compounds for T. brucei, and new HAT therapeutics discovery programs.

  6. 3-(Oxazolo[4,5-b]pyridin-2-yl)anilides as a novel class of potent inhibitors for the kinetoplastid Trypanosoma brucei, the causative agent for human African trypanosomiasis. (United States)

    Ferrins, Lori; Rahmani, Raphaël; Sykes, Melissa L; Jones, Amy J; Avery, Vicky M; Teston, Eliott; Almohaywi, Basmah; Yin, JieXiang; Smith, Jason; Hyland, Chris; White, Karen L; Ryan, Eileen; Campbell, Michael; Charman, Susan A; Kaiser, Marcel; Baell, Jonathan B


    A whole organism high-throughput screen of approximately 87,000 compounds against Trypanosoma brucei brucei led to the recent discovery of several novel compound classes with low micromolar activity against this organism and without appreciable cytotoxicity to mammalian cells. Herein we report a structure-activity relationship (SAR) investigation around one of these hit classes, the 3-(oxazolo[4,5-b]pyridin-2-yl)anilides. Sharp SAR is revealed, with our most active compound (5) exhibiting an IC₅₀ of 91 nM against the human pathogenic strain T.b. rhodesiense and being more than 700 times less toxic towards the L6 mammalian cell line. Physicochemical properties are attractive for many compounds in this series. For the most potent representatives, we show that solubility and metabolic stability are key parameters to target during future optimisation.

  7. In Silico Identification and in Vitro Activity of Novel Natural Inhibitors of Trypanosoma brucei Glyceraldehyde-3-phosphate-dehydrogenase

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    Fabian C. Herrmann


    Full Text Available As part of our ongoing efforts to identify natural products with activity against pathogens causing neglected tropical diseases, we are currently performing an extensive screening of natural product (NP databases against a multitude of protozoan parasite proteins. Within this project, we screened a database of NPs from a commercial supplier, AnalytiCon Discovery (Potsdam, Germany, against Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH, a glycolytic enzyme whose inhibition deprives the parasite of energy supply. NPs acting as potential inhibitors of the mentioned enzyme were identified using a pharmacophore-based virtual screening and subsequent docking of the identified hits into the active site of interest. In a set of 700 structures chosen for the screening, 13 (1.9% were predicted to possess significant affinity towards the enzyme and were therefore tested in an in vitro enzyme assay using recombinant TbGAPDH. Nine of these in silico hits (69% showed significant inhibitory activity at 50 µM, of which two geranylated benzophenone derivatives proved to be particularly active with IC50 values below 10 µM. These compounds also showed moderate in vitro activity against T. brucei rhodesiense and may thus represent interesting starting points for further optimization.

  8. Eleganolone, a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum

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    Anne-Marie Rusig


    Full Text Available Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC50 = 0.53 µg/mL; selectivity index (SI = 11.6. Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC50 = 45.0 µM, SI = 4.0. However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC50 = 7.9 µM, SI = 21.6.

  9. Eleganolone, a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum (United States)

    Gallé, Jean-Baptiste; Attioua, Barthélémy; Kaiser, Marcel; Rusig, Anne-Marie; Lobstein, Annelise; Vonthron-Sénécheau, Catherine


    Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC50 = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC50 = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC50 = 7.9 µM, SI = 21.6). PMID:23442789

  10. Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum.

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    Tanja Wenzler

    Full Text Available Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.

  11. Anti-trypanosomal effect of Peristrophe bicalyculata extract on Trypanosoma brucei brucei-infected rats

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    Abdulazeez Mansurah Abimbola; Ibrahim Abdulrazak Baba; Edibo Zakari Yenusa; Sidali Joseph Omanibe; Idris Habeeb Oladimeji


    Objective: To investigate the in vitro and in vivo effect of whole plant extracts of Peristrophe bicalyculata on Trypanosoma brucei brucei-infected rats. Methods: The experiment was divided into two phases: In the first phase, the anti-trypanosomal activity of the hot water, cold water, methanol and butanol extracts of the whole plant were determined by incubating with Trypanosoma brucei brucei. The cold water extract was partially-purified and the anti-trypanosomal activity of the fractions determined. In the second phase, Trypanosoma brucei brucei-infected rats were treated with fraction 2c for nine days. Packed cell volume (PCV), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (TC), triacylglycerol (TAG), aspartate aminotransferase, alanine aminotransferases (ALT), alkaline phosphatase (ALP), total and direct bilirubin levels were determined at the end of the experiment. Results:Cold water extract immobilized 90%of the parasites after 60 min of incubation, and fraction 2c completely immobilized the parasites after 35 min. It significantly increased PCV in Trypanosoma brucei brucei-infected rats. Decreased TC, TAG, HDL and LDL levels of infected rats increased significantly when rats were treated with the fraction, while elevated levels of total bilirubin and ALT also decreased. The difference in urea, direct bilirubin and ALP was not significant when infected rats were compared to rats in other groups. Conclusions:The ability of the plant to ameliorate the infection-induced biochemical changes calls for detailed investigation of the potentials of the plant for antitrypanosomiasis drug delivery.

  12. Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei. (United States)

    Jamwal, Abhishek; Round, Adam R; Bannwarth, Ludovic; Venien-Bryan, Catherine; Belrhali, Hassan; Yogavel, Manickam; Sharma, Amit


    Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg(2+) and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within TbbVSP1.

  13. Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei* (United States)

    Jamwal, Abhishek; Round, Adam R.; Bannwarth, Ludovic; Venien-Bryan, Catherine; Belrhali, Hassan; Yogavel, Manickam; Sharma, Amit


    Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg2+ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within TbbVSP1. PMID:26494625

  14. Trypanosoma brucei brucei: effects of ferrous iron and heme on ecto-nucleoside triphosphate diphosphohydrolase activity. (United States)

    Leite, Milane S; Thomaz, Rachel; Oliveira, José Henrique M; Oliveira, Pedro L; Meyer-Fernandes, José Roberto


    Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.

  15. A Protein Complex Map of Trypanosoma brucei.

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    Vahid H Gazestani


    Full Text Available The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at:

  16. Genetic control of resistance to Trypanosoma brucei brucei infection in mice.

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    Matyáš Síma


    Full Text Available BACKGROUND: Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped. METHODS: We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem series contains a different random subset of 12.5% genes from the parental "donor" strain STS/A and 87.5% genes from the "background" strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F(2 hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F(2 hybrid mice and their linkage with survival was tested by analysis of variance. RESULTS: We mapped four Tbbr (Trypanosoma brucei brucei response loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3 and Tbbr2 (chromosome 12 have effects on survival independent of inter-genic interactions (main effects. Tbbr3 (chromosome 7 influences survival in interaction with Tbbr4 (chromosome 19. Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes. CONCLUSION: This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F(2 hybrids of inbred strains usually has a precision of 40-80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.

  17. Trypanosoma brucei gambiense group 1 is distinguished by a unique amino acid substitution in the HpHb receptor implicated in human serum resistance.

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    Rebecca E Symula

    Full Text Available Trypanosoma brucei rhodesiense (Tbr and T. b. gambiense (Tbg, causative agents of Human African Trypanosomiasis (sleeping sickness in Africa, have evolved alternative mechanisms of resisting the activity of trypanosome lytic factors (TLFs, components of innate immunity in human serum that protect against infection by other African trypanosomes. In Tbr, lytic activity is suppressed by the Tbr-specific serum-resistance associated (SRA protein. The mechanism in Tbg is less well understood but has been hypothesized to involve altered activity and expression of haptoglobin haemoglobin receptor (HpHbR. HpHbR has been shown to facilitate internalization of TLF-1 in T.b. brucei (Tbb, a member of the T. brucei species complex that is susceptible to human serum. By evaluating the genetic variability of HpHbR in a comprehensive geographical and taxonomic context, we show that a single substitution that replaces leucine with serine at position 210 is conserved in the most widespread form of Tbg (Tbg group 1 and not found in related taxa, which are either human serum susceptible (Tbb or known to resist lysis via an alternative mechanism (Tbr and Tbg group 2. We hypothesize that this single substitution contributes to reduced uptake of TLF and thus may play a key role in conferring serum resistance to Tbg group 1. In contrast, similarity in HpHbR sequence among isolates of Tbg group 2 and Tbb/Tbr provides further evidence that human serum resistance in Tbg group 2 is likely independent of HpHbR function.

  18. Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol.

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    Fabrice E Graf

    Full Text Available The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i have been adapted to axenic in vitro cultivation and (ii mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.

  19. Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol. (United States)

    Graf, Fabrice E; Ludin, Philipp; Wenzler, Tanja; Kaiser, Marcel; Brun, Reto; Pyana, Patient Pati; Büscher, Philippe; de Koning, Harry P; Horn, David; Mäser, Pascal


    The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.

  20. Motility modes of the parasite Trypanosoma brucei (United States)

    Temel, Fatma Zeynep; Qu, Zijie; McAllaster, Michael; de Graffenried, Christopher; Breuer, Kenneth


    The parasitic single-celled protozoan Trypanosoma brucei causes African Sleeping Sickness, which is a fatal disease in humans and animals that threatens more than 60 million people in 36 African countries. Cell motility plays a critical role in the developmental phases and dissemination of the parasite. Unlike many other motile cells such as bacteria Escherichia coli or Caulobacter crescentus, the flagellum of T. brucei is attached along the length of its awl-like body, producing a unique mode of motility that is not fully understood or characterized. Here, we report on the motility of T. brucei, which swims using its single flagellum employing both rotating and undulating propulsion modes. We tracked cells in real-time in three dimensions using fluorescent microscopy. Data obtained from experiments using both short-term tracking within the field of view and long-term tracking using a tracking microscope were analyzed. Motility modes and swimming speed were analyzed as functions of cell size, rotation rate and undulation pattern. Research supported by NSF.

  1. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

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    S Andrea Moreno


    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  2. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes. (United States)

    Moreno, S Andrea; Nava, Mayerly


    Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  3. Cell-penetrating peptide TP10 shows broad-spectrum activity against both Plasmodium falciparum and Trypanosoma brucei brucei. (United States)

    Arrighi, Romanico B G; Ebikeme, Charles; Jiang, Yang; Ranford-Cartwright, Lisa; Barrett, Michael P; Langel, Ulo; Faye, Ingrid


    Malaria and trypanosomiasis are diseases which afflict millions and for which novel therapies are urgently required. We have tested two well-characterized cell-penetrating peptides (CPPs) for antiparasitic activity. One CPP, designated TP10, has broad-spectrum antiparasitic activity against Plasmodium falciparum, both blood and mosquito stages, and against blood-stage Trypanosoma brucei brucei.


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    Georges C. Accrombessi


    Full Text Available The structure of four synthesized thiosemicarbazones, substituted or not, of benzophenone has been confirmed by spectrometrical analysis IR, NMR 1H and 13C. Their anti-trypanosomal activities were evaluated on Trypanosoma brucei brucei. Among these compounds, benzophenone 4 phenyl-3-thiosemicarbazone 4 has the highest activity with the half-inhibitory concentration (IC50 = 8.48 micromolar (µM. Benzophenone 4-methyl-3-thiosemicarbazone 3 and benzophenone thiosemicarbazone 1 showed moderate anti-trypanosomal activity with IC50 values equal to 23.27 µM and 67.17 µM respectively. Benzophenone 2 methyl-3-thiosemicarbazone 2 showed no activity up to IC50 = 371.74 µM.

  5. Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector (United States)

    Mantilla, Brian S.; Dyer, Naomi A.; Biran, Marc; Bringaud, Frédéric; Lehane, Michael J.; Acosta-Serrano, Alvaro


    Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly’s midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut. PMID:28114403

  6. Phenolic Constituents of Medicinal Plants with Activity against Trypanosoma brucei

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    Ya Nan Sun


    Full Text Available Neglected tropical diseases (NTDs affect over one billion people all over the world. These diseases are classified as neglected because they impact populations in areas with poor financial conditions and hence do not attract sufficient research investment. Human African Trypanosomiasis (HAT or sleeping sickness, caused by the parasite Trypanosoma brucei, is one of the NTDs. The current therapeutic interventions for T. brucei infections often have toxic side effects or require hospitalization so that they are not available in the rural environments where HAT occurs. Furthermore, parasite resistance is increasing, so that there is an urgent need to identify novel lead compounds against this infection. Recognizing the wide structural diversity of natural products, we desired to explore and identify novel antitrypanosomal chemotypes from a collection of natural products obtained from plants. In this study, 440 pure compounds from various medicinal plants were tested against T. brucei by in a screening using whole cell in vitro assays. As the result, twenty-two phenolic compounds exhibited potent activity against cultures of T. brucei. Among them, eight compounds—4, 7, 11, 14, 15, 18, 20, and 21—showed inhibitory activity against T. brucei, with IC50 values below 5 µM, ranging from 0.52 to 4.70 μM. Based on these results, we attempt to establish some general trends with respect to structure-activity relationships, which indicate that further investigation and optimization of these derivatives might enable the preparation of potentially useful compounds for treating HAT.

  7. Regulation and spatial organization of PCNA in Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Doris; Gassen, Alwine [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Maiser, Andreas; Leonhardt, Heinrich [University of Munich (LMU), Department Biology II, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Janzen, Christian J., E-mail: [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany)


    Highlights: Black-Right-Pointing-Pointer Characterization of the proliferating cell nuclear antigen in Trypanosoma brucei (TbPCNA). Black-Right-Pointing-Pointer TbPCNA is a suitable marker to detect replication in T. brucei. Black-Right-Pointing-Pointer TbPCNA distribution and regulation is different compared to closely related parasites T. cruzi and Leishmania donovani. -- Abstract: As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.

  8. Effect of ivermectin on Trypanosoma brucei brucei in experimentally infected mice

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    Udensi K. Udensi & A.F. Fagbenro-Beyioku


    Full Text Available Background & objectives: Human and livestock African trypanosomiasis, otherwise known as sleeping sickness,is a neglected tropical disease of public health importance in west and central Africa. In view of the adverse sideeffects of the antitrypanosomal drugs, the relatively few side effects observed in ivermectin use, and becauseboth onchocerciasis and typanosomiasis occur in overlapping foci in Africa, it would be desirable if the ivermectinthat has been used successfully on onchocerciasis management could also be used in the control and treatmentof trypanosomiasis.Method: In this study, prophylactic and therapeutic effects of ivermectin (Mectizan were investigated in albinomice infected with a Nigerian strain of Trypanosoma brucei brucei.Results: A 300 μg/ml/kg dose had the most effective impact because it showed the highest mean survival time of12 days in both the treatment and prophylactic groups of mice. This dose also enhanced the defence capacity ofthe treated groups. It also had positive influence on the packed cell volume (PCV and the state of anaemia inthe trypanosome infected mice, hence, improving their survivability.Interpretation & conclusions: Our report indicates that using the 300 μg/ml/kg dose of ivermectin increases themean survival period from 5 to 12 days. This suggests that ivermectin could be possibly used in the treatment oftrypanosomiasis. Further studies will be required to show whether proper treatment may entail a single dose, asused in this study; an increased number of doses, or combinations with other drugs.

  9. Evaluation of In Vitro Activity of Essential Oils against Trypanosoma brucei brucei and Trypanosoma evansi

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    Nathan Habila


    Full Text Available Essential oils (EOs from Cymbopogon citratus (CC, Eucalyptus citriodora (EC, Eucalyptus camaldulensis (ED, and Citrus sinensis (CS were obtained by hydrodistillation process. The EOs were evaluated in vitro for activity against Trypanosoma brucei brucei (Tbb and Trypanosoma evansi (T. evansi. The EOs were found to possess antitrypanosomal activity in vitro in a dose-dependent pattern in a short period of time. The drop in number of parasite over time was achieved doses of 0.4 g/ml, 0.2 g/mL, and 0.1 g/mL for all the EOs. The concentration of 0.4 g/mL CC was more potent at 3 minutes and 2 minutes for Tbb and T. evansi, respectively. The GC-MS analysis of the EOs revealed presence of Cyclobutane (96.09% in CS, 6-octenal (77.11% in EC, Eucalyptol (75% in ED, and Citral (38.32% in CC among several other organic compounds. The results are discussed in relation to trypanosome chemotherapy.

  10. Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase

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    Paul Smith


    Full Text Available Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1 is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals—including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics—that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s. We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae. Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition.

  11. A tropical tale: how Naja nigricollis venom beats Trypanosoma brucei

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings [1]. During the parasite’s extracellular life in the mammalian host,its outer coat, mainly composed of Variable Surface Glycoproteins (VSGs...

  12. Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents. (United States)

    Lim, Kah Tee; Zahari, Zuriati; Amanah, Azimah; Zainuddin, Zafarina; Adenan, Mohd Ilham


    To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.

  13. In vitro antitrypanosomal activity of plant terpenes against Trypanosoma brucei. (United States)

    Otoguro, Kazuhiko; Iwatsuki, Masato; Ishiyama, Aki; Namatame, Miyuki; Nishihara-Tukashima, Aki; Kiyohara, Hiroaki; Hashimoto, Toshihiro; Asakawa, Yoshinori; Omura, Satoshi; Yamada, Haruki


    During the course of screening to discover antitrypanosomal compounds, 24 known plant terpenes (6 sesquiterpenes, 14 sesquiterpene lactones and 4 diterpenes) were evaluated for in vitro antitrypanosomal activity against Trypanosoma brucei brucei. Among them, 22 terpenes exhibited antitrypanosomal activity. In particular, α-eudesmol, hinesol, nardosinone and 4-peroxy-1,2,4,5-tetrahydro-α-santonin all exhibited selective and potent antitrypanosomal activities in vitro. Detailed here in an in vitro antitrypanosomal properties and cytotoxicities of the 24 terpenes compared with two therapeutic antitrypanosomal drugs (eflornithine and suramin). This finding represents the first report of promising trypanocidal activity of these terpenes. Present results also provide some valuable insight with regard to structure-activity relationships and the possible mode of action of the compounds.

  14. Single molecule analysis of Trypanosoma brucei DNA replication dynamics. (United States)

    Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco; Marques, Catarina A; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L; Elias, Maria Carolina


    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.

  15. Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form

    KAUST Repository

    Acestor, Nathalie


    The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F oF 1-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Intraclonal mating occurs during tsetse transmission of Trypanosoma brucei

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    Ferris Vanessa


    Full Text Available Abstract Background Mating in Trypanosoma brucei is a non-obligatory event, triggered by the co-occurrence of different strains in the salivary glands of the vector. Recombinants that result from intra- rather than interclonal mating have been detected, but only in crosses of two different trypanosome strains. This has led to the hypothesis that when trypanosomes recognize a different strain, they release a diffusible factor or pheromone that triggers mating in any cell in the vicinity whether it is of the same or a different strain. This idea assumes that the trypanosome can recognize self and non-self, although there is as yet no evidence for the existence of mating types in T. brucei. Results We investigated intraclonal mating in T. b. brucei by crossing red and green fluorescent lines of a single strain, so that recombinant progeny can be detected in the fly by yellow fluorescence. For strain 1738, seven flies had both red and green trypanosomes in the salivary glands and, in three, yellow trypanosomes were also observed, although they could not be recovered for subsequent analysis. Nonetheless, both red and non-fluorescent clones from these flies had recombinant genotypes as judged by microsatellite and karyotype analyses, and some also had raised DNA contents, suggesting recombination or genome duplication. Strain J10 produced similar results indicative of intraclonal mating. In contrast, trypanosome clones recovered from other flies showed that genotypes can be transmitted with fidelity. When a yellow hybrid clone expressing both red and green fluorescent protein genes was transmitted, the salivary glands contained a mixture of fluorescent-coloured trypanosomes, but only yellow and red clones were recovered. While loss of the GFP gene in the red clones could have resulted from gene conversion, some of these clones showed loss of heterozygosity and raised DNA contents as in the other single strain transmissions. Our observations suggest

  17. In Vitro Trypanocidal Activity of Antibodies to Bacterially Ex­pressed Trypanosoma brucei Tubulin

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    GW Lubega


    Full Text Available Background: There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacte­rially expressed trypanosome tubulin on T. brucei brucei growth. Methods: T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the ex­pressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450μg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was deter­mined by culturing bloodstream T. brucei brucei in up to 25% of antisera. Results: Low antisera dilutions (25% from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions. Conclusion: Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cul­tured bloodstream trypanosomes.

  18. Biosynthesis and uptake of thiamine (vitamin B1) in bloodstream form Trypanosoma brucei brucei and interference of the vitamin with melarsen oxide activity. (United States)

    Stoffel, Sabine A; Rodenko, Boris; Schweingruber, Anne-Marie; Mäser, Pascal; de Koning, Harry P; Schweingruber, M Ernst


    Bloodstream forms of Trypanosoma brucei brucei were cultivated in the presence and absence of thiamine (vitamin B1) and pyridoxine (vitamin B6). The vitamins do not change growth behaviour, indicating that Trypanosoma brucei is prototrophic for the two vitamins even though in silico no bona-fide thiamine-biosynthetic genes could be identified in the T. brucei genome. Intracellularly, thiamine is mainly present in its diphosphate form. We were unable to detect significant uptake of [3H]thiamine and structural thiamine analogues such as pyrithiamine, oxithiamine and amprolium were not toxic for the bloodstream forms of T. brucei, indicating that the organism does not have an efficient uptake system for thiamine and its analogues. We have previously shown that, in the fission yeast Saccharomyces pombe, the toxicity of melarsen oxide, the pharmacologically active derivative of the frontline sleeping sickness drug melarsoprol, is abolished by thiamine and the drug is taken up by a thiamine-regulated membrane protein which is responsible for the utilization of thiamine. We show here that thiamine also has weak effects on melarsen oxide-induced growth inhibition and lysis in T. brucei. These effects were consistent with a low affinity of thiamine for the P2 adenosine transporter that is responsible for uptake of melaminophenyl arsenicals in African trypanosomes.

  19. Rab23 is a flagellar protein in Trypanosoma brucei

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    Field Mark C


    Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

  20. Selective inhibition of 6-phosphogluconate dehydrogenase from Trypanosoma brucei (United States)

    Bertelli, Massimo; El-Bastawissy, Eman; Knaggs, Michael H.; Barrett, Michael P.; Hanau, Stefania; Gilbert, Ian H.


    A number of triphenylmethane derivatives have been screened against 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Some of these compounds show good inhibition of the enzymes and also selectivity towards the parasite enzyme. Modelling was undertaken to dock the compounds into the active sites of both enzymes. Using a combination of DOCK 3.5 and FLEXIDOCK a correlation was obtained between docking score and both activity for the enzymes and selectivity. Visualisation of the docked structures of the inhibitors in the active sites of the enzymes yielded a possible explanation of the selectivity for the parasite enzyme.

  1. Minimum Information Loss Based Multi-kernel Learning for Flagellar Protein Recognition in Trypanosoma Brucei

    KAUST Repository

    Wang, Jim Jing-Yan


    Trypanosma brucei (T. Brucei) is an important pathogen agent of African trypanosomiasis. The flagellum is an essential and multifunctional organelle of T. Brucei, thus it is very important to recognize the flagellar proteins from T. Brucei proteins for the purposes of both biological research and drug design. In this paper, we investigate computationally recognizing flagellar proteins in T. Brucei by pattern recognition methods. It is argued that an optimal decision function can be obtained as the difference of probability functions of flagella protein and the non-flagellar protein for the purpose of flagella protein recognition. We propose to learn a multi-kernel classification function to approximate this optimal decision function, by minimizing the information loss of such approximation which is measured by the Kull back-Leibler (KL) divergence. An iterative multi-kernel classifier learning algorithm is developed to minimize the KL divergence for the problem of T. Brucei flagella protein recognition, experiments show its advantage over other T. Brucei flagellar protein recognition and multi-kernel learning methods. © 2014 IEEE.

  2. Telomeric expression sites are highly conserved in Trypanosoma brucei.

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    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  3. Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei. (United States)

    Krstin, Sonja; Peixoto, Herbenya Silva; Wink, Michael


    The flagellate Trypanosoma brucei causes sleeping sickness in humans and nagana in animals. Only a few drugs are registered to treat trypanosomiasis, but those drugs show severe side effects. Also, because some pathogen strains have become resistant, new strategies are urgently needed to combat this parasitic disease. An underexplored possibility is the application of combinations of several trypanocidal agents, which may potentiate their trypanocidal activity in a synergistic fashion. In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei. Alkaloids were selected that affect different molecular targets: berberine and chelerythrine (intercalation of DNA), piperine (induction of apoptosis), vinblastine (inhibition of microtubule assembly), emetine (intercalation of DNA, inhibition of protein biosynthesis), homoharringtonine (inhibition of protein biosynthesis), and digitonin (membrane permeabilization and uptake facilitation of polar compounds). Most combinations resulted in an enhanced trypanocidal effect. The addition of digitonin significantly stimulated the activity of almost all alkaloids against trypanosomes. The strongest effect was measured in a combination of digitonin with vinblastine. The highest dose reduction indexes (DRI) were measured in the two-drug combination of digitonin or piperine with vinblastine, where the dose of vinblastine could be reduced 9.07-fold or 7.05-fold, respectively. The synergistic effects of mutual combinations of alkaloids and of alkaloids with digitonin present a new avenue to treat trypanosomiasis but one which needs to be corroborated in future animal experiments.

  4. Antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger on Trypanosoma brucei brucei-infected Wistar mice

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    P. I. Kobo


    Full Text Available Aim: The study was carried out to determine the in vivo antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger in Trypanosoma brucei brucei-infected mice. Materials and Methods: Twenty-five mice were randomly allocated into five groups of five animals each. Group I and II were given Tween 80 (1 ml/kg and diminazene aceturate (3.5 mg/kg to serve as untreated and treated controls, respectively. Groups III-V received the extract at 200, 400 and 800 mg/kg body weight, respectively. All treatments were given for 6 consecutive days and through the oral route. The mean body weight, mean survival period and daily level of parasitaemia were evaluated. Results: Acute toxicity showed the extract to be relatively safe. There was an insignificant increase in body weight and survival rate of mice treated with the extract. The level of parasitaemia in the extract treated groups was decreased. Conclusion: This study shows the in vivo potential of methanolic extract of Z. officinale in the treatment of trypanosomiasis.

  5. Cofactor-independent phosphoglycerate mutase is an essential gene in procyclic form Trypanosoma brucei. (United States)

    Djikeng, Appolinaire; Raverdy, Sylvine; Foster, Jeremy; Bartholomeu, Daniella; Zhang, Yinhua; El-Sayed, Najib M; Carlow, Clotilde


    Glycolysis and gluconeogenesis are, in part, driven by the interconversion of 3- and 2-phosphoglycerate (3-PG and 2-PG) which is performed by phosphoglycerate mutases (PGAMs) which can be cofactor dependant (dPGAM) or cofactor independent (iPGAM). The African trypanosome, Trypanosoma brucei, possesses the iPGAM form which is thought to play an important role in glycolysis. Here, we report on the use of RNA interference to down-regulate the T. brucei iPGAM in procyclic form T. brucei and evaluation of the resulting phenotype. We first demonstrated biochemically that depletion of the steady state levels of iPGM mRNA correlates with a marked reduction of enzyme activity. We further show that iPGAM is required for cell growth in procyclic T. brucei.

  6. Mosaic VSGs and the scale of Trypanosoma brucei antigenic variation.

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    James P J Hall

    Full Text Available A main determinant of prolonged Trypanosoma brucei infection and transmission and success of the parasite is the interplay between host acquired immunity and antigenic variation of the parasite variant surface glycoprotein (VSG coat. About 0.1% of trypanosome divisions produce a switch to a different VSG through differential expression of an archive of hundreds of silent VSG genes and pseudogenes, but the patterns and extent of the trypanosome diversity phenotype, particularly in chronic infection, are unclear. We applied longitudinal VSG cDNA sequencing to estimate variant richness and test whether pseudogenes contribute to antigenic variation. We show that individual growth peaks can contain at least 15 distinct variants, are estimated computationally to comprise many more, and that antigenically distinct 'mosaic' VSGs arise from segmental gene conversion between donor VSG genes or pseudogenes. The potential for trypanosome antigenic variation is probably much greater than VSG archive size; mosaic VSGs are core to antigenic variation and chronic infection.

  7. Pathogenesis of anemia in Trypanosoma brucei-infected mice. (United States)

    Amole, B O; Clarkson, A B; Shear, H L


    The pathogenesis of anemia was studied in trypanosome-infected mice. A strain of Trypanosoma brucei, TREU 667, was used which first produces an acute phase marked by waves of parasitemia. Erythrocytes from infected animals were coated with immunoglobulin M during or just before the waves of anemia and parasitological crises. Erythrocytes from normal animals could be sensitized with "precrisis" sera presumably containing antigen and antibody. These data suggest that anemia during the acute phase is due to sensitization of erythrocytes with immunoglobulin M-antigen complexes. The anemia is partially compensated by a strong erythropoietic response. The acute phase is followed by a chronic phase marked by a constant high parasitemia and immunosuppression. The less marked anemia occurring during this latter phase is due to hemodilution and perhaps a low but significant immune response to the parasites, which causes continuing erythrocyte sensitization by immunoglobulin M-antigen complexes. PMID:7201455

  8. Trypanosoma brucei: a putative RNA polymerase II promoter. (United States)

    Bayele, Henry K


    RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei because gene regulation in the parasite is complex and polycistronic. Here, we describe a putative pol II promoter and its structure-function relationship. The promoter has features of an archetypal eukaryotic pol II promoter including putative canonical CCAAT and TATA boxes, and an initiator element. However, the spatial arrangement of these elements is only similar to yeast pol II promoters. Deletion mapping and transcription assays enabled delineation of a minimal promoter that could drive orientation-independent reporter gene expression suggesting that it may be a bidirectional promoter. In vitro transcription in a heterologous nuclear extract revealed that the promoter can be recognized by the basal eukaryotic transcription complex. This suggests that the transcription machinery in the parasite may be very similar to those of other eukaryotes.

  9. Trypanosoma brucei Invasion and T-Cell Infiltration of the Brain Parenchyma in Experimental Sleeping Sickness: Timing and Correlation with Functional Changes (United States)

    Laperchia, Claudia; Palomba, Maria; Seke Etet, Paul F.; Rodgers, Jean; Bradley, Barbara; Montague, Paul; Grassi-Zucconi, Gigliola; Bentivoglio, Marina


    Background The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. Methodology Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. Principal findings Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. Conclusion These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging. PMID:28002454

  10. Population Vulnerability and Disability in Kenya's Tsetse Fly Habitats


    Grady, Sue C.; Messina, Joseph P.; McCord, Paul F.


    BACKGROUND: Human African Trypanosomiasis (HAT), also referred to as sleeping sickness, and African Animal Trypanosomaisis (AAT), known as nagana, are highly prevalent parasitic vector-borne diseases in sub-Saharan Africa. Humans acquire trypanosomiasis following the bite of a tsetse fly infected with the protozoa Trypanosoma brucei (T.b.) spp. -i.e., T.b. gambiense in West and Central Africa and T.b. rhodesiense in East and Southern Africa. Over the last decade HAT diagnostic capacity to est...

  11. Untreated human infections by Trypanosoma brucei gambiense are not 100% fatal.

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    Vincent Jamonneau

    Full Text Available The final outcome of infection by Trypanosoma brucei gambiense, the main agent of sleeping sickness, has always been considered as invariably fatal. While scarce and old reports have mentioned cases of self-cure in untreated patients, these studies suffered from the lack of accurate diagnostic tools available at that time. Here, using the most specific and sensitive tools available to date, we report on a long-term follow-up (15 years of a cohort of 50 human African trypanosomiasis (HAT patients from the Ivory Coast among whom 11 refused treatment after their initial diagnosis. In 10 out of 11 subjects who continued to refuse treatment despite repeated visits, parasite clearance was observed using both microscopy and polymerase chain reaction (PCR. Most of these subjects (7/10 also displayed decreasing serological responses, becoming progressively negative to trypanosome variable antigens (LiTat 1.3, 1.5 and 1.6. Hence, in addition to the "classic" lethal outcome of HAT, we show that alternative natural progressions of HAT may occur: progression to an apparently aparasitaemic and asymptomatic infection associated with strong long-lasting serological responses and progression to an apparently spontaneous resolution of infection (with negative results in parasitological tests and PCR associated with a progressive drop in antibody titres as observed in treated cases. While this study does not precisely estimate the frequency of the alternative courses for this infection, it is noteworthy that in the field national control programs encounter a significant proportion of subjects displaying positive serologic test results but negative results in parasitological testing. These findings demonstrate that a number of these subjects display such infection courses. From our point of view, recognising that trypanotolerance exists in humans, as is now widely accepted for animals, is a major step forward for future research in the field of HAT.

  12. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model (United States)

    Dauchy, Frédéric-Antoine; Bonhivers, Mélanie; Landrein, Nicolas; Dacheux, Denis; Courtois, Pierrette; Lauruol, Florian; Daulouède, Sylvie


    Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (ptrypanosomiasis. PMID:27855164

  13. MIF Contributes to Trypanosoma brucei Associated Immunopathogenicity Development (United States)

    Stijlemans, Benoît; Leng, Lin; Brys, Lea; Sparkes, Amanda; Vansintjan, Liese; Caljon, Guy; Raes, Geert; Van Den Abbeele, Jan; Van Ginderachter, Jo A.; Beschin, Alain


    African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6Chigh inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity. PMID:25255103

  14. MIF contributes to Trypanosoma brucei associated immunopathogenicity development.

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    Benoît Stijlemans


    Full Text Available African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6C(high inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury, and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.

  15. Exosome secretion affects social motility in Trypanosoma brucei (United States)

    Shaked, Hadassa; Arvatz, Gil; Tkacz, Itai Dov; Binder, Lior; Waldman Ben-Asher, Hiba; Okalang, Uthman; Chikne, Vaibhav; Cohen-Chalamish, Smadar; Michaeli, Shulamit


    Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites. PMID:28257521

  16. Proteins associated with SF3a60 in T. brucei.

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    Benson Nyambega

    Full Text Available Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry.

  17. Proteomics and the Trypanosoma brucei cytoskeleton: advances and opportunities. (United States)

    Portman, Neil; Gull, Keith


    Trypanosoma brucei is the etiological agent of devastating parasitic disease in humans and livestock in sub-saharan Africa. The pathogenicity and growth of the parasite are intimately linked to its shape and form. This is in turn derived from a highly ordered microtubule cytoskeleton that forms a tightly arrayed cage directly beneath the pellicular membrane and numerous other cytoskeletal structures such as the flagellum. The parasite undergoes extreme changes in cellular morphology during its life cycle and cell cycles which require a high level of integration and coordination of cytoskeletal processes. In this review we will discuss the role that proteomics techniques have had in advancing our understanding of the molecular composition of the cytoskeleton and its functions. We then consider future opportunities for the application of these techniques in terms of addressing some of the unanswered questions of trypanosome cytoskeletal cell biology with particular focus on the differences in the composition and organisation of the cytoskeleton through the trypanosome life-cycle.

  18. Protective effect of humus extract against Trypanosoma brucei infection in mice. (United States)

    Kodama, Hiroshi; Denso; Okazaki, Fumi; Ishida, Saeko


    Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms are present. Oral administration of humus extract to mice successfully induced effective protection against experimental challenge by the two subspecies, Trypanosoma brucei brucei and T. brucei gambiense. Mortality was most reduced among mice who received a 3% humus extract for 21 days in drinking water ad libitum. Spleen cells from humus-administered mice exhibited significant non-specific cytotoxic activity against L1210 mouse leukemia target cells. Also, spleen cells produced significantly higher amounts of Interferon-gamma when stimulated in vitro with Concanavalin A than cells from normal controls. These results clearly show that administration to mice of humus extract induced effective resistance against Trypanosoma infection. Enhancement of the innate immune system may be involved in host defense against trypanosomiasis.

  19. A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

    Institute of Scientific and Technical Information of China (English)

    Ranjodh Sandhu; Samantha Sanford; Shrabani Basu; MinA Park; Unnati M Pandya; Bibo Li; Kausik Chakrabarti


    Telomerase is a ribonucleoprotein enzyme typically required for sustained cell proliferation.Although both telomerase activity and the telomerase catalytic protein component,TbTERT,have been identified in the eukaryotic pathogen Trypanosoma brucei,the RNA molecule that dictates telomere synthesis remains unknown.Here,we identify the RNA component of Trypanosoma brucei telomerase,TbTR,and provide phylogenetic and in vivo evidence for TbTR's native folding and activity.We show that TbTR is processed through trans-splicing,and is a capped transcript that interacts and copurifies with TbTERT in vivo.Deletion of TbTR caused progressive shortening of telomeres at a rate of 3-5 bp/population doubling (PD),which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner.Remarkably,introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences,demonstrating that telomere synthesis in T.brucei is dependent on TbTR.We also propose a secondary structure model for TbTR based on phylogenetic analysis and chemical probing experiments,thus defining TbTR domains that may have important functional implications in telomere synthesis.Identification and characterization of TbTR not only provide important insights into T.brucei telomere functions,which have been shown to play important roles in T.brucei pathogenesis,but also offer T.brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes.

  20. Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Alloatti, Andres [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Gupta, Shreedhara; Gualdron-Lopez, Melisa; Nguewa, Paul A. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Altabe, Silvia G. [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Deumer, Gladys; Wallemacq, Pierre [Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, LTAP, Universite Catholique de Louvain, Brussels (Belgium); Michels, Paul A.M. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Uttaro, Antonio D., E-mail: [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina)


    Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

  1. The cell cycle regulated transcriptome of Trypanosoma brucei.

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    Stuart K Archer

    Full Text Available Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a "double-cut" elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.

  2. Isolation and characterization of kinetoplast DNA from the bloodstream form of Trypanosoma brucei.

    NARCIS (Netherlands)

    A.H. Fairlamb; P.O. Weislogel; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)


    textabstractWe have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African Trypanosomiasi

  3. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

    NARCIS (Netherlands)

    Weelden, van S.W.H.; Fast, B.; Vogt, A.; Meer, van der P.; Saas, J.; Hellemond, van J.J.; Tielens, A.G.M.; Boshart, M.


    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene cod

  4. Dynamic Modelling under Uncertainty : The Case of Trypanosoma brucei Energy Metabolism

    NARCIS (Netherlands)

    Achcar, Fiona; Kerkhoven, Eduard J.; Bakker, Barbara M.; Barrett, Michael P.; Breitling, Rainer; Papin, Jason A.


    Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabol

  5. Handling Uncertainty in Dynamic Models : The Pentose Phosphate Pathway in Trypanosoma brucei

    NARCIS (Netherlands)

    Kerkhoven, Eduard J.; Achcar, Fiona; Alibu, Vincent P.; Burchmore, Richard J.; Gilbert, Ian H.; Trybilo, Maciej; Driessen, Nicole N.; Gilbert, David; Breitling, Rainer; Bakker, Barbara M.; Barrett, Michael P.


    Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate

  6. Involvement of lysosomes in the uptake of macromolecular material by bloodstream forms of Trypanosoma brucei. (United States)

    Opperdoes, F R; Van Roy, J


    To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromolecular compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (greater than 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm3 in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and alpha-mannosidase-containing organelles of T. brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function.

  7. Novel molecular mechanism for targeting the parasite Trypanosoma brucei with snake venom toxins

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings. During parasites’extracellular lives in the mammalian host, its outer coat, mainly composedof Variable surface glycoproteins (VSGs)[2], und...

  8. Telomere length affects the frequency and mechanism of antigenic variation in Trypanosoma brucei.

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    Galadriel A Hovel-Miner

    Full Text Available Trypanosoma brucei is a master of antigenic variation and immune response evasion. Utilizing a genomic repertoire of more than 1000 Variant Surface Glycoprotein-encoding genes (VSGs, T. brucei can change its protein coat by "switching" from the expression of one VSG to another. Each active VSG is monoallelically expressed from only one of approximately 15 subtelomeric sites. Switching VSG expression occurs by three predominant mechanisms, arguably the most significant of which is the non-reciprocal exchange of VSG containing DNA by duplicative gene conversion (GC. How T. brucei orchestrates its complex switching mechanisms remains to be elucidated. Recent work has demonstrated that an exogenous DNA break in the active site could initiate a GC based switch, yet the source of the switch-initiating DNA lesion under natural conditions is still unknown. Here we investigated the hypothesis that telomere length directly affects VSG switching. We demonstrate that telomerase deficient strains with short telomeres switch more frequently than genetically identical strains with long telomeres and that, when the telomere is short, switching preferentially occurs by GC. Our data supports the hypothesis that a short telomere at the active VSG expression site results in an increase in subtelomeric DNA breaks, which can initiate GC based switching. In addition to their significance for T. brucei and telomere biology, the findings presented here have implications for the many diverse pathogens that organize their antigenic genes in subtelomeric regions.

  9. Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples

    NARCIS (Netherlands)

    C.M. Mugasa; T. Laurent; G.J. Schoone; P.A. Kager; G.W. Lubega; H.D.F.H. Schallig


    Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control pr

  10. Ribose 5-phosphate isomerase B knockdown compromises Trypanosoma brucei bloodstream form infectivity. (United States)

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana


    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.

  11. The major transcripts of the kinetoplast Trypanosoma brucei are very small ribosomal RNA's.

    NARCIS (Netherlands)

    I.C. Eperon; J.W.G. Janssen; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)


    textabstractThe nucleotide sequence has been determined of a 2.2 kb segment of kinetoplast DNA, which encodes the major mitochondrial transcripts (12S and 9S) of Trypanosoma brucei. The sequence shows that the 12S RNA is a large subunit rRNA, although sufficiently unusual for resistance to chloramph

  12. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System (United States)

    Iribarren, Paula Ana; Berazategui, María Agustina; Cazzulo, Juan José; Alvarez, Vanina Eder


    Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids. PMID:26258470

  13. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model

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    Maina Ngotho


    Full Text Available Human African trypanosomiasis (HAT is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP and polymerase chain reaction (PCR in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF, saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  14. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model. (United States)

    Ngotho, Maina; Kagira, John Maina; Gachie, Beatrice Muthoni; Karanja, Simon Muturi; Waema, Maxwell Wambua; Maranga, Dawn Nyawira; Maina, Naomi Wangari


    Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  15. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology. (United States)

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André


    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

  16. Mutational analysis of Trypanosoma brucei RNA editing ligase reveals regions critical for interaction with KREPA2.

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    Vaibhav Mehta

    Full Text Available The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1 and the closely related T. brucei RNA Editing Ligase 2 (TbREL2. While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1 and KREPA1 (for TbREL2. KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441-444 sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.

  17. T rypanosoma brucei histone H1 inhibits RNA polymerase I transcription and is important for parasite fitness in vivo


    Pena, Ana C.; Pimentel, Mafalda R; Manso, Helena; Vaz-Drago, Rita; Pinto-Neves, Daniel; Aresta-Branco, Francisco; Rijo-Ferreira, Filipa; Guegan, Fabien; Pedro Coelho, Luis; Carmo-Fonseca, Maria; Barbosa-Morais, Nuno L.; Figueiredo, Luisa M.


    T rypanosoma brucei is a unicellular parasite that causes sleeping sickness in humans. Most of its transcription is constitutive and driven by RNA polymerase II. RNA polymerase I (Pol I) transcribes not only ribosomal RNA genes, but also protein-encoding genes, including variant surface glycoproteins (VSGs) and procyclins. In T . brucei, histone H1 (H1) is required for VSG silencing and chromatin condensation. However, whether H1 has a genome-wide role in transcription is unknown. Here, using...

  18. Overview of the Diagnostic Methods Used in the Field for Human African Trypanosomiasis: What Could Change in the Next Years?

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    Julien Bonnet


    Full Text Available Sleeping sickness is a parasitic infection caused by two species of trypanosomes (Trypanosoma brucei gambiense and rhodesiense, transmitted by the tsetse fly. The disease eventually affects the central nervous system, resulting in severe neurological symptoms. Without treatment, death is inevitable. During the first stage of the disease, infected patients are mildly symptomatic and early detection of infection allows safer treatment (administered on an outpatient basis which can avoid death; routine screening of the exposed population is necessary, especially in areas of high endemicity. The current therapeutic treatment of this disease, especially in stage 2, can cause complications and requires a clinical surveillance for several days. A good stage diagnosis of the disease is the cornerstone for delivering the adequate treatment. The task faced by the medical personnel is further complicated by the lack of support from local health infrastructure, which is at best weak, but often nonexistent. Therefore it is crucial to look for new more efficient technics for the diagnosis of stage which are also best suited to use in the field, in areas not possessing high-level health facilities. This review, after an overview of the disease, summarizes the current diagnosis procedures and presents the advances in the field.

  19. Overview of the Diagnostic Methods Used in the Field for Human African Trypanosomiasis: What Could Change in the Next Years? (United States)

    Bonnet, Julien; Boudot, Clotilde; Courtioux, Bertrand


    Sleeping sickness is a parasitic infection caused by two species of trypanosomes (Trypanosoma brucei gambiense and rhodesiense), transmitted by the tsetse fly. The disease eventually affects the central nervous system, resulting in severe neurological symptoms. Without treatment, death is inevitable. During the first stage of the disease, infected patients are mildly symptomatic and early detection of infection allows safer treatment (administered on an outpatient basis) which can avoid death; routine screening of the exposed population is necessary, especially in areas of high endemicity. The current therapeutic treatment of this disease, especially in stage 2, can cause complications and requires a clinical surveillance for several days. A good stage diagnosis of the disease is the cornerstone for delivering the adequate treatment. The task faced by the medical personnel is further complicated by the lack of support from local health infrastructure, which is at best weak, but often nonexistent. Therefore it is crucial to look for new more efficient technics for the diagnosis of stage which are also best suited to use in the field, in areas not possessing high-level health facilities. This review, after an overview of the disease, summarizes the current diagnosis procedures and presents the advances in the field.

  20. Clinical and hematological study of Trypanosoma brucei and Trypanosoma congolense in cattle in Mosul City, Iraq

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    Al-Badrani, B. A


    Full Text Available An out-break of trypanosomosis was reported and subsequently investigated in Mosul city, Iraq. Twenty seven blood samples were received at the Clinical pathology Laboratory of Veterinary Teaching Hospital, University of Mosul, from fifteen cows (2-5 years old and twelve calves (10-12 months old of different farms located around Mosul city. Clinical signs of the infected animals revealed fever, progressive weight loss, anemia, and frequent recumbent position. Identification of trypanosome species were based on their motility using morphological differentiation on Giemsa or Leishman stained that showed trypomastigot which can be classified according to the biometrical data into T. brucei and T. congolense. A decreased was also observed in some blood parameters. To our knowledge, this is the first record of T. brucei and T. congolense infection in cattle. This study also suggested that trypanosomosis is associated with introduction of exotic breeds of cattle into Mosul, Iraq.

  1. Alkaloids Induce Programmed Cell Death in Bloodstream Forms of Trypanosomes (Trypanosoma b. brucei

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    Michael Wink


    Full Text Available The potential induction of a programmed cell death (PCD in Trypanosoma b. brucei by 55 alkaloids of the quinoline, quinolizidine, isoquinoline, indole, terpene, tropane, steroid, and piperidine type was studied by measuring DNA fragmentation and changes in mitochondrial membrane potential. For comparison, the induction of apoptosis by the same alkaloids in human leukemia cells (Jurkat APO-S was tested. Several alkaloids of the isoquinoline, quinoline, indole and steroidal type (berberine, chelerythrine, emetine, sanguinarine, quinine, ajmalicine, ergotamine, harmine, vinblastine, vincristine, colchicine, chaconine, demissidine and veratridine induced programmed cell death, whereas quinolizidine, tropane, terpene and piperidine alkaloids were mostly inactive. Effective PCD induction (EC50 below 10 µM was caused in T. brucei by chelerythrine, emetine, sanguinarine, and chaconine. The active alkaloids can be characterized by their general property to inhibit protein biosynthesis, to intercalate DNA, to disturb membrane fluidity or to inhibit microtubule formation.

  2. An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei.

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    Juan P de Macêdo


    Full Text Available Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug

  3. An essential signal peptide peptidase identified in an RNAi screen of serine peptidases of Trypanosoma brucei.

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    Catherine X Moss

    Full Text Available The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1. This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival.

  4. A transcription-independent epigenetic mechanism is associated with antigenic switching in Trypanosoma brucei


    Aresta-Branco, Francisco; Pimenta, Silvia; Figueiredo, Luisa M.


    Antigenic variation in Trypanosoma brucei relies on periodic switching of variant surface glycoproteins (VSGs), which are transcribed monoallelically by RNA polymerase I from one of about 15 bloodstream expression sites (BES). Chromatin of the actively transcribed BES is depleted of nucleosomes, but it is unclear if this open conformation is a mere consequence of a high rate of transcription, or whether it is maintained by a transcription-independent mechanism. Using an inducible BES-silencin...

  5. Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi


    Parsons, Marilyn; Worthey, Elizabeth A.; Ward, Pauline N; Mottram, Jeremy C.


    Background The trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intr...

  6. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation. (United States)

    van Weelden, Susanne W H; Fast, Beate; Vogt, Achim; van der Meer, Pieter; Saas, Joachim; van Hellemond, Jaap J; Tielens, Aloysius G M; Boshart, Michael


    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.

  7. The extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei. (United States)

    van Hellemond, J J; Opperdoes, F R; Tielens, A G M


    African trypanosomes are parasitic protozoa that cause sleeping sickness and nagana. Trypanosomes are not only of scientific interest because of their clinical importance, but also because these protozoa contain several very unusual biological features, such as their specially adapted mitochondrion and the compartmentalization of glycolytic enzymes in glycosomes. The energy metabolism of Trypanosoma brucei differs significantly from that of their hosts and changes drastically during the life cycle. Despite the presence of all citric acid cycle enzymes in procyclic insect-stage T. brucei, citric acid cycle activity is not used for energy generation. Recent investigations on the influence of substrate availability on the type of energy metabolism showed that absence of glycolytic substrates did not induce a shift from a fermentative metabolism to complete oxidation of substrates. Apparently, insect-stage T. brucei use parts of the citric acid cycle for other purposes than for complete degradation of mitochondrial substrates. Parts of the cycle are suggested to be used for (i) transport of acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, (ii) degradation of proline and glutamate to succinate, (iii) generation of malate, which can then be used for gluconeogenesis. Therefore the citric acid cycle in trypanosomes does not function as a cycle.

  8. Experimental Trypanosoma brucei infection at immediate post partum period:effects on dam and the offspring

    Institute of Scientific and Technical Information of China (English)

    Izuchukwu S Ochiogu; Chukwuka N Uchendu; John I Ihedioha


    Objective:To investigate the effects of immediate post-partum infection with Trypanosoma brucei (T. brucei) on dam and offspring. Methods:Sixty female Albino rats (Rattus norvegicus) weighing between 130-170 g were used as animal model. The animals were divided as follows:25 infected between 1-5 days post partum; 10 infected unbred as positive controls; and 25 uninfected as negative controls. The following parameters were evaluated:packed cell volume (PCV), level of parasitaemia, survival time, litter size and litter weight at birth and on days 7, 14 and 21 post delivery, using conventional methods. Possible trans-mammary transmission of infection to litter through milk was also assessed. Results:The results showed a comparatively (P<0.05) higher mean PCV value for the uninfected negative control on the 8th day post infection compared with the infected groups which corresponded with the increasing level of parasitaemia in the two infected groups. Mean litter size and litter weights were higher (P< 0.05) in the uninfected controls on the 21st day. Survival time in the infected groups were similar. No evidence of trans-mammary transfer of infection was recorded. Conclusion:T. brucei infection during immediate post partum period is detrimental to the dam and impairs growth of the offspring.

  9. Advancing Trypanosoma brucei genome annotation through ribosome profiling and spliced leader mapping. (United States)

    Parsons, Marilyn; Ramasamy, Gowthaman; Vasconcelos, Elton J R; Jensen, Bryan C; Myler, Peter J


    Since the initial publication of the trypanosomatid genomes, curation has been ongoing. Here we make use of existing Trypanosoma brucei ribosome profiling data to provide evidence of ribosome occupancy (and likely translation) of mRNAs from 225 currently unannotated coding sequences (CDSs). A small number of these putative genes correspond to extra copies of previously annotated genes, but 85% are novel. The median size of these novels CDSs is small (81 aa), indicating that past annotation work has excelled at detecting large CDSs. Of the unique CDSs confirmed here, over half have candidate orthologues in other trypanosomatid genomes, most of which were not yet annotated as protein-coding genes. Nonetheless, approximately one-third of the new CDSs were found only in T. brucei subspecies. Using ribosome footprints, RNA-Seq and spliced leader mapping data, we updated previous work to definitively revise the start sites for 414 CDSs as compared to the current gene models. The data pointed to several regions of the genome that had sequence errors that altered coding region boundaries. Finally, we consolidated this data with our previous work to propose elimination of 683 putative genes as protein-coding and arrive at a view of the translatome of slender bloodstream and procyclic culture form T. brucei.

  10. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei. (United States)

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria


    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  11. The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

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    Smiths Lueong

    Full Text Available Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II and without (stage I brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II, 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

  12. Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening. (United States)

    Pedró-Rosa, Laura; Buckner, Frederick S; Ranade, Ranae M; Eberhart, Christina; Madoux, Franck; Gillespie, J Robert; Koh, Cho Yeow; Brown, Steven; Lohse, Jacqueline; Verlinde, Christophe L M; Fan, Erkang; Bannister, Thomas; Scampavia, Louis; Hol, Wim G J; Spicer, Timothy; Hodder, Peter


    Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.

  13. The Centriole Cartwheel Protein SAS-6 in Trypanosoma brucei Is Required for Probasal Body Biogenesis and Flagellum Assembly. (United States)

    Hu, Huiqing; Liu, Yi; Zhou, Qing; Siegel, Sara; Li, Ziyin


    The centriole in eukaryotes functions as the cell's microtubule-organizing center (MTOC) to nucleate spindle assembly, and its biogenesis requires an evolutionarily conserved protein, SAS-6, which assembles the centriole cartwheel. Trypanosoma brucei, an early branching protozoan, possesses the basal body as its MTOC to nucleate flagellum biogenesis. However, little is known about the components of the basal body and their roles in basal body biogenesis and flagellum assembly. Here, we report that the T. brucei SAS-6 homolog, TbSAS-6, is localized to the mature basal body and the probasal body throughout the cell cycle. RNA interference (RNAi) of TbSAS-6 inhibited probasal body biogenesis, compromised flagellum assembly, and caused cytokinesis arrest. Surprisingly, overexpression of TbSAS-6 in T. brucei also impaired probasal body duplication and flagellum assembly, contrary to SAS-6 overexpression in humans, which produces supernumerary centrioles. Furthermore, we showed that depletion of T. brucei Polo-like kinase, TbPLK, or inhibition of TbPLK activity did not abolish TbSAS-6 localization to the basal body, in contrast to the essential role of Polo-like kinase in recruiting SAS-6 to centrioles in animals. Altogether, these results identified the essential role of TbSAS-6 in probasal body biogenesis and flagellum assembly and suggest the presence of a TbPLK-independent pathway governing basal body duplication in T. brucei.

  14. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

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    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  15. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty. (United States)

    Carnes, Jason; Anupama, Atashi; Balmer, Oliver; Jackson, Andrew; Lewis, Michael; Brown, Rob; Cestari, Igor; Desquesnes, Marc; Gendrin, Claire; Hertz-Fowler, Christiane; Imamura, Hideo; Ivens, Alasdair; Kořený, Luděk; Lai, De-Hua; MacLeod, Annette; McDermott, Suzanne M; Merritt, Chris; Monnerat, Severine; Moon, Wonjong; Myler, Peter; Phan, Isabelle; Ramasamy, Gowthaman; Sivam, Dhileep; Lun, Zhao-Rong; Lukeš, Julius; Stuart, Ken; Schnaufer, Achim


    Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  16. Central Nervous System Parasitosis and Neuroinflammation Ameliorated by Systemic IL-10 Administration in Trypanosoma brucei-Infected Mice. (United States)

    Rodgers, Jean; Bradley, Barbara; Kennedy, Peter G E; Sternberg, Jeremy M


    Invasion of the central nervous system (CNS) by African trypanosomes represents a critical step in the development of human African trypanosomiasis. In both clinical cases and experimental mouse infections it has been demonstrated that predisposition to CNS invasion is associated with a type 1 systemic inflammatory response. Using the Trypanosoma brucei brucei GVR35 experimental infection model, we demonstrate that systemic delivery of the counter-inflammatory cytokine IL-10 lowers plasma IFN-γ and TNF-α concentrations, CNS parasitosis and ameliorates neuro-inflammatory pathology and clinical symptoms of disease. The results provide evidence that CNS invasion may be susceptible to immunological attenuation.

  17. The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

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    Nikolay G Kolev

    Full Text Available The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp. and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

  18. Trypanocidal activity of organotin chlorides on Trypanosoma brucei-infected mice

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    Shuaibu M.N.


    Full Text Available The organotin compounds dibulyltin (DBTC and diphenyltin dichlorides (DPTC were tested for trypanocidal activity on a Trypanosoma brucei-infected mice model. At a dose of 10 mg DBTC and 15 mg DPTC/kg/day for five consecutive days, they cleared the parasites from the peripheral blood of the infected mice. Subinoculation of some healthy mice with the homogenates of liver, spleen, kidney, cerebrospinal fluid and blood from the mice considered cured, showed a few cases of relapse. The LD50 of DBTC and DPTC are 90 mg/kg and 75 mg/kg respectively.

  19. A haptoglobin-hemoglobin receptor conveys innate immunity to Trypanosoma brucei in humans

    DEFF Research Database (Denmark)

    Vanhollebeke, Benoit; De Muylder, Géraldine; Nielsen, Marianne J;


    The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which...... receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host...

  20. Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei. (United States)

    Schumann, Gabriela; Kangussu-Marcolino, Monica M; Doiron, Nicholas; Käser, Sandro; de Assis Burle-Caldas, Gabriela; DaRocha, Wanderson D; Teixeira, Santuza M; Roditi, Isabel


    In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.

  1. Structures of aspartate aminotransferases from Trypanosoma brucei, Leishmania major and Giardia lamblia. (United States)

    Abendroth, Jan; Choi, Ryan; Wall, Abigail; Clifton, Matthew C; Lukacs, Christine M; Staker, Bart L; Van Voorhis, Wesley; Myler, Peter; Lorimer, Don D; Edwards, Thomas E


    The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.

  2. Trypanosoma brucei gambiense Type 1 populations from human patients are clonal and display geographical genetic differentiation. (United States)

    Morrison, Liam J; Tait, Andy; McCormack, Gillian; Sweeney, Lindsay; Black, Alana; Truc, Philippe; Likeufack, Anne C L; Turner, C Michael; MacLeod, Annette


    We have rigorously tested the hypothesis that Trypanosoma brucei gambiense Type 1 is composed of genetically homogenous populations by examining the parasite population present in Human African Trypanosomiasis (HAT) patients from the Democratic Republic of Congo (DRC) and Cameroon (CAM). We amplified eight microsatellite markers by PCR directly from blood spots on FTA filters, thereby avoiding the significant parasite selection inherent in the traditional isolation techniques of rodent inoculation or in vitro culture. All microsatellite markers were polymorphic, although for four markers there was only polymorphism between the DRC and CAM populations, not within populations, suggesting very limited genetic exchange. Within the largest population from the DRC, Hardy-Weinberg equilibrium is not evident at any loci. This evidence suggests a clonal population. However, there was significant sub-structuring between the DRC and CAM samples (F(ST) = 0.32), indicating that Trypanosoma brucei gambiense Type 1 has genetically distinct clades. The data combine to indicate that genetic exchange plays a very limited role. The finding of distinct clades in different places suggests the possibility that samples from humans with clinical signs represent clonal expansions from an underlying population that requires identifying and characterising.

  3. KREX2 is not essential for either procyclic or bloodstream form Trypanosoma brucei.

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    Jason Carnes

    Full Text Available BACKGROUND: Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes that catalyze RNA editing but the relative roles of each protein are not known. METHODOLOGY/PRINCIPAL FINDINGS: The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. CONCLUSIONS: KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

  4. Analysis of the Trypanosoma brucei cell cycle by quantitative DAPI imaging. (United States)

    Siegel, T Nicolai; Hekstra, Doeke R; Cross, George A M


    Trypanosoma brucei has two DNA compartments: the nucleus and the kinetoplast. DNA replication of these two compartments only partially coincides. Woodward and Gull [Woodward R, Gull K. Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei. J Cell Sci 1990;95:49-57] comprehensively studied the relative timing of the replication and segregation of nuclear DNA (nDNA) and kinetoplast DNA (kDNA). Others have since assumed the consistency of morphological indicators of cell-cycle stage among strains and conditions. We report the use of quantitative DAPI imaging to determine the cell-cycle stage of individual procyclic cells. Using this approach, we found that kinetoplast elongation occurs mainly during nuclear S phase and not during G2, as previously assumed. We confirmed this finding by sorting cells by DNA content, followed by fluorescence microscopy. In addition, simultaneous quantitative imaging at two wavelengths can be used to determine the abundance of cell-cycle-regulated proteins during the cell cycle. We demonstrate this technique by co-staining for the non-acetylated state of lysine 4 of histone H4 (H4K4), which is enriched during nuclear S phase.

  5. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

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    Adélle Burger


    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  6. Diversity and spation distribution of vectors and hosts of T. brucei gambiense in forest zones of Southern Cameroon: Epidemiological implications

    NARCIS (Netherlands)

    Massussi, J.A.; Mbida Mbida, J.A.; Djieto-Lordon, C.; Njiokou, F.; Laveissière, C.; Ploeg, van der J.D.


    Host and vector distribution of Trypanosoma brucei gambiense was studied in relation to habitat types and seasons. Six (19.35%) of the 31 mammal species recorded in Bipindi were reservoir hosts. Cercopithecus nictitans was confined to the undisturbed forest and the low intensive shifting cultivation

  7. Trypanosoma Brucei Aquaglyceroporins Facilitate the Uptake of Arsenite and Antimonite in a pH Dependent Way

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    Néstor L. Uzcátegui


    Full Text Available Background: Trypanosoma brucei is a primitive parasitic protozoan that thrives in diverse environments such as the midgut of the tsetse fly and the blood of a mammalian host. For an adequate adaptation to these environments, the parasite´s aquaglyceroporins play an important role. Methods and Results: In order to test their ability to transport trivalent arsenic and antimony, we expressed the three known Trypanosoma brucei aquaglyceroporins (TbAQPs in the heterologous systems of yeast null aquaporin mutant and Xenopus laevis oocytes. For both expression systems, we found a pH dependent intracellular accumulation of As(III or Sb(III mediated by all of the three TbAQPs, with the exception of TbAQP1-As(III uptake. Additionally, we observed that Trypanosoma brucei aquaglyceroporins allow the passage of As(III in both directions. Conclusion: Taken together, these results demonstrated that T. brucei aquaglyceroporins can serve as entry routes for As(III and Sb(III into the parasitic cell, and that this uptake is pH sensitive. Therefore, aquaporins of protozoan parasites may be considered useful as a vehicle for drug delivery.

  8. Screening North American plant extracts in vitro against Trypanosoma brucei, the causative agent for Human African Trypanosomiasis (United States)

    Natural products extracts from 522 plants collected from different parts of the North America were screened in vitro against trypamastigote forms of Trypanosoma brucei. The active extracts(150)with >90% inhibition at 20ug/mL concentrations from the plants namely, Alnus rubra, Hoita macrostachya, S...

  9. Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). (United States)

    Kennedy, Peter Ge


    Human African trypanosomiasis, or sleeping sickness, is caused by infection with parasites of the genus Trypanosoma, transmitted by the tsetse fly. The disease has two forms, Trypanosoma brucei (T b) rhodesiense and T b gambiense; and is almost always fatal if untreated. Despite a recent reduction in the number of reported cases, patients with African trypanosomiasis continue to present major challenges to clinicians. Because treatment for CNS-stage disease can be very toxic, diagnostic staging to distinguish early-stage from late-stage disease when the CNS in invaded is crucial but remains problematic. Melarsoprol is the only available treatment for late-stage T b rhodesiense infection, but can be lethal to 5% of patients owing to post-treatment reactive encephalopathy. Eflornithine combined with nifurtimox is the first-line treatment for late-stage T b gambiense. New drugs are in the pipeline for treatment of CNS human African trypanosomiasis, giving rise to cautious optimism.

  10. Murine Models for Trypanosoma brucei gambiense disease progression--from silent to chronic infections and early brain tropism.

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    Christiane Giroud

    Full Text Available Human African trypanosomiasis (HAT caused by Trypanosoma brucei gambiense remains highly prevalent in west and central Africa and is lethal if left untreated. The major problem is that the disease often evolves toward chronic or asymptomatic forms with low and fluctuating parasitaemia producing apparently aparasitaemic serological suspects who remain untreated because of the toxicity of the chemotherapy. Whether the different types of infections are due to host or parasite factors has been difficult to address, since T. b. gambiense isolated from patients is often not infectious in rodents thus limiting the variety of isolates.T. b. gambiense parasites were outgrown directly from the cerebrospinal fluid of infected patients by in vitro culture and analyzed for their molecular polymorphisms. Experimental murine infections showed that these isolates could be clustered into three groups with different characteristics regarding their in vivo infection properties, immune response and capacity for brain invasion. The first isolate induced a classical chronic infection with a fluctuating blood parasitaemia, an invasion of the central nervous system (CNS, a trypanosome specific-antibody response and death of the animals within 6-8 months. The second group induced a sub-chronic infection resulting in a single wave of parasitaemia after infection, followed by a low parasitaemia with no parasites detected by microscope observations of blood but detected by PCR, and the presence of a specific antibody response. The third isolate induced a silent infection characterised by the absence of microscopically detectable parasites throughout, but infection was detectable by PCR during the whole course of infection. Additionally, specific antibodies were barely detectable when mice were infected with a low number of this group of parasites. In both sub-chronic and chronic infections, most of the mice survived more than one year without major clinical symptoms

  11. Chimerization at the AQP2–AQP3 locus is the genetic basis of melarsoprol–pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates

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    Fabrice E. Graf


    Full Text Available Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.

  12. Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates. (United States)

    Graf, Fabrice E; Baker, Nicola; Munday, Jane C; de Koning, Harry P; Horn, David; Mäser, Pascal


    Aquaglyceroporin-2 is a known determinant of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2-AQP3 tandem locus was described from melarsoprol-pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2-aqp3 null T. b. brucei does not. This proves that AQP2-AQP3 chimerization is the cause of melarsoprol-pentamidine cross-resistance in the T. b. gambiense isolates.

  13. Phylogeny of Trypanosoma brucei and Trypanosoma evansi in naturally infected cattle in Nigeria by analysis of repetitive and ribosomal DNA sequences. (United States)

    Takeet, Michael I; Peters, Sunday O; Fagbemi, Benjamin O; De Donato, Marcos; Takeet, Vivian O; Wheto, Mathew; Imumorin, Ikhide G


    In continuing efforts to better understand the genetics of bovine trypanosomosis, we assessed genetic diversity of Trypanosoma brucei and Trypanosoma evansi in naturally infected Nigerian cattle using repetitive DNA and internal transcribed spacer 1 of rDNA sequences and compared these sequences to species from other countries. The length of repetitive DNA sequences in both species ranged from 161 to 244 bp and 239 to 240 bp for T. brucei and T. evansi, respectively, while the ITS1 rDNA sequences length range from 299 to 364 bp. The mean GC content of ITS1 rDNA sequences was 33.57 %, and that of repetitive sequences were 39.9 and 31.1 % for T. brucei and T. evansi, respectively. Result from sequence alignment revealed both T. brucei and T. evansi repetitive DNA sequences to be more polymorphic than ITS1 rDNA sequences, with moderate points of deletion and insertions. T. brucei separated into two clades when subjected to phylogenetic analysis. T. evansi repetitive DNA sequences clustered tightly within the T. brucei clade while the ITS1 rDNA sequences of T. brucei were clearly separated from T. theileri and T. vivax individually used as outgroups. This study suggest that ITS1 rDNA sequences may not be suitable for phylogenetic differentiation of the Trypanozoon group and also suggest that T. evansi may be a phenotypic variant of T. brucei which may have potential implications in designing prevention and therapeutic strategies.

  14. The contribution of chromosomal translocations to antigenic variation in Trypanosoma brucei. (United States)

    Van der Ploeg, L H; Cornelissen, A W


    Genomic rearrangements influencing gene expression occur throughout nature. Several of these rearrangements disrupt normal gene expression, as exemplified by the genetic alterations caused by the mobile genetic elements of maize or Drosophila (see Shapiro 1983). Other rearrangements are part of the normal developmental programme of an organism. An understanding of the control of genomic rearrangements and their effects on gene expression should contribute to our insight into the mechanism of genetic programming and cellular development. The protozoan parasite Trypanosoma brucei exhibits a variety of genomic rearrangements that influence the expression of genes that code for versions of the variant surface glycoprotein (v.s.g.), which makes up the cell surface coat. V.s.g. genes are expressed in a mutually exclusive manner. Several v.s.g. genes are activated by duplicative transposition of the gene to a telomeric expression site where they are transcribed, while others can be activated without detectable genomic rearrangements. Recently we have been able to fractionate the chromosomes of T. brucei in agarose gels (Van der Ploeg et al. 1984 a). This led to the observations that duplicative transpositions occur inter-chromosomally and that the chromosomes of T. brucei are subject to frequent recombinations that displace hundreds of kilobase pairs. At least two and possibly more telomeric expression sites can be used for v.s.g. gene transcription. How these sites are activated and inactivated is still unsolved, but this does not depend on recombinations in the vicinity of the gene. Gross genomic rearrangements occur sometimes in correlation with antigenic switching and this suggests that such rearrangements have a function in regulating the mutually exclusive transcription of the different expression sites. V.s.g. genes consist of two exons. No physical linkage of the 35 nucleotide (n.t.) mini-exon to the v.s.g. gene main exon occurred within 15 kilobase pairs in

  15. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation. (United States)

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard


    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

  16. Sec16 determines the size and functioning of the Golgi in the protist parasite, Trypanosoma brucei. (United States)

    Sealey-Cardona, Marco; Schmidt, Katy; Demmel, Lars; Hirschmugl, Tatjana; Gesell, Tanja; Dong, Gang; Warren, Graham


    The Sec16 homologue in Trypanosoma brucei has been identified and characterized. TbSec16 colocalizes with COPII components at the single endoplasmic reticulum exit site (ERES), which is next to the single Golgi stack in the insect (procyclic) form of this organism. Depletion of TbSec16 reduces the size of the ERES and the Golgi, and slows growth and transport of a secretory marker to the cell surface; conversely, overexpression of TbSec16 increases the size of the ERES and Golgi but has no effect on growth or secretion. Together these data suggest that TbSec16 regulates the size of the ERES and Golgi and this size is set for optimal growth of the organism.

  17. Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice


    Trindade, Sandra; Rijo-Ferreira, Filipa; Carvalho, Tânia; Pinto-Neves, Daniel; Guegan, Fabien; Aresta-Branco, Francisco; Bento, Fabio; Young, Simon A.; Pinto, Andreia; Van Den Abbeele, Jan; Ribeiro, Ruy M.; Dias, Sérgio; Smith, Terry K.; Figueiredo, Luisa M.


    This work was supported by 55007419 (HHMI) and 2151 (EMBO) to L.M.F., D.P.-N., F.B., and F.G.; FCT fellowships to S.T., F.R.-F., and F.A.-B. (SFRH/BPD/89833/2012, SFRH/BD/51286/2010, and SFRH/BD/80718/2011, respectively); Wellcome Trust grant (093228), MRC MR/M020118/1, and European Community Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED) to S.A.Y. and T.K.S.; and PAI 7/41 (Belspo) and ERC-NANOSYM to J.V.D.A. Trypanosoma brucei is an extracellular parasite t...

  18. Contribution of Glucose Transport to the Control of the Glycolytic Flux in Trypanosoma brucei (United States)

    Bakker, Barbara M.; Walsh, Michael C.; Ter Kuile, Benno H.; Mensonides, Femke I. C.; Michels, Paul A. M.; Opperdoes, Fred R.; Westerhoff, Hans V.


    The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions.

  19. Domestic animals as potential reservoir hosts of Trypanosoma brucei gambiense in sleeping sickness foci in Cameroon

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    Njiokou F.


    Full Text Available An explanation of the endemic nature and/or the resurgence of Human African Trypanosomiasis (HAT in the historic foci in West and Central Africa may be the existence of an animal reservoir. In some HAT foci, pigs were found infected by Trypanosoma brucei gambiense but the implication of the other domestic animals was not quite evaluated. This study aims to determine the prevalence of T. b. gambiense in domestic animal species (goat, sheep, pig and dog commonly found in the four active HAT foci in Cameroon (Bipindi, Fontem, Campo and Doumé. Blood samples were collected from 307 pigs, 264 goats, 267 sheep and 37 dogs and used for parasitological (QBC, immunological (LiTat 1.3 CATT and molecular (PCR analyses. QBC detected trypanosomes in 3.88 % domestic animals while 22.7 % were sero-positive with LiTat 1.3 CATT tests. Of the 875 animals analysed, 174 (19.88 % harboured T. brucei s.l. DNA, found in each of the four types of animal and in the four localities. The infection rate significantly differed among the animal species (p < 0.0001 and localities (p < 0.0001. The PCR also revealed T. b. gambiense group 1 DNA in 27 (3.08 % domestic animals. The specific infection rates were as follows: sheep (6.74 %, goats (3.08 %, pigs (0.32 % and dogs (0 %. T. b. gambiense was found in 8 (3.92 % animals from Bipindi, 15 (4.83 % from Campo, 4 (2.59 % from Fontem-Center and none from Doumé. The infection rates significantly differed between the localities, and correlated with the intensity of HAT transmission in the foci.

  20. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment.

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    Corinne S Wilson

    Full Text Available Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei.

  1. Secondary Metabolites from Vietnamese Marine Invertebrates with Activity against Trypanosoma brucei and T. cruzi

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    Nguyen Phuong Thao


    Full Text Available Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 < 5.0 μg/mL. Among the compounds isolated from these extracts, laevigatol B (1 from Lobophytum crassum and L. laevigatum, (24S-ergost-4-ene-3-one (2 from Sinularia dissecta, astropectenol A (3 from Astropecten polyacanthus, and cholest-8-ene-3β,5α,6β,7α-tetraol (4 from Diadema savignyi showed inhibitory activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM. Laevigatol B (1 and 5α-cholest-8(14-ene-3β,7α-diol (5 exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  2. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

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    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)


    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  3. Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella. (United States)

    Gluenz, Eva; Wheeler, Richard John; Hughes, Louise; Vaughan, Sue


    Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites-Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines.

  4. In vitro investigation of Brazilian Cerrado plant extract activity against Plasmodium falciparum, Trypanosoma cruzi and T. brucei gambiense. (United States)

    Charneau, Sébastien; de Mesquita, Mariana Laundry; Bastos, Izabela Marques Dourado; Santana, Jaime Martins; de Paula, José Elias; Grellier, Philippe; Espindola, Laila Salmen


    The threatened Brazilian Cerrado biome is an important biodiversity hotspot but still few explored that constitutes a potential reservoir of molecules to treat infectious diseases. We selected eight Cerrado plant species for screening against the erythrocytic stages of Plasmodium falciparum, human intracellular stages of Trypanosoma cruzi and bloodstream forms of T. brucei gambiense, and for their cytotoxicity upon the rat L6-myoblast cell line. Bioassays were performed with 37 hexane, ethyl acetate and ethanol extracts prepared from different plant organs. Activities against parasites were observed for 24 extracts: 9 with anti-P. falciparum, 4 with anti-T. cruzi and 11 with anti-T. brucei gambiense activities. High anti-protozoal activity (IC50 values Cerrado conservation and sustainable development.

  5. Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei. (United States)

    Turrens, J F; Bickar, D; Lehninger, A L


    The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

  6. TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals. (United States)

    Tetaud, Emmanuel; Lefebvre, Michèle; M'Bang-Benet, Diane-Ethna; Crobu, Lucien; Blancard, Corinne; Sterkers, Yvon; Pages, Michel; Bastien, Patrick; Merlin, Gilles


    TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

  7. Comparative genomics reveals two novel RNAi factors in Trypanosoma brucei and provides insight into the core machinery.

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    Rebecca L Barnes

    Full Text Available The introduction ten years ago of RNA interference (RNAi as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1 protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5. TbRIF4 is a 3'-5' exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.

  8. T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis.

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    Viki Bockstal


    Full Text Available African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB and Follicular B (FoB cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.

  9. A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity.

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    Elizabeth R Sharlow

    Full Text Available BACKGROUND: The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK, an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay. METHODOLOGY/PRINCIPAL FINDINGS: Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were approximately 20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics. CONCLUSIONS/SIGNIFICANCE: The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

  10. Evolutionary consequences of a large duplication event in Trypanosoma brucei: Chromosomes 4 and 8 are partial duplicons

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    Jackson Andrew P


    Full Text Available Abstract Background Gene order along the genome sequence of the human parasite Trypanosoma brucei provides evidence for a 0.5 Mb duplication, comprising the 3' regions of chromosomes 4 and 8. Here, the principal aim was to examine the contribution made by this duplication event to the T. brucei genome sequence, emphasising the consequences for gene content and the evolutionary change subsequently experienced by paralogous gene copies. The duplicated region may be browsed online at Results Comparisons of trypanosomatid genomes demonstrated widespread gene loss from each duplicon, but also showed that 47% of duplicated genes were retained on both chromosomes as paralogous loci. Secreted and surface-expressed genes were over-represented among retained paralogs, reflecting a bias towards important factors at the host-parasite interface, and consistent with a dosage-balance hypothesis. Genetic divergence in both coding and regulatory regions of retained paralogs was bimodal, with a deficit in moderately divergent paralogs; in particular, non-coding sequences were either conserved or entirely remodelled. The conserved paralogs included examples of remarkable sequence conservation, but also considerable divergence of both coding and regulatory regions. Sequence divergence typically displayed strong negative selection; but several features, such as asymmetric evolutionary rates, positively-selected codons and other non-neutral substitutions, suggested that divergence of some paralogs was driven by functional change. The absence of orthologs to retained paralogs in T. congolense indicated that the duplication event was specific to T. brucei. Conclusion The duplication of this chromosomal region doubled the dosage of many genes. Rather than creating 'more of the same', these results show that paralogs were structurally modified according to various evolutionary trajectories. The retention of paralogs, and

  11. Chemopreventive effect of methanolic extract of Azadirachta indica on experimental Trypanosoma brucei induced oxidative stress in dogs

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    Temidayo O Omobowale


    Full Text Available Introduction: The medicinal properties of Azadirachta indica have been harnessed for many years in the treatment of many diseases in both humans and animals. Materials and Methods: Twenty-five apparently healthy dogs weighing between 3 and 8 kg were randomly divided into five groups with five dogs in each group. Ameliorative effect of A. indica on erythrocyte antioxidant status and markers of oxidative stress were assessed. Liver and kidney function tests were also performed. Results: Pre-treatment with methanolic extract of Azadirachta indica (MEAI at different doses did not significantly alter the values of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activity in Trypanosoma brucei infection. Although, serum creatinine significantly (P 0.05 difference compared to the values obtained in pre-treated animals. Pre-treatment with 100 mg/kg and 200 mg/kg of A. indica significantly (P < 0.05 decreased serum myeloperoxidase activity at 2 weeks post-infection with T. brucei. Conclusion: From this study, MEAI showed significant ability to attenuate oxidative stress and inflammation during experimental T. brucei infection.

  12. Changes in blood sugar levels of rats experimentally infected withTrypanosoma brucei and treated with imidocarb dipropionate and diminazene aceturate

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    Nwoha Rosemary Ijeoma Ogechi; Omamegbe Joseph Omalathebu


    Objective:To determine the effect ofTrypanosoma brucei (T. brucei) on blood sugar level of infected rats. Methods: The experiment was done with 42 albino rats grouped into 3 groups of 14 members each. Group A was uninfected (control group), Group B was infected withT. brucei and treated with diminazene aceturate, and Group C was infected withT. brucei and treated with imidocarb dipropionate. Blood samples were collected from the media canthus of the experimental rats on Days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 for the assessment of change in blood sugar levels. The blood sugar levels were determined with a glucometer (Accu-chek active serialNo.GN:10023338). Results: By 4 to 5 days post infection, there was a significant increase (P 0.05) was observed in the groups when compared with the control group till Day 12 of the experiment. Conclusions:T. brucei caused a significant increase in blood sugar of infected rats.

  13. Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

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    Jane Claire Munday


    Full Text Available Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (Berenil®, cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (MPXR is the result of loss of a separate High Affinity Pentamidine Transporter (HAPT1. A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the selectivity region of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this

  14. Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei (United States)

    Munday, Jane C.; Settimo, Luca; de Koning, Harry P.


    Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil®), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the “selectivity region” of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a

  15. Dynamic modelling under uncertainty: the case of Trypanosoma brucei energy metabolism.

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    Fiona Achcar


    Full Text Available Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki ( Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies.

  16. Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach

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    Amine Ghozlane


    Full Text Available Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s (bloodstream form and the insect vector (procyclic form, with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.

  17. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei.

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    Diana Spitznagel

    Full Text Available Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1 that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  18. Identification and characterization of an unusual class I myosin involved in vesicle traffic in Trypanosoma brucei. (United States)

    Spitznagel, Diana; O'Rourke, John F; Leddy, Neal; Hanrahan, Orla; Nolan, Derek P


    Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.

  19. The Role of Folate Transport in Antifolate Drug Action in Trypanosoma brucei* (United States)

    Dewar, Simon; Sienkiewicz, Natasha; Ong, Han B.; Wall, Richard J.; Horn, David


    The aim of this study was to identify and characterize mechanisms of resistance to antifolate drugs in African trypanosomes. Genome-wide RNAi library screens were undertaken in bloodstream form Trypanosoma brucei exposed to the antifolates methotrexate and raltitrexed. In conjunction with drug susceptibility and folate transport studies, RNAi knockdown was used to validate the functions of the putative folate transporters. The transport kinetics of folate and methotrexate were further characterized in whole cells. RNA interference target sequencing experiments identified a tandem array of genes encoding a folate transporter family, TbFT1–3, as major contributors to antifolate drug uptake. RNAi knockdown of TbFT1–3 substantially reduced folate transport into trypanosomes and reduced the parasite's susceptibly to the classical antifolates methotrexate and raltitrexed. In contrast, knockdown of TbFT1–3 increased susceptibly to the non-classical antifolates pyrimethamine and nolatrexed. Both folate and methotrexate transport were inhibited by classical antifolates but not by non-classical antifolates or biopterin. Thus, TbFT1–3 mediates the uptake of folate and classical antifolates in trypanosomes, and TbFT1–3 loss-of-function is a mechanism of antifolate drug resistance. PMID:27703008

  20. Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase (United States)

    Terán, David; Hocková, Dana; Česnek, Michal; Zíková, Alena; Naesens, Lieve; Keough, Dianne T.; Guddat, Luke W.


    Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed. PMID:27786284

  1. Association of a novel preribosomal complex in Trypanosoma brucei determined by fluorescence resonance energy transfer. (United States)

    Wang, Lei; Ciganda, Martin; Williams, Noreen


    We have previously reported that the trypanosome-specific proteins P34 and P37 form a unique preribosomal complex with ribosomal protein L5 and 5S rRNA in the nucleoplasm. We hypothesize that this novel trimolecular complex is necessary for stabilizing 5S rRNA in Trypanosoma brucei and is essential for the survival of the parasite. In vitro quantitative analysis of the association between the proteins L5 and P34 is fundamental to our understanding of this novel complex and thus our ability to exploit its unique characteristics. Here we used in vitro fluorescence resonance energy transfer (FRET) to analyze the association between L5 and P34. First, we demonstrated that FRET can be used to confirm the association between L5 and P34. We then determined that the binding constant for L5 and P34 is 0.60 ± 0.03 μM, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we used FRET to identify the critical regions of L5 and P34 involved in the protein-protein association. We found that the N-terminal APK-rich domain and RNA recognition motif (RRM) of P34 and the L18 domain of L5 are important for the association of the two proteins with each other. These results provide us with the framework for the discovery of ways to disrupt this essential complex.

  2. Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei. (United States)

    Leaver, David J; Patkar, Presheet; Singha, Ujjal K; Miller, Matthew B; Haubrich, Brad A; Chaudhuri, Minu; Nes, W David


    Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Here, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol, and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while having no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min(-1)/0.24 min(-1); partition ratio of 1.08), whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ(24)-sterol is a promising strategy for developing a new treatment for trypanosomiasis.

  3. Parasite development and host responses during the establishment of Trypanosoma brucei infection transmitted by tsetse fly. (United States)

    Barry, J D; Emergy, D L


    Following inoculation of Trypanosoma brucei into large mammals by the tsetse fly a local skin reaction, the 'chancre', develops due to trypanosome proliferation. We have cannulated the afferent and efferent lymphatics of the draining lymph node in goats and examined the onset of a cellular reaction, the emigration of the parasite from the chancre and the development of both antigenic variation and the specific immune response. The chancre first became detectable by day 3 post-infection, peaked by day 6 and then subsided. Lymphocyte output increased 6- to 8-fold by day 10 and the number of lymphoblasts increased 50-fold in this period. Both then declined. Trypanosomes were detected in lymph 1-2 days before the chancre, peaked by days 5-6, declined during development of the chancre and then peaked again. The bloodstream population appeared by days 4-5 and displayed different kinetics from that in lymph. Recirculation of parasites through the lymphatics ensued. Lymph-borne trypanosome populations were highly pleomorphic. Parasites in lymph expressed firstly a mixture of the Variable Antigen Types (VATs) which are found characteristically in the tsetse fly, this being followed by a mixture of other VATs. The two groups overlapped in appearance. In the bloodstream the same sequence of events occurred although 2 or 3 days later. The specific antibody response, as measured by radioimmunoassay and agglutination, arose within a few days of the first detection of each VAT. Activities appeared first in the lymph and then in plasma.

  4. Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei. (United States)

    Ramey-Butler, Kiantra; Ullu, Elisabetta; Kolev, Nikolay G; Tschudi, Christian


    One distinctive feature of the Trypanosoma brucei life cycle is the presence of two discrete populations that are based on differential expression of variant surface glycoproteins (VSGs). Both are adapted to the environmental pressures they face and more importantly, both contribute directly to transmission. Metacyclics in the tsetse fly enable transmission to a new mammalian host, whereas bloodstream trypanosomes must avoid immune destruction to the extent that sufficient numbers are available for transmission, when the insect vector takes a blood meal. At present, there are few investigations on the molecular aspects of parasite biology in the tsetse vector and specifically about the activation of metacyclic VSG gene expression. Here we used an established in vitro differentiation system based on the overexpression of the RNA-binding protein 6 (RBP6), to monitor two metacyclic VSGs (VSG 397 and VSG 653) during development from procyclics to infectious metacyclic forms. We observed that activation of these two mVSGs was simultaneous both at the transcript and protein level, and manifested by the appearance of only one of the mVSGs in individual cells.

  5. Identification of a new EF-hand superfamily member from Trypanosoma brucei (United States)

    Wong, S.; Kretsinger, R. H.; Campbell, D. A.


    We identified several open reading frames between the regions encoding calmodulin and ubiquitin-EP52/1 in the genome of Trypanosoma brucei. One of these, EFH5, encodes a protein 192 amino acids long. The EFH5 transcript is present in poly(A)+ mRNA and is present at similar levels in the mammalian bloodstream form and the insect procyclic form. EFH5 contains four EF-hand homolog domains, two of which are inferred to bind Ca2+ ions. We expressed EFH5 as a fusion protein in Escherichia coli and demonstrated calcium-binding activity of the fusion protein using the 45Ca-overlay technique. The function of EFH5 remains unknown; however, as the fourth EF-hand homolog identified in trypanosomes, it attests to the broad range of functions assumed by calcium functioning as a second messenger. EFH5, which is most closely related to LAV1-2 from Physarum, represents a distinct subfamily among the EF-hand-containing proteins.

  6. Rediscovery of Trypanosoma (Pycnomonas) suis, a tsetse-transmitted trypanosome closely related to T. brucei. (United States)

    Hutchinson, Rachel; Gibson, Wendy


    The African tsetse-transmitted trypanosomes are considered to be a well-known group of parasitic protozoa, but in 2008 a novel and distinctive trypanosome related to Trypanosoma brucei was discovered among tsetse isolates from Msubugwe in Tanzania. The host range, distribution and potential pathogenicity of this new trypanosome remain to be elucidated; such studies would be facilitated by a sensitive and specific identification method. Here, we identified two highly repetitive elements in the genome of the new trypanosome: a 177 bp repeat, which was located predominantly on the highly abundant minichromosomes, and a 138 bp repeat, which was widely dispersed in the genome. A PCR test based on each repeat was specific for the new trypanosome and sensitive to Trypanosoma (Pycnomonas) suis. We also present data on the molecular karyotype and spliced leader (SL, miniexon) repeat of the new trypanosome, both of which distinguish T. suis from other, better-known African tsetse-transmitted trypanosomes. The rediscovery of T. suis opens new lines of research into the evolution and biology of the African trypanosomes.

  7. Astrovirus Diagnostics (United States)

    Pérot, Philippe; Lecuit, Marc; Eloit, Marc


    Various methods exist to detect an astrovirus infection. Current methods include electron microscopy (EM), cell culture, immunoassays, polymerase chain reaction (PCR) and various other molecular approaches that can be applied in the context of diagnostic or in surveillance studies. With the advent of metagenomics, novel human astrovirus (HAstV) strains have been found in immunocompromised individuals in association with central nervous system (CNS) infections. This work reviews the past and current methods for astrovirus detection and their uses in both research laboratories and for medical diagnostic purposes. PMID:28085120

  8. New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle. (United States)

    van Weelden, Susanne W H; van Hellemond, Jaap J; Opperdoes, Fred R; Tielens, Aloysius G M


    We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

  9. Trypanosoma brucei gambiense adaptation to different mammalian sera is associated with VSG expression site plasticity.

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    Carlos Cordon-Obras

    Full Text Available Trypanosoma brucei gambiense infection is widely considered an anthroponosis, although it has also been found in wild and domestic animals. Thus, fauna could act as reservoir, constraining the elimination of the parasite in hypo-endemic foci. To better understand the possible maintenance of T. b. gambiense in local fauna and investigate the molecular mechanisms underlying adaptation, we generated adapted cells lines (ACLs by in vitro culture of the parasites in different mammalian sera. Using specific antibodies against the Variant Surface Glycoproteins (VSGs we found that serum ACLs exhibited different VSG variants when maintained in pig, goat or human sera. Although newly detected VSGs were independent of the sera used, the consistent appearance of different VSGs suggested remodelling of the co-transcribed genes at the telomeric Expression Site (VSG-ES. Thus, Expression Site Associated Genes (ESAGs sequences were analysed to investigate possible polymorphism selection. ESAGs 6 and 7 genotypes, encoding the transferrin receptor (TfR, expressed in different ACLs were characterised. In addition, we quantified the ESAG6/7 mRNA levels and analysed transferrin (Tf uptake. Interestingly, the best growth occurred in pig and human serum ACLs, which consistently exhibited a predominant ESAG7 genotype and higher Tf uptake than those obtained in calf and goat sera. We also detected an apparent selection of specific ESAG3 genotypes in the pig and human serum ACLs, suggesting that other ESAGs could be involved in the host adaptation processes. Altogether, these results suggest a model whereby VSG-ES remodelling allows the parasite to express a specific set of ESAGs to provide selective advantages in different hosts. Finally, pig serum ACLs display phenotypic adaptation parameters closely related to human serum ACLs but distinct to parasites grown in calf and goat sera. These results suggest a better suitability of swine to maintain T. b. gambiense infection

  10. Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate. (United States)

    Dawson, Alice; Gibellini, Federica; Sienkiewicz, Natasha; Tulloch, Lindsay B; Fyfe, Paul K; McLuskey, Karen; Fairlamb, Alan H; Hunter, William N


    The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

  11. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

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    Johanna L Höög


    Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

  12. Genome-wide dissection of the quorum sensing signalling pathway in Trypanosoma brucei. (United States)

    Mony, Binny M; MacGregor, Paula; Ivens, Alasdair; Rojas, Federico; Cowton, Andrew; Young, Julie; Horn, David; Matthews, Keith


    The protozoan parasites Trypanosoma brucei spp. cause important human and livestock diseases in sub-Saharan Africa. In mammalian blood, two developmental forms of the parasite exist: proliferative 'slender' forms and arrested 'stumpy' forms that are responsible for transmission to tsetse flies. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. This response is triggered by an elusive 'stumpy induction factor' (SIF) whose intracellular signalling pathway is also uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains respond inefficiently to SIF but can generate forms with stumpy characteristics when exposed to cell-permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNA interference library screen to identify the signalling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) or 8-pCPT-2'-O-methyl-5'-AMP to select cells that were unresponsive to these signals and hence remained proliferative. Genome-wide Ion Torrent based RNAi target sequencing identified cohorts of genes implicated in each step of the signalling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. Genes at each step were independently validated in cells naturally capable of stumpy formation, confirming their role in density sensing in vivo. The putative RNA-binding protein, RBP7, was required for normal quorum sensing and promoted cell-cycle arrest and transmission competence when overexpressed. This study reveals that quorum sensing signalling in trypanosomes shares similarities to fundamental quiescence pathways in eukaryotic cells, its components providing targets for quorum-sensing interference-based therapeutics.

  13. A monoclonal antibody marker for the exclusion-zone filaments of Trypanosoma brucei

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    Decossas Marion


    Full Text Available Abstract Background Trypanosoma brucei is a haemoflagellate pathogen of man, wild animals and domesticated livestock in central and southern Africa. In all life cycle stages this parasite has a single mitochondrion that contains a uniquely organised genome that is condensed into a flat disk-like structure called the kinetoplast. The kinetoplast is essential for insect form procyclic cells and therefore is a potential drug target. The kinetoplast is unique in nature because it consists of novel structural proteins and thousands of circular, interlocking, DNA molecules (kDNA. Secondly, kDNA replication is critically timed to coincide with nuclear S phase and new flagellum biogenesis. Thirdly, the kinetoplast is physically attached to the flagellum basal bodies via a structure called the tripartite attachment complex (TAC. The TAC consists of unilateral filaments (within the mitochondrion matrix, differentiated mitochondrial membranes and exclusion-zone filaments that extend from the distal end of the basal bodies. To date only one protein, p166, has been identified to be a component of the TAC. Results In the work presented here we provide data based on a novel EM technique developed to label and characterise cytoskeleton structures in permeabilised cells without extraction of mitochondrion membranes. We use this protocol to provide data on a new monoclonal antibody reagent (Mab 22 and illustrate the precise localisation of basal body-mitochondrial linker proteins. Mab 22 binds to these linker proteins (exclusion-zone filaments and provides a new tool for the characterisation of cytoskeleton mediated kinetoplast segregation. Conclusion The antigen(s recognised by Mab 22 are cytoskeletal, insensitive to extraction by high concentrations of non-ionic detergent, extend from the proximal region of basal bodies and bind to the outer mitochondrial membrane. This protein(s is the first component of the TAC exclusion-zone fibres to be identified. Mab 22

  14. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei. (United States)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K; Lübeck, Peter S


    Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities of the enzymes, pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and fumarase (FUM), involved in the reductive tricarboxylic acid (rTCA) branch, were examined and compared in cells harvested from the acid production medium and a complete medium. The results showed that ambient pH had a significant impact on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led to an enhanced production of succinic acid in frd transformants compared with the wild-type in both pH buffered and pH non-buffered conditions with highest amount produced in the pH buffered condition (16.2 ± 0.5 g/L). This study demonstrates the feasibility of increasing succinic acid production through the cytosolic reductive pathway by genetic engineering in A. saccharolyticus.

  15. Human African trypanosomiasis in endemic populations and travellers. (United States)

    Blum, J A; Neumayr, A L; Hatz, C F


    Human African trypanosomiasis (HAT) or sleeping sickness is caused by the protozoan parasites Trypanosoma brucei (T.b.) gambiense (West African form) and T.b. rhodesiense (East African form) that are transmitted by the bite of the tsetse fly, Glossina spp.. Whereas most patients in endemic populations are infected with T.b. gambiense, most tourists are infected with T.b. rhodesiense. In endemic populations, T.b. gambiense HAT is characterized by chronic and intermittent fever, headache, pruritus, and lymphadenopathy in the first stage and by sleep disturbances and neuro-psychiatric disorders in the second stage. Recent descriptions of the clinical presentation of T.b. rhodesiense in endemic populations show a high variability in different foci. The symptomatology of travellers is markedly different from the usual textbook descriptions of African HAT patients. The onset of both infections is almost invariably an acute and febrile disease. Diagnosis and treatment are difficult and rely mostly on old methods and drugs. However, new molecular diagnostic technologies are under development. A promising new drug combination is currently evaluated in a phase 3 b study and further new drugs are under evaluation.

  16. The F(0F(1-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei.

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    Alena Zíková


    Full Text Available The mitochondrial F(0F(1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F(0F(1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F(1 subunits, three to F(0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F(1 alpha subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage cells and are important for the structural integrity of the F(0F(1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.

  17. SAS-4 Protein in Trypanosoma brucei Controls Life Cycle Transitions by Modulating the Length of the Flagellum Attachment Zone Filament. (United States)

    Hu, Huiqing; Zhou, Qing; Li, Ziyin


    The evolutionarily conserved centriole/basal body protein SAS-4 regulates centriole duplication in metazoa and basal body duplication in flagellated and ciliated organisms. Here, we report that the SAS-4 homolog in the flagellated protozoan Trypanosoma brucei, TbSAS-4, plays an unusual role in controlling life cycle transitions by regulating the length of the flagellum attachment zone (FAZ) filament, a specialized cytoskeletal structure required for flagellum adhesion and cell morphogenesis. TbSAS-4 is concentrated at the distal tip of the FAZ filament, and depletion of TbSAS-4 in the trypomastigote form disrupts the elongation of the new FAZ filament, generating cells with a shorter FAZ associated with a longer unattached flagellum and repositioned kinetoplast and basal body, reminiscent of epimastigote-like morphology. Further, we show that TbSAS-4 associates with six additional FAZ tip proteins, and depletion of TbSAS-4 disrupts the enrichment of these FAZ tip proteins at the new FAZ tip, suggesting a role of TbSAS-4 in maintaining the integrity of this FAZ tip protein complex. Together, these results uncover a novel function of TbSAS-4 in regulating the length of the FAZ filament to control basal body positioning and life cycle transitions in T. brucei.

  18. Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression. (United States)

    Valenciano, Ana L; Ramsey, Aaron C; Mackey, Zachary B


    The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

  19. A proteomics approach reveals molecular manipulators of distinct cellular processes in the salivary glands of Glossina m. morsitans in response to Trypanosoma b. brucei infections

    NARCIS (Netherlands)

    Kariithi, Henry M.; Boeren, Sjef; Murungi, Edwin K.; Vlak, Just M.; Abd-Alla, Adly M.M.


    Background: Glossina m. morsitans is the primary vector of the Trypanosoma brucei group, one of the causative agents of African trypanosomoses. The parasites undergo metacyclogenesis, i.e. transformation into the mammalian-infective metacyclic trypomastigote (MT) parasites, in the salivary glands

  20. Analysis of cosmid clones of nuclear DNA from Trypanosome brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

    NARCIS (Netherlands)

    D. Valerio (Dinko); T. de Lange; P. Borst (Piet); F.G. Grosveld (Frank); L.H.T. van der Ploeg


    textabstractTrypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expres

  1. Development of multiplex serological assay for the detection of human African trypanosomiasis. (United States)

    Nzou, Samson Muuo; Fujii, Yoshito; Miura, Masashi; Mwau, Matilu; Mwangi, Anne Wanjiru; Itoh, Makoto; Salam, Md Abdus; Hamano, Shinjiro; Hirayama, Kenji; Kaneko, Satoshi


    Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance. We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.

  2. The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei

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    Ferris Vanessa


    Full Text Available Abstract Background Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy. Results To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP or Red Fluorescent Protein (RFP. Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones. Conclusion The strategy of using production of yellow hybrids

  3. Thyroid diagnostics

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    Scriba, P.C.; Boerner, W.; Emrich, S.; Gutekunst, R.; Herrmann, J.; Horn, K.; Klett, M.; Krueskemper, H.L.; Pfannenstiel, P.; Pickardt, C.R.


    None of the in-vitro and in-vivo methods listed permits on unambiguous diagnosis when applied alone, owing to the fact that similar or even identical findings are obtained for various individual parameters in different thyroid diseases. Further, especially the in-vitro tests are also subject to extrathyroidal effects which may mask the typical findings. The limited and varying specificity and sensitivity of the tests applied, as well as the falsification of results caused by the patients' idiosyncracies and the methodology, make it necessary to interpret and evaluate the in-vivo and in-vitro findings only if the clinical situation (anamnesis and physical examination) is known. For maximum diagnostic quality of the tests, the initial probability of the assumed type of thyroid disease must be increased (formulation of the clinical problem). The concepts of exclusion diagnosis and identification must be distinguished as well as the diagnosis of functional disturbances on the one hand and of thyroid diseases on the other. Both of this requires a qualified, specific and detailed anamnesis and examination procedure, and the clinical examination remains the obligatory basis of clinical diagnostics. In case of inexplicable discrepancies between the clinical manifestations and the findings obtained with specific methods, or between the findings obtained with a specific method, the patient should be referred to an expert institution, or the expert institution should be consulted.

  4. The Aurora Kinase in Trypanosoma brucei plays distinctive roles in metaphase-anaphase transition and cytokinetic initiation.

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    Ziyin Li


    Full Text Available Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC, consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and

  5. Effects of supplementing Erythrina brucei leaf as a substitute for cotton seed meal on growth performance and carcass characteristics of Sidama goats fed basal diet of natural grass hay. (United States)

    Yinnesu, Asmamaw; Nurfeta, Ajebu


    The replacement value of dried Erythrina brucei leaf for cotton seed meal (CSM) on growth performance and carcass characteristics was evaluated. Twenty-five yearling buck goats (15.8 ± 1.4 kg) were assigned into five treatments in a randomized complete block design: natural grass hay alone (T1) or supplemented with 100% CSM (T2), 67% CSM + 33% E. brucei (T3), 33% CSM + 67% E. brucei (T4), and 100% E. brucei (T5) on dry matter (DM) basis. Supplemented goats consumed more (P  0.05) by the proportion of the supplements. The highest (P goats supplemented with CSM alone, whereas the lowest intake was observed in the non-supplemented group. Total CP intake decreased (P goats gained more (P goats than in the non-supplemented ones, but similar (P > 0.05) among the supplemented group. The digestibility of CP was higher (P goats, except in those goats fed E. brucei alone, than the non-supplemented group. Slaughter weight, empty body weight, hot carcass weight, dressing percentage, rib eye muscle area, and total edible offals were higher (P goats than for the non-supplemented ones. It could be concluded that E. brucei could be used as a substitute to CSM under smallholder production systems.

  6. Rotorcraft Diagnostics (United States)

    Haste, Deepak; Azam, Mohammad; Ghoshal, Sudipto; Monte, James


    Health management (HM) in any engineering systems requires adequate understanding about the system s functioning; a sufficient amount of monitored data; the capability to extract, analyze, and collate information; and the capability to combine understanding and information for HM-related estimation and decision-making. Rotorcraft systems are, in general, highly complex. Obtaining adequate understanding about functioning of such systems is quite difficult, because of the proprietary (restricted access) nature of their designs and dynamic models. Development of an EIM (exact inverse map) solution for rotorcraft requires a process that can overcome the abovementioned difficulties and maximally utilize monitored information for HM facilitation via employing advanced analytic techniques. The goal was to develop a versatile HM solution for rotorcraft for facilitation of the Condition Based Maintenance Plus (CBM+) capabilities. The effort was geared towards developing analytic and reasoning techniques, and proving the ability to embed the required capabilities on a rotorcraft platform, paving the way for implementing the solution on an aircraft-level system for consolidation and reporting. The solution for rotorcraft can he used offboard or embedded directly onto a rotorcraft system. The envisioned solution utilizes available monitored and archived data for real-time fault detection and identification, failure precursor identification, and offline fault detection and diagnostics, health condition forecasting, optimal guided troubleshooting, and maintenance decision support. A variant of the onboard version is a self-contained hardware and software (HW+SW) package that can be embedded on rotorcraft systems. The HM solution comprises components that gather/ingest data and information, perform information/feature extraction, analyze information in conjunction with the dependency/diagnostic model of the target system, facilitate optimal guided troubleshooting, and offer

  7. Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes. (United States)

    Canning, Peter; Rea, Dean; Morty, Rory E; Fülöp, Vilmos


    Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

  8. Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes.

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    Peter Canning

    Full Text Available Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

  9. Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei. (United States)

    Kafková, Lucie; Ammerman, Michelle L; Faktorová, Drahomíra; Fisk, John C; Zimmer, Sara L; Sobotka, Roman; Read, Laurie K; Lukes, Julius; Hashimi, Hassan


    A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

  10. Mathematical modelling of polyamine metabolism in bloodstream-form Trypanosoma brucei: an application to drug target identification.

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    Xu Gu

    Full Text Available We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT and ornithine production (OrnPt have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.

  11. Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen.

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    Nathaniel G Jones


    Full Text Available The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2, depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

  12. The orthologue of Sjogren's syndrome nuclear autoantigen 1 (SSNA1 in Trypanosoma brucei is an immunogenic self-assembling molecule.

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    Helen P Price

    Full Text Available Primary Sjögren's Syndrome (PSS is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14 is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13 and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.

  13. Crystal Structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.

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    Iain D Kerr

    Full Text Available BACKGROUND: Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei. METHODS AND FINDINGS: We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002. CONCLUSIONS: The mature domain of our TbCat*CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat*CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain*K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

  14. Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole.

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    Chiung-Kuang Chen

    Full Text Available BACKGROUND: Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14alpha-demethylase (CYP51, which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding. METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 A and 2.27 A, and from the related pathogen T. brucei (resolutions of 2.7 A and 2.6 A, co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180 degrees depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target. CONCLUSIONS/SIGNIFICANCE: The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and

  15. Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.

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    Stuart K Archer


    Full Text Available Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9--LIGKA, PNT1 and PNT2--were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3' UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3' UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.

  16. Diagnostic thoracoscopy

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    Plavec Goran


    Full Text Available Diagnostic thoracoscopy in patients with pleural effusion of unclear origin mostly provides the correct diagnosis. Results from published reports of previous researches are not uniform. In 47 male and 20 female patients with pleural effusion of unknown etiology, after receiving negative results obtained from cytological finding of pleural effusion and percutaneous needle biopsy, thoracoscopy with biopsy of one or both pleurae was performed. Procedure was done in local anesthesia using Stortz rigid thoracoscope. In 37 patients with malignant disease (primary or metastatic diagnosis was confirmed histopathologically in 31 patient (81.12%. In 27 patients with inflammatory pleural disease diagnosis was confirmed histopathologically in 22 patients (81.4%. Among 11 patients with specific pleural effusions, tuberculosis was confirmed in 10 (90.91%. Normal finding in cases of spontaneous pneumothorax and pulmonary embolism was taken as a positive result. Total number of positive findings was 55 (82.10%. In one patient, the third spontaneous pneumothorax was the indication for thoracoscopy, and after numerous bullae were seen during the procedure, talcum powder pleurodesis was done. In four patients low intensity subcutaneous emphysema occurred one day after thoracoscopy. It can be concluded that thoracoscopy in local anesthesia out of the operating room is good and practical method for solving the unclear pleural effusions, with neglectable rate of complications.

  17. Effect of experimental single Ancylostoma caninum and mixed infections of Trypanosoma brucei and Trypanosoma congolense on the humoural immune response to anti-rabies vaccination in dogs

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    Nwoha Rosemary Ijeoma Ogechi; Anene Boniface Maduka


    Objective:To determine the effect of Ancylostoma caninum (A. caninum) and trypanosome parasites on the immune response to vaccination in dogs in endemic environments. Methods:Sixteen dogs for the experiment were grouped into 4 of 4 members each. Group I was the uninfected control one, and GPII was infected with A. caninum; GPIII was infected with A. caninum/Trypanosoma congolense (T. congolense), and GPIV was infected with Trypanosoma brucei (T. brucei)/A. caninum. The dogs were first vaccinated with antirabies vaccine before infecting GPII, GPIII and GPIV with A. caninum which were done 4 weeks after vaccination. By 2-week post-vaccination, trypanosome parasites were superimposed on both GPIII and GPIV. A secondary vaccination was given to GPI, GPII, GPIII, and GPIV by Week 12 of the experiment (4 weeks post treatment). Results:The prepatent period was (3.00 ± 1.40) days, in the conjunct infection of T. brucei/A. caninum. It was (9.00 ± 1.10) days, in conjunct T. congolense/A. caninum. The prepatent period of A. caninum was (14.0 ± 2.0) days in the single A. caninum group and (13.0 ± 1.0) days in the conjunct trypanosome/A. caninum. At the 1st week after vaccination, the antibody titer in all the vaccinated groups (GPI, GPII, GPIII, and GPIV) significantly increased (P Conclusions:It was therefore concluded that A. caninum, T. brucei and T. congolense induced immunosuppression in antirabies vaccination in dogs.

  18. Parasite-based screening and proteome profiling reveal orlistat, an FDA-approved drug, as a potential anti Trypanosoma brucei agent. (United States)

    Yang, Peng-Yu; Wang, Min; Liu, Kai; Ngai, Mun Hong; Sheriff, Omar; Lear, Martin J; Sze, Siu Kwan; He, Cynthia Y; Yao, Shao Q


    Trypanosoma brucei is a parasite that causes African sleeping sickness in humans and nagana in livestock and is transmitted by the tsetse fly. There is an urgent need for the development of new drugs against African trypanosomiasis due to the lack of vaccines and effective drugs. Orlistat (also called tetrahydrolipstatin or THL) is an FDA-approved antiobesity drug targeting primarily the pancreatic and gastric lipases within the gastrointestinal tract. It shows potential activities against tumors, mycobacteria, and parasites. Herein, we report the synthesis and evaluation of an expanded set of orlistat-like compounds, some of which showed highly potent trypanocidal activities in both the bloodstream form (BSF) and the procyclic form (PCF) of T. brucei. Subsequent in situ parasite-based proteome profiling was carried out to elucidate potential cellular targets of the drug in both forms. Some newly identified targets were further validated by the labeling of recombinantly expressed enzymes in Escherichia coli lysates. Bioimaging experiments with a selected compound were carried out to study the cellular uptake of the drug in T. brucei. Results indicated that orlistat is much more efficiently taken up by the BSF than the PCF of T. brucei and has clear effects on the morphology of mitochondria, glycosomes, and the endoplasmic reticulum in both BSF and PCF cells. These results support specific effects of orlistat on these organelles and correlate well with our in situ proteome profiling. Given the economic challenges of de novo drug development for neglected diseases, we hope that our findings will stimulate further research towards the conversion of orlistat-like compounds into new trypanocidal drugs.

  19. Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi


    Ward Pauline N; Worthey Elizabeth A; Parsons Marilyn; Mottram Jeremy C


    Abstract Background The trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordi...

  20. Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery.

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    Jan Mani

    Full Text Available Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs. GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are

  1. A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

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    E DeJesus

    Full Text Available Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1. This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT, escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

  2. Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

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    Darren J Creek


    Full Text Available Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

  3. Molecular Evidence of a Trypanosoma brucei gambiense Sylvatic Cycle in the Human African Trypanosomiasis Foci of Equatorial Guinea

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    Carlos eCordon-Obras


    Full Text Available Gambiense trypanosomiasis is considered an anthroponotic disease. Consequently, control programs are generally aimed at stopping transmission of Trypanosoma brucei gambiense (T. b. gambiense by detecting and treating human cases. However, the persistence of numerous foci despite efforts to eliminate this disease questions this strategy as unique tool to pursue the eradication. The role of animals as a reservoir of T. b. gambiense is still controversial, but could partly explain maintenance of the infection at hypo-endemic levels. In the present study, we evaluated the presence of T. b. gambiense in wild animals in Equatorial Guinea. The infection rate ranged from 0.8% in the insular focus of Luba to more than 12% in Mbini, a focus with a constant trickle of human cases. The parasite was detected in a wide range of animal species including four species never described previously as putative reservoirs. Our study comes to reinforce the hypothesis that animals may play a role in the persistence of T. b. gambiense transmission, being particularly relevant in low transmission settings. Under these conditions the integration of sustained vector control and medical interventions should be considered to achieve the elimination of Gambiense trypanosomiasis.

  4. A global comparison of the human and T. brucei degradomes gives insights about possible parasite drug targets.

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    Susan T Mashiyama

    Full Text Available We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of Homo sapiens and the human parasite Trypanosoma brucei. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups ("M32" and "C51" that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at

  5. Human African trypanosomiasis: a review of non-endemic cases in the past 20 years. (United States)

    Migchelsen, Stephanie J; Büscher, Philippe; Hoepelman, Andy I M; Schallig, Henk D F H; Adams, Emily R


    Human African trypanosomiasis (HAT) is caused by sub-species of the parasitic protozoan Trypanosoma brucei and is transmitted by tsetse flies, both of which are endemic only to sub-Saharan Africa. Several cases have been reported in non-endemic areas, such as North America and Europe, due to travelers, ex-patriots or military personnel returning from abroad or due to immigrants from endemic areas. In this paper, non-endemic cases reported over the past 20 years are reviewed; a total of 68 cases are reported, 19 cases of Trypanosoma brucei gambiense HAT and 49 cases of Trypanosoma brucei rhodesiense HAT. Patients ranged in age from 19 months to 72 years and all but two patients survived. Physicians in non-endemic areas should be aware of the signs and symptoms of this disease, as well as methods of diagnosis and treatment, especially as travel to HAT endemic areas increases. We recommend extension of the current surveillance systems such as TropNetEurop and maintaining and promotion of existing reference centers of diagnostics and expertise. Important contact information is also included, should physicians require assistance in diagnosing or treating HAT.

  6. Discovery of Infection Associated Metabolic Markers in Human African Trypanosomiasis. (United States)

    Lamour, Sabrina D; Gomez-Romero, Maria; Vorkas, Panagiotis A; Alibu, Vincent P; Saric, Jasmina; Holmes, Elaine; Sternberg, Jeremy M


    Human African trypanosomiasis (HAT) remains a major neglected tropical disease in Sub-Saharan Africa. As clinical symptoms are usually non-specific, new diagnostic and prognostic markers are urgently needed to enhance the number of identified cases and optimise treatment. This is particularly important for disease caused by Trypanosoma brucei rhodesiense, where indirect immunodiagnostic approaches have to date been unsuccessful. We have conducted global metabolic profiling of plasma from T.b.rhodesiense HAT patients and endemic controls, using 1H nuclear magnetic resonance (NMR) spectroscopy and ultra-performance liquid chromatography, coupled with mass spectrometry (UPLC-MS) and identified differences in the lipid, amino acid and metabolite profiles. Altogether 16 significantly disease discriminatory metabolite markers were found using NMR, and a further 37 lipid markers via UPLC-MS. These included significantly higher levels of phenylalanine, formate, creatinine, N-acetylated glycoprotein and triglycerides in patients relative to controls. HAT patients also displayed lower concentrations of histidine, sphingomyelins, lysophosphatidylcholines, and several polyunsaturated phosphatidylcholines. While the disease metabolite profile was partially consistent with previous data published in experimental rodent infection, we also found unique lipid and amino acid profile markers highlighting subtle but important differences between the host response to trypanosome infections between animal models and natural human infections. Our results demonstrate the potential of metabolic profiling in the identification of novel diagnostic biomarkers and the elucidation of pathogenetic mechanisms in this disease.

  7. Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei.

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    David Reynolds


    Full Text Available Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V, at sites involved in RNA Polymerase (RNAP II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase. As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination.

  8. Diagnostic Algorithm Benchmarking (United States)

    Poll, Scott


    A poster for the NASA Aviation Safety Program Annual Technical Meeting. It describes empirical benchmarking on diagnostic algorithms using data from the ADAPT Electrical Power System testbed and a diagnostic software framework.

  9. Thioaptamer Diagnostic System Project (United States)

    National Aeronautics and Space Administration — AM Biotechnologies (AM) will develop a diagnostic system in response to SBIR Topic X10.01 Reusable Diagnostic Lab Technology that will simultaneously detect and...

  10. Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei.

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    Christal Worthen

    Full Text Available BACKGROUND: The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage. METHODOLOGY/PRINCIPAL FINDINGS: We therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D(2, quercetin, etoposide, camptothecin, and a tetrahydroquinoline and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds. CONCLUSIONS/SIGNIFICANCE: Parasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or

  11. Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei.

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    Rommie E Amaro

    Full Text Available RNA editing ligase 1 (TbREL1 is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

  12. Genetic character of drug resistance in Trpanosoma brucei%锥虫抗药性的遗传特性

    Institute of Scientific and Technical Information of China (English)

    廖党金; 沈杰


    @@ 锥虫病是由锥虫寄生于人和动物的血液而引起人和动物的一种疾病.锥虫抗药性的产生,可能给人和家畜带来较严重的后果[1],国外于1908年已注意到锥虫的抗药性问题[2、3],而我国于1988年第一次口头报道在云南用厂方推荐的安锥赛治疗剂量难以治愈家畜锥虫病,1991年沈杰等正式报道了我国伊氏锥虫云南株和安徽株对安锥赛已产生抗药性[4].迄今,报告产生抗药性的锥虫虫种有布氏锥虫(Trypanosoma brucei)等常见的8个种,其它未报告的锥虫并不意味着不产生抗药性.已报告产生抗药性的药物有苏拉明(Suramine)等30多种抗锥虫药物.抗药性是能遗传的,对锥虫抗药性的遗传特性的了解,能帮助我们正确地选择和使用药物、减少和避免抗药性的产生及怎样消除已有的抗药性具有极其重要的作用,其次也有助于锥虫的生理、生化及生物学研究.

  13. Crystal Structures of Trypanosoma brucei Sterol 14[alpha]-Demethylase and Implications for Selective Treatment of Human Infections

    Energy Technology Data Exchange (ETDEWEB)

    Lepesheva, Galina I.; Park, Hee-Won; Hargrove, Tatiana Y.; Vanhollebeke, Benoit; Wawrzak, Zdzislaw; Harp, Joel M.; Sundaramoorthy, Munirathinam; Nes, W. David; Pays, Etienne; Chaudhuri, Minu; Villalta, Fernando; Waterman, Michael R. (ULdB); (Vanderbilt); (TTU); (Toronto); (NWU); (Meharry)


    Sterol 14{alpha}-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 {angstrom} resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.

  14. A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei. (United States)

    Hu, Huiqing; Zhou, Qing; Li, Ziyin


    The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.

  15. Nucleolar accumulation of RNA binding proteins induced by Actinomycin D is functional in Trypanosoma cruzi and Leishmania mexicana but not in T. brucei.

    Directory of Open Access Journals (Sweden)

    Ezequiel Názer

    Full Text Available We have recently shown in T. cruzi that a group of RNA Binding Proteins (RBPs, involved in mRNA metabolism, are accumulated into the nucleolus in response to Actinomycin D (ActD treatment. In this work, we have extended our analysis to other members of the trypanosomatid lineage. In agreement with our previous study, the mechanism seems to be conserved in L. mexicana, since both endogenous RBPs and a transgenic RBP were relocalized to the nucleolus in parasites exposed to ActD. In contrast, in T. brucei, neither endogenous RBPs (TbRRM1 and TbPABP2 nor a transgenic RBP from T. cruzi were accumulated into the nucleolus under such treatment. Interestingly, when a transgenic TbRRM1 was expressed in T. cruzi and the parasites exposed to ActD, TbRRM1 relocated to the nucleolus, suggesting that it contains the necessary sequence elements to be targeted to the nucleolus. Together, both experiments demonstrate that the mechanism behind nucleolar localization of RBPs, which is present in T. cruzi and L. mexicana, is not functional in T. brucei, suggesting that it has been lost or retained differentially during the evolution of the trypanosomatid lineage.

  16. Deletion of a novel protein kinase with PX and FYVE-related domains increases the rate of differentiation of Trypanosoma brucei. (United States)

    Vassella, E; Krämer, R; Turner, C M; Wankell, M; Modes, C; van den Bogaard, M; Boshart, M


    Growth control of African trypanosomes in the mammalian host is coupled to differentiation of a non-dividing life cycle stage, the stumpy bloodstream form. We show that a protein kinase with novel domain architecture is important for growth regulation. Zinc finger kinase (ZFK) has a kinase domain related to RAC and S6 kinases flanked by a FYVE-related zinc finger and a phox (PX) homology domain. To investigate the function of the kinase during cyclical development, a stable transformation procedure for bloodstream forms of differentiation-competent (pleomorphic) Trypanosoma brucei strains was established. Deletion of both allelic copies of ZFK by homologous recombination resulted in reduced growth of bloodstream-form parasites in culture, which was correlated with an increased rate of differentiation to the non-dividing stumpy form. Growth and differentiation rates were returned to wild-type level by ectopic ZFK expression. The phenotype is stage-specific, as growth of procyclic (insect form) trypanosomes was unaffected, and Deltazfk/Deltazfk clones were able to undergo full cyclical development in the tsetse fly vector. Deletion of ZFK in a differentiation-defective (monomorphic) strain of T. brucei did not change its growth rate in the bloodstream stage. This suggests a function of ZFK associated with the trypanosomes' decision between either cell cycle progression, as slender bloodstream form, or differentiation to the non-dividing stumpy form.

  17. Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A. (United States)

    Kirkham, Justin K; Park, Sung Hee; Nguyen, Tu N; Lee, Ju Huck; Günzl, Arthur


    Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

  18. Evaluation of Antigens for Development of a Serological Test for Human African Trypanosomiasis (United States)

    Biéler, Sylvain; Waltenberger, Harald; Barrett, Michael P.; McCulloch, Richard; Mottram, Jeremy C.; Carrington, Mark; Schwaeble, Wilhelm; McKerrow, James; Phillips, Margaret A.; Michels, Paul A.; Büscher, Philippe; Sanchez, Jean-Charles; Bishop, Richard; Robinson, Derrick R.; Bangs, James; Ferguson, Michael; Nerima, Barbara; Albertini, Audrey; Michel, Gerd; Radwandska, Magdalena; Ndung’u, Joseph Mathu


    Background Control and elimination of human African trypanosomiasis (HAT) can be accelerated through the use of diagnostic tests that are more accurate and easier to deploy. The goal of this work was to evaluate the immuno-reactivity of antigens and identify candidates to be considered for development of a simple serological test for the detection of Trypanosoma brucei gambiense or T. b. rhodesiense infections, ideally both. Methodology/Principal Findings The reactivity of 35 antigens was independently evaluated by slot blot and ELISA against sera from both T. b. gambiense and T. b. rhodesiense infected patients and controls. The antigens that were most reactive by both tests to T. b. gambiense sera were the membrane proteins VSG LiTat 1.3, VSG LiTat 1.5 and ISG64. Reactivity to T. b. rhodesiense sera was highest with VSG LiTat 1.3, VSG LiTat 1.5 and SRA, although much lower than with T. b. gambiense samples. The reactivity of all possible combinations of antigens was also calculated. When the slot blot results of 2 antigens were paired, a VSG LiTat 1.3- ISG75 combination performed best on T. b. gambiense sera, while a VSG LiTat 1.3-VSG LiTat 1.5 combination was the most reactive using ELISA. A combination of SRA and either VSG LiTat 1.3 or VSG LiTat 1.5 had the highest reactivity on T. b. rhodesiense sera according to slot blot, while in ELISA, pairing SRA with either GM6 or VSG LiTat 1.3 yielded the best results. Conclusions This study identified antigens that were highly reactive to T. b. gambiense sera, which could be considered for developing a serological test for gambiense HAT, either individually or in combination. Antigens with potential for inclusion in a test for T. b. rhodesiense HAT were also identified, but because their reactivity was comparatively lower, a search for additional antigens would be required before developing a test for this form of the disease. PMID:27936225

  19. Identification and Characterization of FTY720 for the Treatment of Human African Trypanosomiasis (United States)

    Kaiser, Marcel; Avery, Vicky M.


    The screening of a focused library identified FTY720 (Fingolimod; Gilenya) as a potent selective antitrypanosomal compound active against Trypanosoma brucei gambiense and T. brucei rhodesiense, the causative agents of human African trypanosomiasis (HAT). This is the first report of trypanocidal activity for FTY720, an oral drug registered for the treatment of relapsing multiple sclerosis, and the characterization of sphingolipids as a potential new class of compounds for HAT. PMID:26666915

  20. Diagnostic Development on NSTX

    Energy Technology Data Exchange (ETDEWEB)

    A.L. Roquemore; D. Johnson; R. Kaita; et al


    Diagnostics are described which are currently installed or under active development for the newly commissioned NSTX device. The low aspect ratio (R/a less than or equal to 1.3) and low toroidal field (0.1-0.3T) used in this device dictate adaptations in many standard diagnostic techniques. Technical summaries of each diagnostic are given, and adaptations, where significant, are highlighted.

  1. LAMP for human African trypanosomiasis: a comparative study of detection formats.

    Directory of Open Access Journals (Sweden)

    Sally L Wastling

    Full Text Available Loop-mediated isothermal amplification (LAMP is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT. Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i visualization of turbidity; (ii addition of hydroxynaphthol blue before incubation; (iii addition of calcein with MnCl₂ before incubation and (iv addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl₂, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl₂ adversely affects reaction sensitivity and was unpopular with several observers.

  2. A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Michelle L Ammerman

    Full Text Available Uridine insertion/deletion RNA editing is a unique and vital process in kinetoplastids, required for creation of translatable open reading frames in most mitochondrially-encoded RNAs. Emerging as a key player in this process is the mitochondrial RNA binding 1 (MRB1 complex. MRB1 comprises an RNA-independent core complex of at least six proteins, including the GAP1/2 guide RNA (gRNA binding proteins. The core interacts in an RNA-enhanced or -dependent manner with imprecisely defined TbRGG2 subcomplexes, Armadillo protein MRB10130, and additional factors that comprise the dynamic MRB1 complex. Towards understanding MRB1 complex function in RNA editing, we present here functional characterization of the pentein domain-containing MRB1 core protein, MRB11870. Inducible RNAi studies demonstrate that MRB11870 is essential for proliferation of both insect vector and human infective stage T. brucei. MRB11870 ablation causes a massive defect in RNA editing, affecting both pan-edited and minimally edited mRNAs, but does not substantially affect mitochondrial RNA stability or processing of precursor transcripts. The editing defect in MRB1-depleted cells occurs at the initiation stage of editing, as pre-edited mRNAs accumulate. However, the gRNAs that direct editing remain abundant in the knockdown cells. To examine the contribution of MRB11870 to MRB1 macromolecular interactions, we tagged core complexes and analyzed their composition and associated proteins in the presence and absence of MRB11870. These studies demonstrated that MRB11870 is essential for association of GAP1/2 with the core, as well as for interaction of the core with other proteins and subcomplexes. Together, these data support a model in which the MRB1 core mediates functional interaction of gRNAs with the editing machinery, having GAP1/2 as its gRNA binding constituents. MRB11870 is a critical component of the core, essential for its structure and function.

  3. Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei (United States)

    Liu, Shuo; Ji, Debin; Cliffe, Laura; Sabatini, Robert; Wang, Yinsheng


    β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2'-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2'-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the

  4. Diagnostics of Nanodusty Plasma (United States)

    Greiner, Franko; Groth, Sebastian; Tadsen, Bejamin; Piel, Alexander


    The diagnostic of nanodusty plasmas, i.e. plasmas including nano-sized dust particles, is a challenging task. For both, the diagnostic of the nanodusty plasma itself, and the in-situ diagnostic of the nanoparticles, no standard diagnostic exist. Nanodust particle size and density can be estimated using light scattering techniques, namely kinetic Mie ellipsometry and extinction measurements. The charge of the nanoparticles can be estimated from the analysis of dust density waves (DDW). Parameters like the electron density, which give information about the plasma itself, may be deduced from the DDW analysis. We present detailed investigations on nanodust in a reactive Argon-Acetylene plasma created in an rf-driven parallel plate reactor at low pressure using the above mentioned portfolio of diagnostic. Funded by DFG under contract SFB TR-24/A2.

  5. Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

    NARCIS (Netherlands)

    Pieretti, Simone; Haanstra, Jurgen R.; Mazet, Muriel; Perozzo, Remo; Bergamini, Christian; Prati, Federica; Fato, Romana; Lenaz, Giorgio; Capranico, Giovanni; Brun, Reto; Bakker, Barbara M.; Michels, Paul A. M.; Scapozza, Leonardo; Bolognesi, Maria Laura; Cavalli, Andrea


    Background and Methodology: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a s

  6. Multiorgan dysfunction caused by travel-associated African trypanosomiasis. (United States)

    Cottle, Lucy E; Peters, Joanna R; Hall, Alison; Bailey, J Wendi; Noyes, Harry A; Rimington, Jane E; Beeching, Nicholas J; Squire, S Bertel; Beadsworth, Mike B J


    We describe a case of multiorgan dysfunction secondary to Trypanosoma brucei rhodesiense infection acquired on safari in Zambia. This case was one of several recently reported to ProMED-mail in persons who had traveled to this region. Trypanosomiasis remains rare in travelers but should be considered in febrile patients who have returned from trypanosomiasis-endemic areas of Africa.

  7. Diagnostics in critical conditions

    Directory of Open Access Journals (Sweden)



    Full Text Available The purpose of research: improvement of quality of diagnostics at the patients in a critical condition in intensive care unit. Material and methods. In total have analyzed 1957 medical cards of the patients who have died in ICU»s. At the first stage studied the factors influencing on diagnostics of critically ill patients (medical cards of 1557 patients; at the second stage investigated influence of the diagnostic standards in ICU»s practice on improvement of quality of diag- nostics of critically ill patients (400 medical cards of the patients who have died. Entry criterions were standards and algorithm of diagnostics. Techniques of research: average bed-day in groups, first-day lethality, quantity of the carried out laboratory tests and tool methods of research, level of consciousness of the patients (Glasgow come score, severity of disease by ICU»s patients (APACHE II scores. Results. Quality of diagnostics depend on carried out laboratory tests and tool methods of research, level of consciousness of the patients (Glasgow come score, severity of disease by ICU»s patients (APACHE II score. The conclusion. The laboratory tests and tool methods of research conforming to the standards of diagnostics are necessary for improvement of quality of diagnostics, it is necessary to take into account an altered level of consciousness (Glasgow come score and severity of disease by ICU»s patients (APACHE II scores

  8. 3-(3-amino-3-carboxypropyl)-5,6-Dihydrouridine is one of two novel post-transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Krog, Jesper Schak; Español, Yaiza; Giessing, Anders M B;


    was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence......tRNA is the most heavily modified of all RNA types, with typically 10-20% of the residues being post-transcriptionally altered. Unravelling the modification pattern of a tRNA is a challenging task; there are 92 currently known tRNA modifications [1], many of which are chemically similar....... Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys) (UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step...


    Institute of Scientific and Technical Information of China (English)

    原丽红; 林本夫; 王祥生; 李莲瑞


    布氏锥虫(Trypanosoma brucei)是一种原虫性寄生虫,通过采采蝇(tsets fly)的传播感染人和其它哺乳动物,导致人的昏睡病和家畜的那卡那病。布氏锥虫有两个截然不同的生活阶段,即血液期(bloodstream stage,寄生于哺乳动物血细胞内)和昆虫期(insect stage,又叫循环期或感染期,寄生于采采蝇的中肠内)。

  10. Cable Diagnostic Focused Initiative

    Energy Technology Data Exchange (ETDEWEB)

    Hartlein, R.A.; Hampton, R.N.


    This report summarizes an extensive effort made to understand how to effectively use the various diagnostic technologies to establish the condition of medium voltage underground cable circuits. These circuits make up an extensive portion of the electric delivery infrastructure in the United States. Much of this infrastructure is old and experiencing unacceptable failure rates. By deploying efficient diagnostic testing programs, electric utilities can replace or repair circuits that are about to fail, providing an optimal approach to improving electric system reliability. This is an intrinsically complex topic. Underground cable systems are not homogeneous. Cable circuits often contain multiple branches with different cable designs and a range of insulation materials. In addition, each insulation material ages differently as a function of time, temperature and operating environment. To complicate matters further, there are a wide variety of diagnostic technologies available for assessing the condition of cable circuits with a diversity of claims about the effectiveness of each approach. As a result, the benefits of deploying cable diagnostic testing programs have been difficult to establish, leading many utilities to avoid the their use altogether. This project was designed to help address these issues. The information provided is the result of a collaborative effort between Georgia Tech NEETRAC staff, Georgia Tech academic faculty, electric utility industry participants, as well as cable system diagnostic testing service providers and test equipment providers. Report topics include: •How cable systems age and fail, •The various technologies available for detecting potential failure sites, •The advantages and disadvantages of different diagnostic technologies, •Different approaches for utilities to employ cable system diagnostics. The primary deliverables of this project are this report, a Cable Diagnostic Handbook (a subset of this report) and an online

  11. The response of trypanosomes and other eukaryotes to ER stress and the spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei. (United States)

    Michaeli, Shulamit


    The unfolded protein response (UPR) is induced when the quality control machinery of the cell is overloaded with unfolded proteins or when one of the functions of the endoplasmic reticulum (ER) is perturbed. Here, I describe UPR in yeast and mammals, and compare it to what we know about pathogenic fungi and the parasitic protozoans from the order kinetoplastida, focusing on the novel pathway the spliced leader silencing (SLS) in Trypanosoma brucei. Trypanosomes lack conventional transcription regulation, and thus, lack most of the UPR machinery present in other eukaryotes. Trypanosome genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon known as the spliced leader (SL) is added to all mRNAs from a small RNA, the SL RNA. Under severe ER stress, T. brucei elicits the SLS pathway. In SLS, the transcription of the SL RNA gene is extinguished, and the entire transcription complex dissociates from the SL RNA promoter. Induction of SLS is mediated by an ER-associated kinase (PK3) that migrates to the nucleus, where it phosphorylates the TATA-binding protein (TRF4), leading shut-off of SL RNA transcription. As a result, trans-splicing is inhibited and the parasites activate a programmed cell death (PCD) pathway. Despite the ability to sense the ER stress, the different eukaryotes, especially unicellular parasites and pathogenic fungi, developed a variety of unique and different ways to sense and adjust to this stress in a manner different from their host.

  12. Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

    KAUST Repository

    Nguyen, T. N.


    Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite\\'s ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro, as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7.

  13. The role of B-cells and IgM antibodies in parasitemia, anemia, and VSG switching in Trypanosoma brucei-infected mice.

    Directory of Open Access Journals (Sweden)

    Stefan Magez

    Full Text Available African trypanosomes are extracellular parasitic protozoa, predominantly transmitted by the bite of the haematophagic tsetse fly. The main mechanism considered to mediate parasitemia control in a mammalian host is the continuous interaction between antibodies and the parasite surface, covered by variant-specific surface glycoproteins. Early experimental studies have shown that B-cell responses can be strongly protective but are limited by their VSG-specificity. We have used B-cell (microMT and IgM-deficient (IgM(-/- mice to investigate the role of B-cells and IgM antibodies in parasitemia control and the in vivo induction of trypanosomiasis-associated anemia. These infection studies revealed that that the initial setting of peak levels of parasitemia in Trypanosoma brucei-infected microMT and IgM(-/- mice occurred independent of the presence of B-cells. However, B-cells helped to periodically reduce circulating parasites levels and were required for long term survival, while IgM antibodies played only a limited role in this process. Infection-associated anemia, hypothesized to be mediated by B-cell responses, was induced during infection in microMT mice as well as in IgM(-/- mice, and as such occurred independently from the infection-induced host antibody response. Antigenic variation, the main immune evasion mechanism of African trypanosomes, occurred independently from host antibody responses against the parasite's ever-changing antigenic glycoprotein coat. Collectively, these results demonstrated that in murine experimental T. brucei trypanosomiasis, B-cells were crucial for periodic peak parasitemia clearance, whereas parasite-induced IgM antibodies played only a limited role in the outcome of the infection.

  14. Structures of Trypanosoma brucei methionyl-tRNA synthetase with urea-based inhibitors provide guidance for drug design against sleeping sickness.

    Directory of Open Access Journals (Sweden)

    Cho Yeow Koh


    Full Text Available Methionyl-tRNA synthetase of Trypanosoma brucei (TbMetRS is an important target in the development of new antitrypanosomal drugs. The enzyme is essential, highly flexible and displaying a large degree of changes in protein domains and binding pockets in the presence of substrate, product and inhibitors. Targeting this protein will benefit from a profound understanding of how its structure adapts to ligand binding. A series of urea-based inhibitors (UBIs has been developed with IC50 values as low as 19 nM against the enzyme. The UBIs were shown to be orally available and permeable through the blood-brain barrier, and are therefore candidates for development of drugs for the treatment of late stage human African trypanosomiasis. Here, we expand the structural diversity of inhibitors from the previously reported collection and tested for their inhibitory effect on TbMetRS and on the growth of T. brucei cells. The binding modes and binding pockets of 14 UBIs are revealed by determination of their crystal structures in complex with TbMetRS at resolutions between 2.2 Å to 2.9 Å. The structures show binding of the UBIs through conformational selection, including occupancy of the enlarged methionine pocket and the auxiliary pocket. General principles underlying the affinity of UBIs for TbMetRS are derived from these structures, in particular the optimum way to fill the two binding pockets. The conserved auxiliary pocket might play a role in binding tRNA. In addition, a crystal structure of a ternary TbMetRS•inhibitor•AMPPCP complex indicates that the UBIs are not competing with ATP for binding, instead are interacting with ATP through hydrogen bond. This suggests a possibility that a general 'ATP-engaging' binding mode can be utilized for the design and development of inhibitors targeting tRNA synthetases of other disease-causing pathogen.

  15. Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux. (United States)

    Allmann, Stefan; Morand, Pauline; Ebikeme, Charles; Gales, Lara; Biran, Marc; Hubert, Jane; Brennand, Ana; Mazet, Muriel; Franconi, Jean-Michel; Michels, Paul A M; Portais, Jean-Charles; Boshart, Michael; Bringaud, Frédéric


    All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/(RNAi)PGI double mutant when compared with the single mutants, and (iii) the (13)C enrichment of glycolytic and PPP intermediates from cells incubated with [U-(13)C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.

  16. Thioaptamer Diagnostic System Project (United States)

    National Aeronautics and Space Administration — AM Biotechnologies (AM) in partnership with Sandia National Laboratories will develop a Thioaptamer Diagnostic System (TDS) in response to Topic X10.01 Reusable...

  17. Rotorcraft Diagnostics Project (United States)

    National Aeronautics and Space Administration — Under this SBIR program, Ridgetop will introduce the first low-cost, low-power, and lightweight data monitoring solution for rotorcraft diagnostics. The solution is...

  18. National Convective Weather Diagnostic (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Current convective hazards identified by the National Convective Weather Detection algorithm. The National Convective Weather Diagnostic (NCWD) is an automatically...

  19. Prenatal Genetic Diagnostic Tests (United States)

    ... are offered to all pregnant women. What is amniocentesis? Amniocentesis is a diagnostic test. It usually is done ... a very small chance of pregnancy loss with amniocentesis. Leakage of amniotic fluid and slight bleeding can ...

  20. Optics/Optical Diagnostics Laboratory (United States)

    Federal Laboratory Consortium — The Optics/Optical Diagnostics Laboratory supports graduate instruction in optics, optical and laser diagnostics and electro-optics. The optics laboratory provides...

  1. [Diagnostics in osteology]. (United States)

    Jakob, F; Genest, F; Seefried, L; Tsourdi, E; Lapa, C; Hofbauer, L C


    Clinical diagnostics in metabolic bone diseases cover a broad spectrum of conventional and state of the art methods ranging from the medical history and clinical examination to molecular imaging. Patient treatment is carried out in an interdisciplinary team due to the multiple interactions of bone with other organ systems. Diagnosis of osteoporosis is supported by high level national guidelines. A paradigm shift concerning the clinical relevance of bone mineral density measurement renders this now to be a strong risk factor rather than a diagnostic parameter, while strengthening the value of other clinical factors for risk assessment. The impact of parameters for muscle mass, structure and function is steadily increasing in all age groups. In order to identify underlying diseases that influence bone metabolism a panel of general laboratory diagnostic parameters is recommended. Markers for bone formation and resorption and specific parameters for the regulation of calcium and phosphate metabolism should be evaluated by specialists because they require diligence in preanalytics and experience in interpretation. Genetic diagnosis is well established for rare bone diseases while diagnostic panels are not yet available for routine diagnostics in polygenetic diseases such as osteoporosis. Conventional radiology is still very important to identify, e. g. fractures, osteolytic and osteoblastic lesions and extraosseous calcifications; however tomography-based methods which combine, e. g. scintigraphy or positron emission technologies with anatomical imaging are of increasing significance. Clinical diagnostics in osteology require profound knowledge and are subject to a dynamic evolution.

  2. Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain. (United States)

    McShan, Danielle; Kathman, Stefan; Lowe, Brittiney; Xu, Ziyang; Zhan, Jennifer; Statsyuk, Alexander; Ogungbe, Ifedayo Victor


    Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic assays will further expand the use of the covalent tethering method, a simple fragment-based drug discovery technique to discover covalent drug leads.

  3. Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme. (United States)

    Carvalho, Alcione Silva de; Salomão, Kelly; Castro, Solange Lisboa de; Conde, Taline Ramos; Zamith, Helena Pereira da Silva; Caffarena, Ernesto Raúl; Hall, Belinda Suzette; Wilkinson, Shane Robert; Boechat, Núbia


    Megazol (7) is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8) in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR) enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7) for nitrogen (in the triazole in 8), the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

  4. Development of Companion Diagnostics. (United States)

    Mankoff, David A; Edmonds, Christine E; Farwell, Michael D; Pryma, Daniel A


    The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient's cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods as companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has "hit" the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2-targeted therapy. The review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.

  5. MJO Simulation Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Waliser, D; Sperber, K; Hendon, H; Kim, D; Maloney, E; Wheeler, M; Weickmann, K; Zhang, C; Donner, L; Gottschalck, J; Higgins, W; Kang, I; Legler, D; Moncrieff, M; Schubert, S; Stern, W; Vitart, F; Wang, B; Wang, W; Woolnough, S


    The Madden-Julian Oscillation (MJO) interacts with, and influences, a wide range of weather and climate phenomena (e.g., monsoons, ENSO, tropical storms, mid-latitude weather), and represents an important, and as yet unexploited, source of predictability at the subseasonal time scale. Despite the important role of the MJO in our climate and weather systems, current global circulation models (GCMs) exhibit considerable shortcomings in representing this phenomenon. These shortcomings have been documented in a number of multi-model comparison studies over the last decade. However, diagnosis of model performance has been challenging, and model progress has been difficult to track, due to the lack of a coherent and standardized set of MJO diagnostics. One of the chief objectives of the US CLIVAR MJO Working Group is the development of observation-based diagnostics for objectively evaluating global model simulations of the MJO in a consistent framework. Motivation for this activity is reviewed, and the intent and justification for a set of diagnostics is provided, along with specification for their calculation, and illustrations of their application. The diagnostics range from relatively simple analyses of variance and correlation, to more sophisticated space-time spectral and empirical orthogonal function analyses. These diagnostic techniques are used to detect MJO signals, to construct composite life-cycles, to identify associations of MJO activity with the mean state, and to describe interannual variability of the MJO.

  6. Pitfalls in diagnostic radiology

    Energy Technology Data Exchange (ETDEWEB)

    Peh, Wilfred C.G. (ed.) [Khoo Teck Puat Hospital (Singapore). Dept. of Diagnostic Radiology


    Only textbook to focus primarily on the topic of pitfalls in diagnostic radiology. Highlights the pitfalls in a comprehensive and systematic manner. Written by experts in different imaging modalities and subspecialties from reputable centers across the world. The practice of diagnostic radiology has become increasingly complex, with the use of numerous imaging modalities and division into many subspecialty areas. It is becoming ever more difficult for subspecialist radiologists, general radiologists, and residents to keep up with the advances that are occurring year on year, and this is particularly true for less familiar topics. Failure to appreciate imaging pitfalls often leads to diagnostic error and misinterpretation, and potential medicolegal problems. Diagnostic errors may be due to various factors such as inadequate imaging technique, imaging artifacts, failure to recognize normal structures or variants, lack of correlation with clinical and other imaging findings, and poor training or inexperience. Many, if not most, of these factors are potentially recognizable, preventable, or correctable. This textbook, written by experts from reputable centers across the world, systematically and comprehensively highlights the pitfalls that may occur in diagnostic radiology. Both pitfalls specific to different modalities and techniques and those specific to particular organ systems are described with the help of numerous high-quality illustrations. Recognition of these pitfalls is crucial in helping the practicing radiologist to achieve a more accurate diagnosis.

  7. Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment. (United States)

    Mukadi, Pierre; Lejon, Veerle; Barbé, Barbara; Gillet, Philippe; Nyembo, Christophe; Lukuka, Albert; Likwela, Joris; Lumbala, Crispin; Mbaruku, Justin; Vander Veken, Wim; Mumba, Dieudonné; Lutumba, Pascal; Muyembe, Jean-Jacques; Jacobs, Jan


    The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013-April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported "malaria" or "Plasmodium falciparum" (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied "malaria" or "Plasmodium falciparum" (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.

  8. ORION laser target diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Bentley, C. D.; Edwards, R. D.; Andrew, J. E.; James, S. F.; Gardner, M. D.; Comley, A. J.; Vaughan, K.; Horsfield, C. J.; Rubery, M. S.; Rothman, S. D.; Daykin, S.; Masoero, S. J.; Palmer, J. B.; Meadowcroft, A. L.; Williams, B. M.; Gumbrell, E. T.; Fyrth, J. D.; Brown, C. R. D.; Hill, M. P.; Oades, K. [Plasma Physics Department, Atomic Weapons Establishment, Aldermaston, Reading, Berkshire RG7 4PR (United Kingdom); and others


    The ORION laser facility is one of the UK's premier laser facilities which became operational at AWE in 2010. Its primary mission is one of stockpile stewardship, ORION will extend the UK's experimental plasma physics capability to the high temperature, high density regime relevant to Atomic Weapons Establishment's (AWE) program. The ORION laser combines ten laser beams operating in the ns regime with two sub ps short pulse chirped pulse amplification beams. This gives the UK a unique combined long pulse/short pulse laser capability which is not only available to AWE personnel but also gives access to our international partners and visiting UK academia. The ORION laser facility is equipped with a comprehensive suite of some 45 diagnostics covering optical, particle, and x-ray diagnostics all able to image the laser target interaction point. This paper focuses on a small selection of these diagnostics.

  9. Beamlet laser diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Burkhart, S.C.; Behrendt, W.C.; Smith, I.


    Beamlet is instrumented extensively to monitor the performance of the overall laser system and many of its subsystems. Beam diagnostics, installed in key locations, are used to fully characterize the beam during its propagation through the multipass cavity and the laser`s output section. This article describes the diagnostics stations located on Beamlet and discusses the design, calibration, and performance of the Beamlet calorimeters. The authors used Nova`s diagnostics packages to develop the Beamlet design to determine beam energy, spatial profile, temporal profile, and other beam parameters. Technologic improvements within the last several years in controls, charge-coupled device (CCD) cameras, and fast oscilloscopes have allowed the authors to obtain more accurate measurements on the Beamlet laser system. They briefly cover some of these techniques, including a description of their LabVIEW based data acquisition system.

  10. ORION laser target diagnostics. (United States)

    Bentley, C D; Edwards, R D; Andrew, J E; James, S F; Gardner, M D; Comley, A J; Vaughan, K; Horsfield, C J; Rubery, M S; Rothman, S D; Daykin, S; Masoero, S J; Palmer, J B; Meadowcroft, A L; Williams, B M; Gumbrell, E T; Fyrth, J D; Brown, C R D; Hill, M P; Oades, K; Wright, M J; Hood, B A; Kemshall, P


    The ORION laser facility is one of the UK's premier laser facilities which became operational at AWE in 2010. Its primary mission is one of stockpile stewardship, ORION will extend the UK's experimental plasma physics capability to the high temperature, high density regime relevant to Atomic Weapons Establishment's (AWE) program. The ORION laser combines ten laser beams operating in the ns regime with two sub ps short pulse chirped pulse amplification beams. This gives the UK a unique combined long pulse/short pulse laser capability which is not only available to AWE personnel but also gives access to our international partners and visiting UK academia. The ORION laser facility is equipped with a comprehensive suite of some 45 diagnostics covering optical, particle, and x-ray diagnostics all able to image the laser target interaction point. This paper focuses on a small selection of these diagnostics.

  11. Diagnostic hematology of reptiles. (United States)

    Stacy, Nicole I; Alleman, A Rick; Sayler, Katherine A


    The hematologic evaluation of reptiles is an indispensable diagnostic tool in exotic veterinary practice. The diversity of reptile species, their characteristic physiologic features, and effects of intrinsic and extrinsic factors present unique challenges for accurate interpretation of the hemogram. Combining the clinical presentation with hematologic findings provides valuable information in the diagnosis and monitoring of disease and helps guide the clinician toward therapy and further diagnostic testing. This article outlines the normal and pathologic morphology of blood cells of reptile species. The specific comparative aspects of reptiles are emphasized, and structural and functional abnormalities in the reptilian hemogram are described.

  12. [Molecular diagnostics and imaging]. (United States)

    Fink, Christian; Fisseler-Eckhoff, Annette; Huss, Ralf; Nestle, Ursula


    Molecular diagnostic methods and biological imaging techniques can make a major contribution to tailoring patients' treatment needs with regard to medical, ethical and pharmaco-economic aspects. Modern diagnostic methods are already being used to help identify different sub-groups of patients with thoracic tumours who are most likely to benefit significantly from a particular type of treatment. This contribution looks at the most recent developments that have been made in the field of thoracic tumour diagnosis and analyses the pros and cons of new molecular and other imaging techniques in day-to-day clinical practice.

  13. Beamlet focal plane diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    Caird, J.A.; Nielsen, N.D.; Patton, H.G.; Seppala, L.G.; Thompson, C.E.; Wegner, P.J.


    This paper describes the major optical and mechanical design features of the Beamlet Focal Plane Diagnostic system as well as measurements of the system performance, and typical data obtained to date. We also discuss the NIF requirements on the focal spot that we are interested in measuring, and some of our plans for future work using this system.

  14. Diagnostic and interventional radiology

    Energy Technology Data Exchange (ETDEWEB)

    Vogl, Thomas J. [Klinikum der Johann Wolfgang Goethe-Universitaet, Frankfurt am Main (Germany). Inst. fuer Diagnostische und Interventionelle Radiologie; Reith, Wolfgang [Universitaetsklinikum des Saarlandes, Homburg/Saar (Germany). Klinik fuer Diagnostische und Interventionelle Neuroradiologie; Rummeny, Ernst J. (ed.) [Technische Univ. Muenchen Klinikum rechts der Isar, Muenchen (Germany). Inst. fuer Radiologie


    This exceptional book covers all aspects of diagnostic and interventional radiology within one volume, at a level appropriate for the specialist. From the basics through diagnosis to intervention: the reader will find a complete overview of all areas of radiology. The clear, uniform structure, with chapters organized according to organ system, facilitates the rapid retrieval of information. Features include: Presentation of the normal radiological anatomy Classification of the different imaging procedures according to their diagnostic relevance Imaging diagnosis with many reference images Precise description of the interventional options The inclusion of many instructive aids will be of particular value to novices in decision making: Important take home messages and summaries of key radiological findings smooth the path through the jungle of facts Numerous tables on differential diagnosis and typical findings in the most common diseases offer a rapid overview and orientation Diagnostic flow charts outline the sequence of diagnostic evaluation All standard procedures within the field of interventional radiology are presented in a clinically relevant and readily understandable way, with an abundance of illustrations. This is a textbook, atlas, and reference in one: with more than 2500 images for comparison with the reader's own findings. This comprehensive and totally up-to-date book provides a superb overview of everything that the radiology specialist of today needs to know.

  15. Cardiovascular modeling and diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Kangas, L.J.; Keller, P.E.; Hashem, S.; Kouzes, R.T. [Pacific Northwest Lab., Richland, WA (United States)


    In this paper, a novel approach to modeling and diagnosing the cardiovascular system is introduced. A model exhibits a subset of the dynamics of the cardiovascular behavior of an individual by using a recurrent artificial neural network. Potentially, a model will be incorporated into a cardiovascular diagnostic system. This approach is unique in that each cardiovascular model is developed from physiological measurements of an individual. Any differences between the modeled variables and the variables of an individual at a given time are used for diagnosis. This approach also exploits sensor fusion to optimize the utilization of biomedical sensors. The advantage of sensor fusion has been demonstrated in applications including control and diagnostics of mechanical and chemical processes.

  16. Gene Disease Diagnostic System

    Institute of Scientific and Technical Information of China (English)

    黄国亮; 张腾飞; 程京; 周玉祥; 刘诚迅; 金国藩; 邬敏贤; 严瑛白; 杨蓉


    Binary optics, where the optical element can be fabricated on a thin glass plate with micro-ion-etching film layer, has been widely applied in recent years. A novel optical scanning system for gene disease diagnostics described in this paper has four kinds of optical devices, including beam splitters, an array lens, an array filter and detection arrays. A software was developed to design the binary optics system using an iterative method. Two beam splitters were designed and fabricated, which can divide a beam into a 9×9 array or into a 13×13 array. The beam splitters have good diffraction efficiencies (>70%) and an even energy distribution. The gene disease diagnostic system is a portable biochip and binary optics technology. The binary optical devices in the non-confocal scanning system can raise the fluorescence detection sensitivity of the micro-array hybrid biochip.

  17. A comparative proteomic analysis reveals a new bi-lobe protein required for bi-lobe duplication and cell division in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Qing Zhou

    Full Text Available A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

  18. The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control. (United States)

    Millerioux, Yoann; Ebikeme, Charles; Biran, Marc; Morand, Pauline; Bouyssou, Guillaume; Vincent, Isabel M; Mazet, Muriel; Riviere, Loïc; Franconi, Jean-Michel; Burchmore, Richard J S; Moreau, Patrick; Barrett, Michael P; Bringaud, Frédéric


    The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonine's contribution being ∼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end-products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC, is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC shows a 1.7-fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8-fold reduction in threonine-derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.

  19. The glycosylphosphatidylinositol-PLC in Trypanosoma brucei forms a linear array on the exterior of the flagellar membrane before and after activation. (United States)

    Hanrahan, Orla; Webb, Helena; O'Byrne, Robert; Brabazon, Elaine; Treumann, Achim; Sunter, Jack D; Carrington, Mark; Voorheis, H Paul


    Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC.

  20. `In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase. (United States)

    Zhong, Wenhe; Morgan, Hugh P; McNae, Iain W; Michels, Paul A M; Fothergill-Gilmore, Linda A; Walkinshaw, Malcolm D


    The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg(2+) triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg(2+) and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg(2+) occupies a position that is distinct from the two canonical Mg(2+)-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg(2+) in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg(2+) cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.

  1. Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei (United States)

    Levy, Gabriela V.; Moretti, Georgina; Tekiel, Valeria S.; Sánchez, Daniel O.


    Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family. PMID:26284933

  2. PROcess Based Diagnostics PROBE (United States)

    Clune, T.; Schmidt, G.; Kuo, K.; Bauer, M.; Oloso, H.


    Many of the aspects of the climate system that are of the greatest interest (e.g., the sensitivity of the system to external forcings) are emergent properties that arise via the complex interplay between disparate processes. This is also true for climate models most diagnostics are not a function of an isolated portion of source code, but rather are affected by multiple components and procedures. Thus any model-observation mismatch is hard to attribute to any specific piece of code or imperfection in a specific model assumption. An alternative approach is to identify diagnostics that are more closely tied to specific processes -- implying that if a mismatch is found, it should be much easier to identify and address specific algorithmic choices that will improve the simulation. However, this approach requires looking at model output and observational data in a more sophisticated way than the more traditional production of monthly or annual mean quantities. The data must instead be filtered in time and space for examples of the specific process being targeted.We are developing a data analysis environment called PROcess-Based Explorer (PROBE) that seeks to enable efficient and systematic computation of process-based diagnostics on very large sets of data. In this environment, investigators can define arbitrarily complex filters and then seamlessly perform computations in parallel on the filtered output from their model. The same analysis can be performed on additional related data sets (e.g., reanalyses) thereby enabling routine comparisons between model and observational data. PROBE also incorporates workflow technology to automatically update computed diagnostics for subsequent executions of a model. In this presentation, we will discuss the design and current status of PROBE as well as share results from some preliminary use cases.

  3. Idiopathic chondrolysis - diagnostic difficulties

    Energy Technology Data Exchange (ETDEWEB)

    Kozlowski, K.; Scougall, J.


    Four cases of idiopathic chondrolysis of the hip in three white girls and one Maori girl are reported. The authors stress the causes why a disease with characteristic clinical and radiographic appearances and normal biochemical findings presents diagnostic difficulties. It is suspected that idiopathic chondrolysis is a metabolic disorder of chondrocytes, triggered by environment circumstances in susceptible individuals. Idiopathic chondrolysis is probably one of the most common causes of coxarthrosis in women.

  4. STELLA Experiment - Microbunch Diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    He, P.; Liu, Y.; Cline, D. B.; Babzien, M.; Gallardo, J. C.; Kusche, K. P.; Pogorelsky, I. V.; Skaritka, J.; van Steenbergen, A.; Yakimenko, V.; Kimura, W. D.


    A microbunch diagnostic system is built at the Accelerator Test Facility (ATF) of Brookhaven National Laboratory for monitoring microbunches (10-fs bunch length) produced by the Inverse Free Electron Laser accelerator in Staged Electron Laser Acceleration experiment. It is similar to one already demonstrated at the ATF. With greatly improved beam optics conditions higher order harmonic coherent transition radiation will be measurable to determine the microbunch length and shape.

  5. SNS Diagnostics Timing Integration

    CERN Document Server

    Long, Cary D; Murphy, Darryl J; Pogge, James; Purcell, John D; Sundaram, Madhan


    The Spallation Neutron Source (SNS) accelerator systems will deliver a 1.0 GeV, 1.4 MW proton beam to a liquid mercury target for neutron scattering research. The accelerator complex consists of a 1 GeV linear accelerator, an accumulator ring and associated transport lines. The SNS diagnostics platform is PC-based running Windows XP Embedded for its OS and LabVIEW as its programming language. Coordinating timing among the various diagnostics instruments with the generation of the beam pulse is a challenging task that we have chosen to divide into three phases. First, timing was derived from VME based systems. In the second phase, described in this paper, timing pulses are generated by an in house designed PCI timing card installed in ten diagnostics PCs. Using fan-out modules, enough triggers were generated for all instruments. This paper describes how the Timing NAD (Network Attached Device) was rapidly developed using our NAD template, LabVIEW's PCI driver wizard, and LabVIEW Channel Access library. The NAD...

  6. [Histopathological meniscus diagnostic]. (United States)

    Fisseler-Eckhoff, A; Müller, K-M


    Menisci fulfill many functions within the complex biomechanics of the knee joint. In the case of meniscus lesions, sparing arthroscopic resections and operative refixation are the treatments of choice. With regard to diagnostics, this means that in general terms, the histopathologic diagnostics are carried out on detached meniscus fragments of between 5 mm and 2 cm in size. An experienced pathologist's knowledge of physiologically possible cellular and fibrous histological meniscus damage, as opposed to nonphysiological change regarded as normal with respect to age, is essential during a diagnostic meniscus evaluation. The clinician expects clear statements from the pathologist regarding the severity of previous or secondary degenerative meniscus damage, the age and type of traumatic tears, and appraisal of the relationship between trauma and meniscus damage from an insurance point of view. Close cooperation between the clinician and the pathologist allows for fast and unambiguous correlation of anamnesis, the clinical picture, and morphological reporting so that cases involving insurance problems - which are numerous, often long-term, and often unsatisfactory - can be clarified quickly.

  7. Diagnostics for Developing Countries

    Directory of Open Access Journals (Sweden)

    Ruth McNerney


    Full Text Available Improving the availability of high quality diagnostic tests for infectious diseases is a global priority. Lack of access by people living in low income countries may deprive them of life saving treatment and reduces opportunities to prevent onward transmission and spread of the disease. Diagnostic laboratories are often poorly resourced in developing countries, and sparsely distributed. Improved access may be achieved by using tests that do not require laboratory support, including rapid tests for use at the point-of-care. Despite increased interest, few new in vitro diagnostic (IVD products reach the majority populations in low income countries. Barriers to uptake include cost and lack of robustness, with reduced test performances due to environmental pressures such as high ambient temperatures or dust. In addition to environmental factors test developers must consider the local epidemiology. Confounding conditions such as immunosuppression or variations in antigen presentation or genotype can affect test performance. Barriers to product development include access to finance to establish manufacturing capacity and cover the costs of market entry for new devices. Costs and delays may be inflated by current regulatory preregistration processes to ensure product safety and quality, and more harmonized approaches are needed.

  8. DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide. (United States)

    Shoda, L K; Kegerreis, K A; Suarez, C E; Roditi, I; Corral, R S; Bertot, G M; Norimine, J; Brown, W C


    The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423-5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-alpha, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli > or = T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations, E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.

  9. Invasive mycoses: diagnostic challenges. (United States)

    Ostrosky-Zeichner, Luis


    Despite the availability of newer antifungal drugs, outcomes for patients with invasive fungal infections (IFIs) continue to be poor, in large part due to delayed diagnosis and initiation of appropriate antifungal therapy. Standard histopathologic diagnostic techniques are often untenable in at-risk patients, and culture-based diagnostics typically are too insensitive or nonspecific, or provide results after too long a delay for optimal IFI management. Newer surrogate markers of IFIs with improved sensitivity and specificity are needed to enable earlier diagnosis and, ideally, to provide prognostic information and/or permit therapeutic monitoring. Surrogate assays should also be accessible and easy to implement in the hospital. Several nonculture-based assays of newer surrogates are making their way into the medical setting or are currently under investigation. These new or up-and-coming surrogates include antigens/antibodies (mannan and antimannan antibodies) or fungal metabolites (d-arabinitol) for detection of invasive candidiasis, the Aspergillus cell wall component galactomannan used to detect invasive aspergillosis, or the fungal cell wall component and panfungal marker β-glucan. In addition, progress continues with use of polymerase chain reaction- or other nucleic acid- or molecular-based assays for diagnosis of either specific or generic IFIs, although the various methods must be better standardized before any of these approaches can be more fully implemented into the medical setting. Investigators are also beginning to explore the possibility of combining newer surrogate markers with each other or with more standard diagnostic approaches to improve sensitivity, specificity, and capacity for earlier diagnosis, at a time when fungal burden is still relatively low and more responsive to antifungal therapy.

  10. Diagnostic electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Dickersin, G.R.


    In this book the author presents a comprehensive reference text on diagnostic electron microscopy. Throughout the book he illustrates how ultrastructural identification can be helpful for the recognition of cell type and the identification of mechanisms of pathogenesis in various diseases. In addition to electron microscopy photographs, there are also numerous light microscopy photographs for comparison. This text presents the classification of neoplasms in the order and arrangement most familiar to the pathologist. Contents: Introduction; Diagram of a Normal Cell; Normal Cell Function; Embryology; Neoplasms; Infectious Agents; Metabolic Diseases; Renal Diseases; Skeletal Muscle and Peripheral Nerve Diseases; Index.

  11. Diagnostic approaches for cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)


    Cholangiocarcinomas arise from the epithelial cells of the bile ducts and are associated with poor prognosis. Despite new diagnostic approaches, the definite diagnosis of this malignancy continues to be challenging. Cholangiocarcinomas often grow longitudinally along the bile duct rather than in a radial direction. Thus, large tumor masses are frequently absent and imaging techniques, including ultrasound, CT, and MRI have only limited sensitivity. Tissue collection during endoscopic (ERCP) and/or percutaneous transhepatic (PTC) procedures are usually used to confirm a definitive diagnosis of cholangiocarcinoma. However, forceps biopsy and brush cytology provide positive results for malignancy in about only 50% of patients. Percutaneous and peroral cholangioscopy using fiber-optic techniques were therefore developed for direct visualization of the biliary tree, yielding additional information about endoscopic appearance and tumor extension, as well as a guided biopsy acquistion. Finally, endoscopic ultrasonography (EUS) complements endoscopic and percutaneous approaches and may provide a tissue diagnosis of tumors in the biliary region through fine- needle aspiration. In the future, new techniques allowing for early detection, including molecular markers, should be developed to improve the diagnostic sensitivity in this increasing tumor entity.

  12. Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor. (United States)

    Bacchi, Cyrus J; Barker, Robert H; Rodriguez, Aixa; Hirth, Bradford; Rattendi, Donna; Yarlett, Nigel; Hendrick, Clifford L; Sybertz, Edmund


    Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

  13. Trypanosoma brucei RNA binding proteins p34 and p37 mediate NOPP44/46 cellular localization via the exportin 1 nuclear export pathway. (United States)

    Hellman, Kristina; Prohaska, Kimberly; Williams, Noreen


    We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.

  14. Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Ghazal Peter


    Full Text Available Abstract Background Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. Results In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry, thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. Conclusion Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition.

  15. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    Energy Technology Data Exchange (ETDEWEB)

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH


    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  16. Micro RNA expression profiles in peripheral blood cells of rats that were experimentally infected with Trypanosoma congolense and different Trypanosoma brucei subspecies. (United States)

    Simo, Gustave; Lueong, Smiths; Grebaut, Pascal; Guny, Gerard; Hoheisel, Jörg D


    To identify miRNAs whose expression are differentially regulated during trypanosome infections a microarray targeting more than 600 rat miRNA was used to analyze the miRNA expression profiles between uninfected rats and animals infected by Trypanosoma congolense and Trypanosoma brucei s.l. The potential targets of dysregulated miRNAs as well as their biological pathways and functions were predicted using several bioinformatics software tools. Irrespective of the infecting trypanosome species, eight miRNAs (seven up- and one down-regulated) were dysregulated during infections. Moreover, other miRNAs were differentially regulated in rats infected by specific trypanosome species. Functional analyses of differentially regulated miRNAs indicated their involvement in diverse biological processes. Among these, transcription repressor activity, gene expression control as well as protein transporter activity were predominant. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of dysregulated miRNAs revealed their involvement in several biological pathways and disease conditions. This suggests possible modulation of such pathways following trypanosome infection; for example, the MAPK signaling pathway which is known to play vital roles in apoptosis, innate immune response and response to viral infections was highly affected. Axon guidance was equally highly impacted and may indicate a cross reactivity between pathogen proteins and guidance molecules representing one pathological mechanism as it has been observed with influenza HA. Furthermore, Ingenuity pathway analyses of dysregulated miRNAs and potential targets indicated strong association with inflammatory responses, cell death and survival as well as infectious diseases. The data generated here provide valuable information to understand the regulatory function of miRNAs during trypanosome infections. They improved our knowledge on host-parasite cross-talks and provide a framework for investigations to

  17. Diagnostics for Hypersonic Engine Control (United States)


    AFRL-RQ-WP-TR-2015-0037 DIAGNOSTICS FOR HYPERSONIC ENGINE CONTROL Michael S. Brown and Jeffrey M. Donbar Hypersonic Sciences Branch...DD-MM-YY) 2. REPORT TYPE 3. DATES COVERED (From - To) February 2015 Interim 01 March 2013 – 24 February 2015 4. TITLE AND SUBTITLE DIAGNOSTICS FOR...SUPPLEMENTARY NOTES PA Case Number: 88ABW-2015-0636; Clearance Date: 23 Feb 2015. 14. ABSTRACT The overall goal of the research is to find diagnostic

  18. Diagnostic and forensic toxicology. (United States)

    Galey, F D


    In most competent veterinary diagnostic laboratories, analytical findings are interpreted by the veterinary toxicologist to determine the significance of the finding in view of historic, clinical, and pathologic findings. A veterinary toxicologist also will provide consultation about possible toxic rule-outs for a case, treatment of affected animals, and prevention of additional cases. Once all of the information is available, a complete summary of the findings can be provided to the client. When the procedures outlined are followed, including a systematic approach to collecting all the evidence (historic, clinical, pathologic, and analytic), proper sampling techniques, and good communication between the clinician and the client and laboratory, the usefulness of the toxicology investigation will be maximized.

  19. Diagnostics and Microelectronics

    Energy Technology Data Exchange (ETDEWEB)

    Balch, J.W.


    This report discusses activities of the Diagnostics and Microelectronics thrust area which conducts activities in semiconductor devices and semiconductor fabrication technology for programs at Lawrence Livermore National Laboratory. Our multidisciplinary engineering and scientific staff use modern computational tools and semi-conductor microfabrication equipment to develop high-performance devices. Our work concentrates on three broad technologies of semiconductor microdevices: (1) silicon on III-V semiconductor microeletronics, (2) lithium niobate-based and III-V semiconductor-based photonics, and (3) silicon-based micromaching for application to microstructures and microinstruments. In FY-92, we worked on projects in seven areas, described in this report: novel photonic detectors; a wideband phase modulator; an optoelectronic terahertz beam system; the fabrication of microelectrode electrochemical sensors; diamond heatsinks; advanced micromachining technologies; and electrophoresis using silicon microchannels.

  20. Diagnostics procedures in rabies

    Directory of Open Access Journals (Sweden)

    Malovrh Tadej


    Full Text Available Rabies is a major zoonosis for which diagnostic techniques can only be performed in the laboratory. Laboratory techniques are preferably oriented on tissue removed from the cranium: hippocampus (Ammon's horn, cerebellum and the medulla oblongata or tissue liquids. Clinical observation may only lead to a suspicion of rabies. The only way to perform a reliable diagnosis of the disease is to identify the virus or some of its specific components using laboratory tests such as histological identification of characteristic cell lesions, immunochemical identification of rabies virus antigen and virus isolation. Serological tests are rarely used in epidemiological surveys but much more frequently in control of the vaccination programs (e.g. oral vaccination. Most commonly used serological tests are the virus neutralization test on cell culture (FAVN, virus neutralization in mice and ELISA.

  1. Diagnostic Challenges at SNS

    CERN Document Server

    Plum, M A


    The Spallation Neutron Source now being built in Oak Ridge, Tennessee, USA, accelerates an H- ion beam to 1000 MeV with an average power of 1.4 MW. The H- beam is then stripped to H+, compressed in a storage ring to a pulse length of 695 ns, and then directed onto a mercury neutron spallation target. Most of the acceleration is accomplished with superconducting rf cavities. The presence of these cavities, the high average beam power, and the large range of beam intensity in the storage ring, provide unique challenges to the beam diagnostics systems. In this talk we will discuss these challenges and some of our solutions, including the laser profile monitor system, the residual gas ionization profile monitors, and network attached devices. Measurements performed using prototype instrumentation will also be presented.

  2. Oral vs. salivary diagnostics (United States)

    Marques, Joana; Corby, Patricia M.; Barber, Cheryl A.; Abrams, William R.; Malamud, Daniel


    The field of "salivary diagnostics" includes studies utilizing samples obtained from a variety of sources within the oral cavity. These samples include; whole unstimulated saliva, stimulated whole saliva, duct saliva collected directly from the parotid, submandibular/sublingual glands or minor salivary glands, swabs of the buccal mucosa, tongue or tonsils, and gingival crevicular fluid. Many publications state "we collected saliva from subjects" without fully describing the process or source of the oral fluid. Factors that need to be documented in any study include the time of day of the collection, the method used to stimulate and collect the fluid, and how much fluid is being collected and for how long. The handling of the oral fluid during and post-collection is also critical and may include addition of protease or nuclease inhibitors, centrifugation, and cold or frozen storage prior to assay. In an effort to create a standard protocol for determining a biomarker's origin we carried out a pilot study collecting oral fluid from 5 different sites in the mouth and monitoring the concentrations of pro- and anti-inflammatory cytokines detected using MesoScaleDiscovery (MSD) electrochemiluminesence assays. Our data suggested that 3 of the cytokines are primarily derived from the submandibular gland, while 7 of the cytokines come from a source other than the major salivary glands such as the minor salivary glands or cells in the oral mucosae. Here we review the literature on monitoring biomarkers in oral samples and stress the need for determining the blood/saliva ratio when a quantitative determination is needed and suggest that the term oral diagnostic be used if the source of an analyte in the oral cavity is unknown.

  3. Diagnostics development plan for ZR.

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, David Lester


    The Z Refurbishment (ZR) Project is a program to upgrade the Z machine at SNL with modern durable pulsed power technology, providing additional shot capacity and improved reliability as well as advanced capabilities for both pulsed x-ray production and high pressure generation. The development of enhanced diagnostic capabilities is an essential requirement for ZR to meet critical mission needs. This report presents a comprehensive plan for diagnostic instrument and infrastructure development for the first few years of ZR operation. The focus of the plan is on: (1) developing diagnostic instruments with high spatial and temporal resolution, capable of low noise operation and survival in the severe EMP, bremsstrahlung, and blast environments of ZR; and (2) providing diagnostic infrastructure improvements, including reduced diagnostic trigger signal jitter, more and flexible diagnostic line-of-sight access, and the capability for efficient exchange of diagnostics with other laboratories. This diagnostic plan is the first step in an extended process to provide enhanced diagnostic capabilities for ZR to meet the diverse programmatic needs of a broad range of defense, energy, and general science programs of an international user community into the next decade.

  4. Cognitive Diagnostic Assessment via Bayesian Evaluation of Informative Diagnostic Hypotheses

    NARCIS (Netherlands)

    Hoitink, Herbert; Beland, Sebastien; Vermeulen, Jorine A.


    There exist diverse approaches that can be used for cognitive diagnostic assessment, such as mastery testing, constrained latent class analysis, rule space methodology, diagnostic cognitive modeling, and person-fit analysis. Each of these approaches can be used within 1 of the 4 psychometric perspec

  5. Cognitive diagnostic assessment via Bayesian evaluation of informative diagnostic hypotheses.

    NARCIS (Netherlands)

    Hoijtink, Herbert; Béland, Sébastien; Vermeulen, Jorine A.


    There exist diverse approaches that can be used for cognitive diagnostic assessment, such as mastery testing, constrained latent class analysis, rule space methodology, diagnostic cognitive modeling, and person-fit analysis. Each of these approaches can be used within 1 of the 4 psychometric perspec

  6. Plasma diagnostics discharge parameters and chemistry

    CERN Document Server

    Auciello, Orlando


    Plasma Diagnostics, Volume 1: Discharge Parameters and Chemistry covers seven chapters on the important diagnostic techniques for plasmas and details their use in particular applications. The book discusses optical diagnostic techniques for low pressure plasmas and plasma processing; plasma diagnostics for electrical discharge light sources; as well as Langmuir probes. The text also describes the mass spectroscopy of plasmas, microwave diagnostics, paramagnetic resonance diagnostics, and diagnostics in thermal plasma processing. Electrical engineers, nuclear engineers, microwave engineers, che

  7. Dual Processing and Diagnostic Errors (United States)

    Norman, Geoff


    In this paper, I review evidence from two theories in psychology relevant to diagnosis and diagnostic errors. "Dual Process" theories of thinking, frequently mentioned with respect to diagnostic error, propose that categorization decisions can be made with either a fast, unconscious, contextual process called System 1 or a slow, analytical,…

  8. Pyomyositis tropicans: a diagnostic dilemma. (United States)

    Chaitow, J; Martin, H C; Knight, P; Buchanan, N


    Pyomyositis tropicans is a rare disease in non-tropical climates and thus presents diagnostic difficulties. Two children with single staphylococcal psoas muscle abscesses were recently successfully treated. Computerized axial tomography was found to be a useful diagnostic aid, allowing exact localization of the lesion. The diagnosis and therapy of these abscesses are discussed.

  9. Motor neurone disease: diagnostic pitfalls. (United States)

    Williams, Timothy L


    The misdiagnosis of MND (particularly of the ALS phenotype), is uncommon. Atypical presentations, particularly of focal onset and with pure LMN or UMN signs, present a more difficult diagnostic challenge, although perhaps reassuringly, treatable mimics are rare. A working knowledge of potential alternative conditions and MND diagnostic pitfalls should help to reduce the misdiagnosis rate, particularly if the key points are considered.

  10. Status of US ITER Diagnostics (United States)

    Stratton, B.; Delgado-Aparicio, L.; Hill, K.; Johnson, D.; Pablant, N.; Barnsley, R.; Bertschinger, G.; de Bock, M. F. M.; Reichle, R.; Udintsev, V. S.; Watts, C.; Austin, M.; Phillips, P.; Beiersdorfer, P.; Biewer, T. M.; Hanson, G.; Klepper, C. C.; Carlstrom, T.; van Zeeland, M. A.; Brower, D.; Doyle, E.; Peebles, A.; Ellis, R.; Levinton, F.; Yuh, H.


    The US is providing 7 diagnostics to ITER: the Upper Visible/IR cameras, the Low Field Side Reflectometer, the Motional Stark Effect diagnostic, the Electron Cyclotron Emission diagnostic, the Toroidal Interferometer/Polarimeter, the Core Imaging X-Ray Spectrometer, and the Diagnostic Residual Gas Analyzer. The front-end components of these systems must operate with high reliability in conditions of long pulse operation, high neutron and gamma fluxes, very high neutron fluence, significant neutron heating (up to 7 MW/m3) , large radiant and charge exchange heat flux (0.35 MW/m2) , and high electromagnetic loads. Opportunities for repair and maintenance of these components will be limited. These conditions lead to significant challenges for the design of the diagnostics. Space constraints, provision of adequate radiation shielding, and development of repair and maintenance strategies are challenges for diagnostic integration into the port plugs that also affect diagnostic design. The current status of design of the US ITER diagnostics is presented and R&D needs are identified. Supported by DOE contracts DE-AC02-09CH11466 (PPPL) and DE-AC05-00OR22725 (UT-Battelle, LLC).

  11. Verification of Loop Diagnostics (United States)

    Winebarger, A.; Lionello, R.; Mok, Y.; Linker, J.; Mikic, Z.


    Many different techniques have been used to characterize the plasma in the solar corona: density-sensitive spectral line ratios are used to infer the density, the evolution of coronal structures in different passbands is used to infer the temperature evolution, and the simultaneous intensities measured in multiple passbands are used to determine the emission measure. All these analysis techniques assume that the intensity of the structures can be isolated through background subtraction. In this paper, we use simulated observations from a 3D hydrodynamic simulation of a coronal active region to verify these diagnostics. The density and temperature from the simulation are used to generate images in several passbands and spectral lines. We identify loop structures in the simulated images and calculate the loop background. We then determine the density, temperature and emission measure distribution as a function of time from the observations and compare with the true temperature and density of the loop. We find that the overall characteristics of the temperature, density, and emission measure are recovered by the analysis methods, but the details of the true temperature and density are not. For instance, the emission measure curves calculated from the simulated observations are much broader than the true emission measure distribution, though the average temperature evolution is similar. These differences are due, in part, to inadequate background subtraction, but also indicate a limitation of the analysis methods.

  12. Diagnostic of Horndeski Theories

    CERN Document Server

    Perenon, Louis; Piazza, Federico


    We study the effects of Horndeski models of dark energy on the observables of the large-scale structure in the late time universe. A novel classification into {\\it Late dark energy}, {\\it Early dark energy} and {\\it Early modified gravity} scenarios is proposed, according to whether such models predict deviations from the standard paradigm persistent at early time in the matter domination epoch. We discuss the physical imprints left by each specific class of models on the effective Newton constant $\\mu$, the gravitational slip parameter $\\eta$, the light deflection parameter $\\Sigma$ and the growth function $f\\sigma_8$ and demonstrate that a convenient way to dress a complete portrait of the viability of the Horndeski accelerating mechanism is via two, redshift-dependent, diagnostics: the $\\mu(z)-\\Sigma(z)$ and the $f\\sigma_8(z)-\\Sigma(z)$ planes. If future, model-independent, measurements point to either $\\Sigma-10$ at high redshifts or $\\mu-1>0$ with $\\Sigma-11.5$ then Early dark energy models are definitel...

  13. Diagnostic of Horndeski theories (United States)

    Perenon, Louis; Marinoni, Christian; Piazza, Federico


    We study the effects of Horndeski models of dark energy on the observables of the large-scale structure in the late time universe. A novel classification into Late dark energy, Early dark energy and Early modified gravity scenarios is proposed, according to whether such models predict deviations from the standard paradigm persistent at early time in the matter domination epoch. We discuss the physical imprints left by each specific class of models on the effective Newton constant μ, the gravitational slip parameter η, the light deflection parameter Σ and the growth function fσ8 and demonstrate that a convenient way to dress a complete portrait of the viability of the Horndeski accelerating mechanism is via two, redshift-dependent, diagnostics: the μ(z) ‑ Σ(z) and the fσ8(z) ‑ Σ(z) planes. If future, model-independent, measurements point to either Σ ‑ 1 zero or μ ‑ 1 0 at high redshifts or μ ‑ 1 > 0 with Σ ‑ 1 1.5 then Early dark energy models are definitely ruled out. On the opposite case, Late dark energy models are rejected by data if Σ 1, only Early modifications of gravity provide a viable framework to interpret data.

  14. Gonorrhoea diagnostics: An update

    Directory of Open Access Journals (Sweden)

    R Verma


    Full Text Available Diagnosis of gonorrhoea is an ongoing challenge. The organism is fastidious requiring meticulous collection and transport for successful cultivation. Asymptomatic infections are common which go undetected by conventional methods thereby leading to continued transmission and the risk of complications. The nucleic acid amplification tests, now increasingly used in developed countries, offer improved sensitivity compared to bacterial culture. However, these continue to suffer sequence related problems leading to false positive and false negative results. Further, these cannot be used for generation of data on antibiotic susceptibility because genetic markers of antibiotic resistance to recommended therapies have not been fully characterised. They are unaffordable in a setting like ours where reliance is placed on syndromic approach for sexually transmitted infection (STI management. The use of syndromic approach has resulted in a considerable decline in the number of Neisseria gonorrhoeae isolates that have been cultured for diagnostic purposes. Many laboratories formerly doing so are no longer performing culture for gonococci, and the basic skills have been lost. There is a need to not only revive this skill but also adopt newer technologies that can aid in accurate diagnosis in a cost-effective manner. There is room for innovation that can facilitate the development of a point-of-care test for this bacterial STI.

  15. Plasma diagnostics for tokamaks and stellarators

    Energy Technology Data Exchange (ETDEWEB)

    Stott, P. E.; Sanchez, J.


    A collection of papers on plasma diagnostics is presented. The papers show the state of the art developments in a series of techniques: Magnetic diagnostics, Edge diagnostics, Langmuir probes, Spectroscopy, Microwave and FIR diagnostics as well as Thomson Scattering. Special interest was focused on those diagnostics oriented to fluctuations measurements in the plasma. (Author) 451 refs.

  16. Melarsoprol sensitivity profile of Trypanosoma brucei gambiense isolates from cured and relapsed sleeping sickness patients from the Democratic Republic of the Congo.

    Directory of Open Access Journals (Sweden)

    Patient Pyana Pati


    Full Text Available Sleeping sickness caused by Trypanosoma brucei (T.b. gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba.Forty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1.We conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.

  17. [Novel methods for dementia diagnostics]. (United States)

    Wiltfang, J


    Novel diagnostic methods, such as cerebrospinal fluid-based neurochemical dementia diagnostics (CSF-NDD) and [18F] amyloid positron emission tomography (PET) are meanwhile recommended for specific indications by international guidelines for the improved early and differential diagnostics of multigenic (sporadic) Alzheimer's dementia (AD). In the case of CSF-NDD the German neuropsychiatric guidelines have already been validated on the S3 level of evidence ( and the additional consideration of [18F] amyloid-PET in the current update of the guidelines is to be expected. By means of CSF-NDD and/or [18F] amyloid-PET a predictive diagnosis of incipient (preclinical) AD is also possible for patients at high risk for AD who are in prodromal stages, such as mild cognitive impairment (MCI). As accompanying (secondary) preventive therapy of AD cannot be offered a predictive molecular dementia diagnostics is not recommended by the German neuropsychiatric dementia guidelines ( However, novel diagnostic approaches, which offer molecular positive diagnostics of AD have already gained high relevance in therapy research as they allow promising preventive treatment avenues to be validated directly in the clinical trial. Moreover, future blood-based dementia diagnostics by means of multiplex assays is becoming increasingly more feasible; however, so far corresponding proteomic or epigenetic assays could not be consistently validated in independent studies.

  18. [BMW diagnostic criteria for IBS]. (United States)

    Matsueda, Kei


    Rome I diagnostic criteria for IBS was published in 1992 and it became a global diagnostic criteria. However, the criteria was not practical and somewhat complicated. Moreover, its symptomatic duration was too long (defined as more than 3 months) to be introduced in clinical practice. Therefore, Japanese member of BMW(Bowel Motility Workshop) tried to develop a new diagnostic criteria for IBS and it was established in 1995 by way of the Delphi method. The criteria was named as BMW diagnostic criteria and it was shown below: BMW diagnostic criteria for IBS (1995) At least one month or more of repetitive symptoms of the following 1) and 2) and no evidence of organic disease that likely to explain the symptoms. 1) Existence of abdominal pain, abdominal discomfort or abdominal distension 2) Existence of abnormal bowel movement (diarrhea, constipation) Abnormal bowel movement includes at least one of the below; (1) Abnormal stool frequency (2) Abnormal stool form (lumpy/hard or loose/wartery stool) Moreover, the following test should be performed as a rule to exclude organic diseases. (1) Urinalysis, fecal occult blood testing, CBC, chemistry (2) Barium enema or colonofiberscopic examination The other diagnostic criteria for IBS was also reviewed and their characteristics were compared with BMW diagnostic criteria.

  19. Diagnostic Challenges in AIH

    Directory of Open Access Journals (Sweden)

    A Taghavi Ardakani


    Full Text Available Autoimmune hepatitis is a chronic hepatitis that occurs in children and adults of all ages. Diagnosis is based upon characteristic serologic and histologic findings and the exclusion of other forms of chronic liver disease. Guidelines issued by the AASLD suggest the following diagnostic considerations: The diagnosis should be made in patients with compatible clinical signs, symptoms, and laboratory abnormalities. Other conditions that can cause chronic hepatitis should be excluded. In unclear cases a standardized scoring system should be used in the assessment. In those who are negative for conventional autoantibodies, additional autoantibodies should be sought. All patients with autoimmune hepatitis and inflammatory bowel disease should undergo cholangiographic studies to exclude primary sclerosing cholangitis.   Scoring systems- A scoring system developed and subsequently revised by the International Autoimmune Hepatitis Group to standardize the diagnosis with using simplified criteria based upon titers of autoantibodies, IgG levels, liver histology, and the exclusion of viral hepatitis. Autoantibodies: assign one point if the ANA or SMA are 1:40 OR assign two points if the ANA or SMA are ≥1:80 (OR if the LKM ≥1:40 OR if the SLA is positive. IgG: assign one point if the IgG is > the upper limit of normal OR assign two points if the IgG is >1.10 times the upper limit of normal. Liver histology: assign one point if the histological features are compatible with autoimmune hepatitis OR two points if the histological features are typical of autoimmune hepatitis. Absence of viral hepatitis A probable diagnosis of autoimmune hepatitis is made if the total points are six, while a definite diagnosis is made if the total points are ≥seven.  

  20. Industrial applications of laser diagnostics

    CERN Document Server

    Deguchi, Yoshihiro


    Tighter regulations of harmful substances such as NOx, CO, heavy metals, particles, emissions from commercial plants and automobiles reflect a growing demand for lowering the anthropogenic burdens on the environment. It is equally important to monitor controlling factors to improve the operation of industrial machinery and plants. Among the many methods for doing this, laser diagnostics stands out. Taking a practical approach, Industrial Applications of Laser Diagnostics discusses how to apply laser diagnostics to engines, gas turbines, thermal and chemical plant systems, and disposal faciliti

  1. Evolution of the serum resistance-associated SRA gene in African trypanosomes

    Institute of Scientific and Technical Information of China (English)

    LAI DeHua; WANG QiaoPing; LI Zhi; JULIUS Luke(s); LUN ZhaoRong


    Serum resistance-associated (SRA) protein, a protein unique for Trypanosoma brucei rhodesiense, is responsible for resistance of this parasite to the lysis by normal human serum (NHS) and is a vital molecular marker to distinguish this species from other African trypanosomes. We cloned and se-quenced the SRA basic copy (SRAbc) gene from T. b. rhodesiense and related species and found that this gene is confined to the subgenus Trypanozoon. The average 82% identity among the sequenced SRAbc genes indicates that they may have a common origin and are highly conserved. Since SRAbc coexists in the T. b. rhodesiense genome with SRA, we propose that SRAbc might be the 'donor VSG', which after duplication became inserted into the expression site by recombination. Under natural se-lection, SRAbc could reform into SRA following mosaic formation.

  2. FEL-accelerator related diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Kevin Jordan; David Douglas; Stephen V. Benson; Pavel Evtuschenko


    Free Electron Lasers (FEL) present a unique set of beam parameters to the diagnostics suite. The FEL requires characterization of the full six dimensional phase space of the electron beam at the wiggler and accurate alignment of the electron beam to the optical mode of the laser. In addition to the FEL requirements on the diagnostics suite, the Jefferson Lab FEL is operated as an Energy Recovered Linac (ERL) which imposes additional requirements on the diagnostics. The ERL aspect of the Jefferson Lab FEL requires that diagnostics operate over a unique dynamic range and operate with simultaneous transport of the accelerated and energy recovered beams. This talk will present how these challenges are addressed at the Jefferson Lab FEL.

  3. The architecture of diagnostic research

    DEFF Research Database (Denmark)

    Colli, Agostino; Fraquelli, Mirella; Casazza, Giovanni


    The diagnostic research process can be divided into five phases, designed to establish the clinical utility of a new diagnostic test - the index test. The aim of the present review is to illustrate the study designs that are appropriate for each diagnostic phase, using clinical examples regarding...... the present strategy. Phase 4 comprises large surveillance cohort studies that aim to assess the effectiveness of the new diagnostic-therapeutic strategy in clinical practice. As common in clinical research, putting excessive weight on the results of single studies and trials is likely to divert from...... the totality of evidence reached through the systematic reviews of these studies, conducted with rigorous methodology and statistical methods. (Hepatology 2013;)....

  4. Saliva Preservative for Diagnostic Purposes (United States)

    Pierson, Duane L.; Mehta, Satish K.


    Saliva is an important body fluid for diagnostic purposes. Glycoproteins, glucose, steroids, DNA, and other molecules of diagnostic value are found in saliva. It is easier to collect as compared to blood or urine. Unfortunately, saliva also contains large numbers of bacteria that can release enzymes, which can degrade proteins and nucleic acids. These degradative enzymes destroy or reduce saliva s diagnostic value. This innovation describes the formulation of a chemical preservative that prevents microbial growth and inactivates the degradative enzymes. This extends the time that saliva can be stored or transported without losing its diagnostic value. Multiple samples of saliva can be collected if needed without causing discomfort to the subject and it does not require any special facilities to handle after it is collected.

  5. Diagnostic Process of Company Productivity

    Directory of Open Access Journals (Sweden)

    Emese Tokarˇcíková


    Full Text Available This paper deals with an actual topic of how key factors of enterprise diagnostics can help to increase company productivity. Recognition and use of relevant internal and external information in this field determines the success of the enterprise. Application of the general diagnostic model of company productivity to the net income has been a frequent problem of company practice. This problem is of profit showing, which is an inevitable precondition for long-term company development and growth. Diagnostic access of company productivity allows recognition of specific problems in greater detail, which results from the activity of each company. This article also presents an introduction to the researched area of enterprise diagnostics, which opens opportunities for other publishing activities and can lead to information exchange.

  6. Molecular diagnostics of myeloproliferative neoplasms

    DEFF Research Database (Denmark)

    Langabeer, S. E.; Andrikovics, H.; Asp, J.;


    identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation...... of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic...

  7. Synthesis and antiprotozoal activity of pyridyl analogues of pentamidine. (United States)

    Bakunova, Svetlana M; Bakunov, Stanislav A; Wenzler, Tanja; Barszcz, Todd; Werbovetz, Karl A; Brun, Reto; Tidwell, Richard R


    A series of novel pyridyl analogues 1-18 of antiprotozoal drug 1,5-bis(4-amidinophenoxy)pentane (pentamidine) has been synthesized and tested for in vitro activities against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, and for cytotoxicity against mammalian cells. Antiprotozoal properties of compounds 1-18 depended on the placement of cationic moieties on the pyridine rings as well as the nature of substituents on the amidine groups. Diamidine 6 with cationic moieties adjacent to pyridine nitrogen atoms was the most promising compound in the series showing superior in vitro activities against T. brucei rhodesiense, P. falciparum, and L. donovani compared to pentamidine. An oral prodrug of diamidine 6, diamidoxime 9, administered at 25 mg/kg daily for 4 days, exhibited excellent antitrypanosomal efficacy in vivo curing all infected animals in the STIB900 acute mouse model of trypanosomiasis.

  8. In vitro and in vivo activities of 2-aminopyrazines and 2-aminopyridines in experimental models of human African trypanosomiasis. (United States)

    Vodnala, Suman K; Lundbäck, Thomas; Sjöberg, Birger; Svensson, Richard; Rottenberg, Martin E; Hammarström, Lars G J


    New drugs for the treatment of human African trypanosomiasis are urgently needed. A number of 2-aminopyrazines/2-aminopyridines were identified as promising leads following a focused screen of 5,500 compounds for Trypanosoma brucei subsp. brucei viability. Described compounds are trypanotoxic in the submicromolar range and show comparably low cytotoxicity on representative mammalian cell lines. Specifically, 6-([6-fluoro-3,4-dihydro-2H-1-benzopyran-4-yl)]oxy)-N-(piperidin-4-yl)pyrazin-2-amine (CBK201352) is trypanotoxic for T. brucei subsp. brucei, T. brucei subsp. gambiense, and T. brucei subsp. rhodesiense and is nontoxic to mammalian cell lines, and in vitro preclinical assays predict promising pharmacokinetic parameters. Mice inoculated intraperitoneally (i.p.) with 25 mg/kg CBK201352 twice daily for 10 days, starting on the day of infection with T. brucei subsp. brucei, show complete clearance of parasites for more than 90 days. Thus, CBK201352 and related analogs are promising leads for the development of novel treatments for human African trypanosomiasis.

  9. Diagnostic indices for vertiginous diseases

    Directory of Open Access Journals (Sweden)

    Warninghoff Jan-Christian


    Full Text Available Abstract Background Vertigo and dizziness are symptoms which are reported frequently in clinical practice. We aimed to develop diagnostic indices for four prevalent vertiginous diseases: benign paroxysmal positional vertigo (BPPV, Menière's disease (MD, vestibular migraine (VM, and phobic postural vertigo (PPV. Methods Based on a detailed questionnaire handed out to consecutive patients presenting for the first time in our dizziness clinic we preselected a set of seven questions with desirable diagnostic properties when compared with the final diagnosis after medical workup. Using exact logistic regression analysis diagnostic scores, each comprising of four to six items that can simply be added up, were built for each of the four diagnoses. Results Of 193 patients 131 questionnaires were left after excluding those with missing consent or data. Applying the suggested cut-off points, sensitivity and specificity were 87.5 and 93.5% for BPPV, 100 and 87.4% for MD, 92.3 and 83.7% for VM, 73.7 and 84.1% for PPV, respectively. By changing the cut-off points sensitivity and specificity can be adjusted to meet diagnostic needs. Conclusions The diagnostic indices showed promising diagnostic properties. Once further validated, they could provide an ease to use and yet flexible tool for screening vertigo in clinical practice and epidemiological research.

  10. Stochastic Engine Convergence Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Glaser, R


    ;'burn-in'' period. The MCMC process begins at a particular state, which is selected at random or by design, according to the wish of the user of the engine. After the burn-in period, the chain has essentially forgotten where it started. Moreover, the sample x{sup (t{sub 0})}, x{sup (T{sub 0}+1)},... can be used for most purposes as a random sample from f, even though the x{sup (T{sub 0}+t)}, because of Markovian dependency, are not independent. For example, averages involving x{sup (t{sub 0})}, x{sup (t{sub 0}+1)},... may have an approximate normal distribution. The purpose of this note is to discuss the monitoring techniques currently in place in the stochastic engine software that addresses the issues of burn-in, stationarity, and normality. They are loosely termed ''convergence diagnostics'', in reference to the underlying Markov chains, which converge asymptotically to the desired posterior distribution.

  11. Lead optimization of a pyrazole sulfonamide series of Trypanosoma brucei N-myristoyltransferase inhibitors: identification and evaluation of CNS penetrant compounds as potential treatments for stage 2 human African trypanosomiasis. (United States)

    Brand, Stephen; Norcross, Neil R; Thompson, Stephen; Harrison, Justin R; Smith, Victoria C; Robinson, David A; Torrie, Leah S; McElroy, Stuart P; Hallyburton, Irene; Norval, Suzanne; Scullion, Paul; Stojanovski, Laste; Simeons, Frederick R C; van Aalten, Daan; Frearson, Julie A; Brenk, Ruth; Fairlamb, Alan H; Ferguson, Michael A J; Wyatt, Paul G; Gilbert, Ian H; Read, Kevin D


    Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood-brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT.

  12. Human African trypanosomiasis in a Belgian traveller returning from the Masai Mara area, Kenya, February 2012. (United States)

    Clerinx, J; Vlieghe, E; Asselman, V; Van de Casteele, S; Maes, M B; Lejon, V


    A Belgian traveller was diagnosed with human African trypanosomiasis (HAT) due to Trypanosoma brucei rhodesiense nine days after visiting the Masai Mara area in Kenya. He presented with an inoculation chancre and was treated with suramin within four days of fever onset. Two weeks earlier, HAT was also reported in a German traveller who had visited the Masai Mara area. Because no cases have occurred in the area for over 12 years, this may indicate a focal cluster of HAT.

  13. Synthesis, DNA binding and antitrypanosomal activity of benzimidazole analogues of DAPI. (United States)

    Farahat, Abdelbasset A; Bennett-Vaughn, Cheree; Mineva, Ekaterina M; Kumar, Arvind; Wenzler, Tanja; Brun, Reto; Liu, Yang; Wilson, W David; Boykin, David W


    A series of novel benzimidazole diamidines were prepared from the corresponding dicyano analogues either by applying Pinner methodology (5a-c, 10 and 13a) or by making amidoximes intermediates that were reduced to the corresponding amidines (15a-c). The new amidines were evaluated in vitro against the protozoan parasite Trypanosoma brucei rhodesiense (T. b. r.). The thiophene analogue 5b and the N-methyl compound 15a showed superior antitrypanosomal activity compared to that of the parent I.

  14. Synthesis and antiprotozoal properties of pentamidine congeners bearing the benzofuran motif. (United States)

    Bakunov, Stanislav A; Bakunova, Svetlana M; Bridges, Arlene S; Wenzler, Tanja; Barszcz, Todd; Werbovetz, Karl A; Brun, Reto; Tidwell, Richard R


    Forty-eight cationically substituted pentamidine congeners possessing benzofuran rings were synthesized by a copper mediated heteroannulation of substituted o-iodophenols with phenyl acetylenes. Activities of compounds 1-48 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and cytotoxicities for mammalian cells were influenced by the nature of cationic substituents, placement of the benzofuran fragment, and the length of the carbon linker between aromatic moieties. Several dications exhibited superior antiplasmodial and antileishmanial potencies compared to pentamidine.

  15. Evolution of the primate trypanolytic factor APOL1. (United States)

    Thomson, Russell; Genovese, Giulio; Canon, Chelsea; Kovacsics, Daniella; Higgins, Matthew K; Carrington, Mark; Winkler, Cheryl A; Kopp, Jeffrey; Rotimi, Charles; Adeyemo, Adebowale; Doumatey, Ayo; Ayodo, George; Alper, Seth L; Pollak, Martin R; Friedman, David J; Raper, Jayne


    ApolipoproteinL1 (APOL1) protects humans and some primates against several African trypanosomes. APOL1 genetic variants strongly associated with kidney disease in African Americans have additional trypanolytic activity against Trypanosoma brucei rhodesiense, the cause of acute African sleeping sickness. We combined genetic, physiological, and biochemical studies to explore coevolution between the APOL1 gene and trypanosomes. We analyzed the APOL1 sequence in modern and archaic humans and baboons along with geographic distribution in present day Africa to understand how the kidney risk variants evolved. Then, we tested Old World monkey, human, and engineered APOL1 variants for their ability to kill human infective trypanosomes in vivo to identify the molecular mechanism whereby human trypanolytic APOL1 variants evade T. brucei rhodesiense virulence factor serum resistance-associated protein (SRA). For one APOL1 kidney risk variant, a two-residue deletion of amino acids 388 and 389 causes a shift in a single lysine residue that mimics the Old World monkey sequence, which augments trypanolytic activity by preventing SRA binding. A second human APOL1 kidney risk allele, with an amino acid substitution that also restores sequence alignment with Old World monkeys, protected against T. brucei rhodesiense due in part to reduced SRA binding. Both APOL1 risk variants induced tissue injury in murine livers, the site of transgenic gene expression. Our study shows that both genetic variants of human APOL1 that protect against T. brucei rhodesiense have recapitulated molecular signatures found in Old World monkeys and raises the possibility that APOL1 variants have broader innate immune activity that extends beyond trypanosomes.

  16. Far infrared fusion plasma diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Luhmann, N.C. Jr.; Peebles, W.A.


    Over the last several years, reflectometry has grown in importance as a diagnostic for both steady-state density Profiles as well as for the investigation of density fluctuations and turbulence. As a diagnostic for density profile measurement, it is generally believed to be well understood in the tokamak environment. However, its use as a fluctuation diagnostic is hampered by a lack of quantitative experimental understanding of its wavenumber sensitivity and spatial resolution. Several researchers, have theoretically investigated these questions. However, prior to the UCLA laboratory investigation, no group has experimentally investigated these questions. Because of the reflectometer's importance to the world effort in understanding plasma turbulence and transport, UCLA has, over the last year, made its primary Task IIIA effort the resolution of these questions. UCLA has taken the lead in a quantitative experimental understanding of reflectometer data as applied to the measurement of density fluctuations. In addition to this, work has proceeded on the design, construction, and installation of a reflectometer system on UCLA's CCT tokamak. This effort will allow a comparison between the improved confinement regimes (H-mode) observed on both the DIII-D and CCT machines with the goal of achieving a physics understanding of the phenomena. Preliminary investigation of a new diagnostic technique to measure density profiles as a function of time has been initiated at UCLA. The technique promises to be a valuable addition to the range of available plasma diagnostics. Work on advanced holographic reflectometry technique as applied to fluctuation diagnostics has awaited a better understanding of the reflectometer signal itself as discussed above. Efforts to ensure the transfer of the diagnostic developments have continued with particular attention devoted to the preliminary design of a multichannel FIR interferometer for MST.

  17. Diagnostics for the ATA beam propagation experiments

    Energy Technology Data Exchange (ETDEWEB)

    Fessenden, T.J.; Atchison, W.L.; Barletta, W.A.


    This report contains a discussion of the diagnostics required for the beam propagation experiment to be done with the ATA accelerator. Included are a list of the diagnostics needed; a description of the ATA experimental environment; the status of beam diagnostics available at Livermore including recent developments, and a prioritized list of accelerator and propagation diagnostics under consideration or in various stages of development.

  18. Microarray Technologies in Fungal Diagnostics. (United States)

    Rupp, Steffen


    Microarray technologies have been a major research tool in the last decades. In addition they have been introduced into several fields of diagnostics including diagnostics of infectious diseases. Microarrays are highly parallelized assay systems that initially were developed for multiparametric nucleic acid detection. From there on they rapidly developed towards a tool for the detection of all kind of biological compounds (DNA, RNA, proteins, cells, nucleic acids, carbohydrates, etc.) or their modifications (methylation, phosphorylation, etc.). The combination of closed-tube systems and lab on chip devices with microarrays further enabled a higher automation degree with a reduced contamination risk. Microarray-based diagnostic applications currently complement and may in the future replace classical methods in clinical microbiology like blood cultures, resistance determination, microscopic and metabolic analyses as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel nucleic acid based biomarkers. Here I focus an microarray technologies in diagnostics and as research tools, based on nucleic acid-based arrays.

  19. Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human African trypanosomiasis.

    Directory of Open Access Journals (Sweden)

    Lauren Sullivan


    Full Text Available The diagnosis of human African trypanosomiasis (HAT caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT. There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2 and a specificity of 83.3% (95% CI: 76.4 to 88.9 for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9 and specificity of 97.3% (95% CI: 90.7 to 99.7. However, using one or both positive antigen band(s as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4 with a specificity of 85.3% (95% CI: 75.3 to 92.4. These results encourage further development of the dual-antigen device for clinical use.

  20. Optical diagnostics of intermittent flows

    DEFF Research Database (Denmark)

    Okulov, V.L.; Naumov, I.V.; Sørensen, Jens Nørkær


    The efficiency of combined use of different optical techniques for flow diagnostics is demonstrated with the practically important case of intense swirling flows. It is shown that, when applied separately, commonly used optical measuring techniques, such as laser Doppler anemometry and particle...... image velocimetry, frequently give erroneous results, especially for the transition flow and developed nonstationary flow. However, their combined use in diagnostics of unsteady (intermittent) flows significantly improves both the temporal and spatial resolution of measurements. Such a complex approach...... is for the first time applied for diagnostics of the flow pattern in a closed cylinder with a rotating end face with the aim of studying the changeover from the steady axisymmetric to unsteady asymmetric flow over a wide range of flow parameters. It is found that such a transition is notable for azimuthal...

  1. Diagnostic accuracy in virtual dermatopathology

    DEFF Research Database (Denmark)

    Mooney, E.; Kempf, W.; Jemec, G.B.E.;


    Background Virtual microscopy is used for teaching medical students and residents and for in-training and certification examinations in the United States. However, no existing studies compare diagnostic accuracy using virtual slides and photomicrographs. The objective of this study was to compare...... slides and photomicrographs with corresponding clinical photographs and information in a self-assessment examination format. Descriptive data analysis and comparison of groups were performed using a chi-square test. Results Diagnostic accuracy in dermatopathology using virtual dermatopathology...... represented a useful tool for learning; 90% felt that virtual dermatopathology is useful tool for teaching dermatopathology. Conclusion No significant difference was observed in diagnostic accuracy using virtual dermatopathology compared to photomicrographs. Most participants felt virtual dermatopathology...

  2. A Diagnostic Approach to Hemochromatosis

    Directory of Open Access Journals (Sweden)

    Anthony S Tavill


    Full Text Available In the present clinical review, a diagnostic approach to hemochromatosis is discussed from the perspective of two clinicians with extensive experience in this area. The introduction of genetic testing and large-scale population screening studies have broadened our understanding of the clinical expression of disease and the utility of biochemical iron tests for the detection of disease and for the assessment of disease severity. Liver biopsy has become more of a prognostic test than a diagnostic test. The authors offer a stepwise, diagnostic algorithm based on current evidence-based data, that they regard as most cost-effective. An early diagnosis can lead to phlebotomy therapy to prevent the development of cirrhosis.

  3. Enhanced NIF neutron activation diagnostics. (United States)

    Yeamans, C B; Bleuel, D L; Bernstein, L A


    The NIF neutron activation diagnostic suite relies on removable activation samples, leading to operational inefficiencies and a fundamental lower limit on the half-life of the activated product that can be observed. A neutron diagnostic system measuring activation of permanently installed samples could remove these limitations and significantly enhance overall neutron diagnostic capabilities. The physics and engineering aspects of two proposed systems are considered: one measuring the (89)Zr/(89 m)Zr isomer ratio in the existing Zr activation medium and the other using potassium zirconate as the activation medium. Both proposed systems could improve the signal-to-noise ratio of the current system by at least a factor of 5 and would allow independent measurement of fusion core velocity and fuel areal density.

  4. Molecular diagnostics of neurodegenerative disorders

    Directory of Open Access Journals (Sweden)

    Megha eAgrawal


    Full Text Available Molecular diagnostics provide a powerful method to detect and diagnose various neurological diseases such as Alzheimer’s and Parkinson’s disease. The confirmation of such diagnosis allows early detection and subsequent medical counseling that help specific patients to undergo clinically important drug trials. This provides a medical pathway to have better insight of neurogenesis and eventual cure of the neurodegenerative diseases. In this short review, we present recent advances in molecular diagnostics especially biomarkers and imaging spectroscopy for neurological diseases. We describe advances made in Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis and Huntington’s disease, and finally present a perspective on the future directions to provide a framework for further developments and refinements of molecular diagnostics to combat neurodegenerative disorders.

  5. Audiologic diagnostics of vestibular schwannoma

    Directory of Open Access Journals (Sweden)

    Komazec Zoran


    Full Text Available Introduction Vestibular schwannoma (acoustic neuroma is a rare, but important cause of sensorineural hearing loss. Patients with asymmetric hearing loss, or unilateral tinnitus should be evaluated expeditiously, to prevent further neurological damage. Audiologic diagnostics Audiologic diagnostics represents the basic diagnosis for early detection of vestibular schwannoma. Patients with vestibular schwannomas may present with a variety of clinical features, including retrocochlear pattern of sensorineural hearing loss. Supraliminary audiometry, tympano- metry, stapedius reflex and otoacoustic emissions as well as vestibular response to caloric testing are methods for selection of patients with suspicion of this tumor. Conclusion The golden standard for audiologic diagnostics of vestibular schwannoma is BAEP (Brainstem Auditory Evoked Potentials. Patients with pathological findings of BAEP should undergo MRI of the posterior fossa. Gadolinium-enhanced magnetic resonance imaging is the best and final tool for making a diagnosis of vestibular schwannoma.

  6. Imaging Techniques for Microwave Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Donne, T. [FOM-Institute for Plasma Physics Rijnhuizen, Trilateral Euregio Cluster, PO Box 1207, 3430 BE Nieuwegein (Netherlands); Luhmann Jr, N.C. [University of California, Davis, CA 95616 (United States); Park, H.K. [POSTECH, Pohang, Gyeongbuk 790-784 (Korea, Republic of); Tobias, B.


    Advances in microwave technology have made it possible to develop a new generation of microwave imaging diagnostics for measuring the parameters of magnetic fusion devices. The most prominent of these diagnostics is electron cyclotron emission imaging (ECE-I). After the first generation of ECE-I diagnostics utilized at the TEXT-U, RTP and TEXTOR tokamaks and the LHD stellarator, new systems have recently come into operation on ASDEX-UG and DIII-D, soon to be followed by a system on KSTAR. The DIII-D and KSTAR systems feature dual imaging arrays that observe different parts of the plasma. The ECE-I diagnostic yields two-dimensional movies of the electron temperature in the plasma and has given already new insights into the physics of sawtooth oscillations, tearing modes and edge localized modes. Microwave Imaging Reflectometry (MIR) is used on LHD to measure electron density fluctuations. A pilot MIR system has been tested at TEXTOR and, based on the promising results, a new system is now under design for KSTAR. The system at TEXTOR was used to measure the plasma rotation velocity. The system at KSTAR and also the one on LHD will be/are used for measuring the profile of the electron density fluctuations in the plasma. Other microwave imaging diagnostics are phase imaging interferometry, and imaging microwave scattering. The emphasis in this paper will be largely focused on ECE-I. First an overview of the advances in microwave technology are discussed, followed by a description of a typical ECE-I system along with some typical experimental results. Also the utilization of imaging techniques in other types of microwave diagnostics will be briefly reviewed. This document is composed of the slides of the presentation. (authors)

  7. [The diagnostic possibilities of saliva]. (United States)

    Kochurova, E V; Kozlov, S V


    Saliva is a clinically informative biological fluid which contains multitude of bio-markers. This characteristic makes it possible to carry out numerous analyzes for developing mode to test patient in situ, express-tests included. The diagnostic by saliva is a new area of more simple application both markers and analyzers that can be useful in diagnostic of diseases of oral cavity, oncological diseases included. The using of saliva expands perspectives for making clinical diagnosis and establishment of dynamics and monitoring of disease.

  8. First Wall and Operational Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Lasnier, C; Allen, S; Boedo, J; Groth, M; Brooks, N; McLean, A; LaBombard, B; Sharpe, J; Skinner, C; Whyte, D; Rudakov, D; West, W; Wong, C


    In this chapter we review numerous diagnostics capable of measurements at or near the first wall, many of which contribute information useful for safe operation of a tokamak. There are sections discussing infrared cameras, visible and VUV cameras, pressure gauges and RGAs, Langmuir probes, thermocouples, and erosion and deposition measurements by insertable probes and quartz microbalance. Also discussed are dust measurements by electrostatic detectors, laser scattering, visible and IR cameras, and manual collection of samples after machine opening. In each case the diagnostic is discussed with a view toward application to a burning plasma machine such as ITER.

  9. Diagnostic testing for Giardia infections. (United States)

    Heyworth, Martin F


    The traditional method for diagnosing Giardia infections involves microscopic examination of faecal specimens for Giardia cysts. This method is subjective and relies on observer experience. From the 1980s onwards, objective techniques have been developed for diagnosing Giardia infections, and are superseding diagnostic techniques reliant on microscopy. Detection of Giardia antigen(s) by immunoassay is the basis of commercially available diagnostic kits. Various nucleic acid amplification techniques (NAATs) can demonstrate DNA of Giardia intestinalis, and have the potential to become standard approaches for diagnosing Giardia infections. Of such techniques, methods involving either fluorescent microspheres (Luminex) or isothermal amplification of DNA (loop-mediated isothermal amplification; LAMP) are especially promising.

  10. Diagnostic imaging in bovine orthopedics. (United States)

    Kofler, Johann; Geissbühler, Urs; Steiner, Adrian


    Although a radiographic unit is not standard equipment for bovine practitioners in hospital or field situations, ultrasound machines with 7.5-MHz linear transducers have been used in bovine reproduction for many years, and are eminently suitable for evaluation of orthopedic disorders. The goal of this article is to encourage veterinarians to use radiology and ultrasonography for the evaluation of bovine orthopedic disorders. These diagnostic imaging techniques improve the likelihood of a definitive diagnosis in every bovine patient but especially in highly valuable cattle, whose owners demand increasingly more diagnostic and surgical interventions that require high-level specialized techniques.

  11. Human and animal Trypanosomes in Cote d'Ivoire form a single breeding population.

    Directory of Open Access Journals (Sweden)

    Paul Capewell

    Full Text Available BACKGROUND: Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits. METHODOLOGY/PRINCIPAL FINDINGS: A collection of sympatric T. brucei isolates from Côte d'Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, FST, Hardy-Weinberg equilibrium and linkage disequilibrium. CONCLUSIONS/SIGNIFICANCE: Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.

  12. Immunosensors in Clinical Laboratory Diagnostics. (United States)

    Justino, Celine I L; Duarte, Armando C; Rocha-Santos, Teresa A P


    The application of simple, cost-effective, rapid, and accurate diagnostic technologies for detection and identification of cardiac and cancer biomarkers has been a central point in the clinical area. Biosensors have been recognized as efficient alternatives for the diagnostics of various diseases due to their specificity and potential for application on real samples. The role of nanotechnology in the construction of immunological biosensors, that is, immunosensors, has contributed to the improvement of sensitivity, since they are based in the affinity between antibody and antigen. Other analytes than biomarkers such as hormones, pathogenic bacteria, and virus have also been detected by immunosensors for clinical point-of-care applications. In this chapter, we first introduced the various types of immunosensors and discussed their applications in clinical diagnostics over the recent 6 years, mainly as point-of-care technologies for the determination of cardiac and cancer biomarkers, hormones, pathogenic bacteria, and virus. The future perspectives of these devices in the field of clinical diagnostics are also evaluated.

  13. Local Sensitivity and Diagnostic Tests

    NARCIS (Netherlands)

    Magnus, J.R.; Vasnev, A.L.


    In this paper we confront sensitivity analysis with diagnostic testing.Every model is misspecified, but a model is useful if the parameters of interest (the focus) are not sensitive to small perturbations in the underlying assumptions. The study of the e ect of these violations on the focus is calle

  14. Diagnostic Ultrasound in Gynecologic Diagnosis


    Coates, C. F.


    Diagnostic ultrasound is helpful in the assessment of many common gynecological conditions from early teens to the late postmenopause. Female pelvic anatomy and pathology are readily imaged through a distended urinary bladder. In gynecologic cancer, examination of peritoneal cavity, kidneys and liver can suggest metastatic spread, which aids tumor staging.

  15. Diagnostic Criteria for Pediatric MS

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap


    Full Text Available Investigators at Northwestern University Feinberg School of Medicine and Ann & Robert H. Lurie Children’s Hospital of Chicago review the diagnostic criteria for pediatric multiple sclerosis, the differential diagnosis, the 2010 McDonald criteria, and Callen criteria.

  16. Cognitive Diagnostic Modeling Using R (United States)

    Ravand, Hamdollah


    Cognitive diagnostic models (CDM) have been around for more than a decade but their application is far from widespread for mainly two reasons: (1) CDMs are novel, as compared to traditional IRT models. Consequently, many researchers lack familiarity with them and their properties, and (2) Software programs doing CDMs have been expensive and not…


    Directory of Open Access Journals (Sweden)

    Khrapov Y. N.


    Full Text Available The article presents a technical diagnostics of a car as a complex of goals and tasks connected with trouble-shooting of mechanisms and systems in order to eliminate them. We have considered the stages of computer diagnostics of different automobile systems such as diagnosing the engine, the brake system, steering and suspension. We have analyzed their components, the ways of troubleshooting and elimination recommendations. The article presents the main troubles transferred from the electronic control unit. The article also presents the stages of diagnosing the engine including external examination, listening to abnormal noises, checking the operating fluids and the engine management system, diagnosing the basic engine systems and checking the cylinders being filled. The article contains the list of main troubles and their reasons. One can also see diagnosing the brake system, its defects and remedies. The article presents diagnostics and repair of the suspender and graphics describing the check of the dismantled shock strut at the stand and tests of the shock strut without being dismantled. We have analyzed computer diagnostics and the problems it solves

  18. A guide for diagnostic evaluations

    NARCIS (Netherlands)

    R.W. Peeling; P.G. Smith; P.M.M. Bossuyt


    Accurate diagnostic tests have a key role in patient management and the control of most infectious diseases. Unfortunately, in many developing countries, clinical care is often critically compromised by the lack of regulatory controls on the quality of these tests. The information available on the p

  19. A guide for diagnostic evaluations

    NARCIS (Netherlands)

    R.W. Peeling; P.G. Smith; P.M.M. Bossuyt


    Accurate diagnostic tests have a key role in patient management and the control of most infectious diseases. Unfortunately, in many developing countries, clinical care is often critically compromised by the lack of regulatory controls on the quality of these tests. The information available on the p

  20. Diagnostic errors in pediatric radiology

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, George A.; Voss, Stephan D. [Children' s Hospital Boston, Department of Radiology, Harvard Medical School, Boston, MA (United States); Melvin, Patrice R. [Children' s Hospital Boston, The Program for Patient Safety and Quality, Boston, MA (United States); Graham, Dionne A. [Children' s Hospital Boston, The Program for Patient Safety and Quality, Boston, MA (United States); Harvard Medical School, The Department of Pediatrics, Boston, MA (United States)


    Little information is known about the frequency, types and causes of diagnostic errors in imaging children. Our goals were to describe the patterns and potential etiologies of diagnostic error in our subspecialty. We reviewed 265 cases with clinically significant diagnostic errors identified during a 10-year period. Errors were defined as a diagnosis that was delayed, wrong or missed; they were classified as perceptual, cognitive, system-related or unavoidable; and they were evaluated by imaging modality and level of training of the physician involved. We identified 484 specific errors in the 265 cases reviewed (mean:1.8 errors/case). Most discrepancies involved staff (45.5%). Two hundred fifty-eight individual cognitive errors were identified in 151 cases (mean = 1.7 errors/case). Of these, 83 cases (55%) had additional perceptual or system-related errors. One hundred sixty-five perceptual errors were identified in 165 cases. Of these, 68 cases (41%) also had cognitive or system-related errors. Fifty-four system-related errors were identified in 46 cases (mean = 1.2 errors/case) of which all were multi-factorial. Seven cases were unavoidable. Our study defines a taxonomy of diagnostic errors in a large academic pediatric radiology practice and suggests that most are multi-factorial in etiology. Further study is needed to define effective strategies for improvement. (orig.)

  1. Diagnostic discrepancies in clinical practice (United States)

    Issa, Victor Sarli; Dinardi, Layara Fernanda Lipari; Pereira, Thiago Vicente; de Almeida, Lyna Kyria Rodrigues; Barbosa, Thaisa Silveira; Benvenutti, Luiz Alberto; Ayub-Ferreira, Silvia Moreira; Bocchi, Edimar Alcides


    Abstract Autopsies are the gold standard for diagnostic accuracy; however, no recent study has analyzed autopsies in heart failure (HF). We reviewed 1241 autopsies (January 2000–May 2005) and selected 232 patients with HF. Clinical and autopsy diagnoses were analyzed and discrepancies categorized according to their importance regarding therapy and prognosis. Mean age was 63.3 ± 15.9 years; 154 (66.4%) patients were male. The causes of death at autopsy were end-stage HF (40.9%), acute myocardial infarction (17.2%), infection (15.9), and pulmonary embolism 36 (15.5). Diagnostic discrepancies occurred in 191 (82.3%) cases; in 56 (24.1%), discrepancies were related to major diagnoses with potential influence on survival or treatment; pulmonary embolism was the cause of death for 24 (42.9%) of these patients. In 35 (15.1%), discrepancies were related to a major diagnosis with equivocal influence on survival or treatment; in 100 (43.1%), discrepancies did not influence survival or treatment. In multivariate analysis, age (OR: 1.03, 95% CI: 1.008–1.052, P = 0.007) and presence of diabetes mellitus (OR: 0.359, 95% CI: 0.168–0.767, P = 0.008) influenced the occurrence discrepancies. Diagnostic discrepancies with a potential impact on prognosis are frequent in HF. These findings warrant reconsideration in diagnostic and therapeutic practices with HF patients. PMID:28121951

  2. [Diagnostic dyspraxia and frontal syndrome]. (United States)

    Donnet, A; Schmitt, A; Poncet, M


    A 27-year-old ambidexter woman experienced a clinical and psychometric frontal syndrome associated with a partial callosal syndrome following transcallosal surgery for an intraventricular neurocytoma. She also complained of difficulties with her left hand which realized a particular form of diagnostic dyspraxia: there were specific features of an isolated dysfunction of the control of the realization of a program.

  3. When Diagnostic Labels Mask Trauma (United States)

    Foltz, Robert; Dang, Sidney; Daniels, Brian; Doyle, Hillary; McFee, Scott; Quisenberry, Carolyn


    A growing body of research shows that many seriously troubled children and adolescents are reacting to adverse life experiences. Yet traditional diagnostic labels are based on checklists of surface symptoms. Distracted by disruptive behavior, the common response is to medicate, punish, or exclude rather than respond to needs of youth who have…

  4. Laser Diagnostics for Reacting Flows (United States)


    absorption diagnostic for vapor-phase measurements in an evaporating n-decane aerosol,” Appied Physics B. 97, 215-225, (2009). 30. J.M. Porter, J.B...fluorescence of toluene for time- resolved imaging of gaseous flows,” Appied Physics B, 2010, in press. 35. J.M. Porter, J.B. Jeffries and R.K. Hanson

  5. ITER diagnostics ex-vessel engineering services

    Energy Technology Data Exchange (ETDEWEB)

    Arumugam, A.P., E-mail:; Walker, C.I.; Andrew, P.; Barnsley, R.; Beltran, D.; Bertalot, L.; Dammann, A.; Direz, M.F.; Drevon, J.M.; Encheva, A.; Giacomin, T.; Hourtoule, J.; Kuehn, I.; Lanza, R.; Levesy, B.; Maquet, P.; Patel, K.M.; Patisson, L.; Pitcher, C.S.; Portales, M.; and others


    Highlights: • This paper describes about the ITER diagnostics ex-vessel engineering services. • It describes various diagnostics systems, its location and its environment. • Diagnostics interfaces with other services such as the buildings, HVAC, electrical services, cooling water, vacuum, liquid and gas distribution. • All the interfaces with these services are identified and defined. • Buildings services for diagnostics, such as penetrations, local shielding, embedment and temperature control are discussed. -- Abstract: Extensive diagnostics systems will be installed on the ITER machine to provide the measurements necessary to control, evaluate and optimize plasma performance in ITER and to further the understanding of plasma physics. These include measurements of temperature, density, impurity concentration, and particle and energy confinement times. ITER diagnostic systems extend from the center of the Tokamak to the various diagnostic areas, where they are controlled and acquired data is processed. This mainly includes the areas such as ports, port cells, gallery, diagnostics enclosures and cubicle areas. The diagnostics port plugs encloses the front end of the diagnostic systems and the diagnostics building houses the diagnostics equipment, instrumentation and control cubicles. There are several systems providing services to diagnostics. These mainly include ITER buildings, electrical power services, cooling water services, Heating Ventilation and Air Conditioning (HVAC), vacuum services, liquid and gas distribution services, cable engineering, de-tritiation systems, control cubicles, etc. Requirements of these service systems have to be defined, even though many of the diagnostics are at an early stage of development. It is a real challenge to define and to design diagnostics systems considering the constraints imposed by these service systems. This paper summarizes the provision of these services to the individual diagnostics and diagnostics areas

  6. A diagnostic approach to test priorization

    NARCIS (Netherlands)

    Gonzalez-Sanchez, A.; Abreu, R.; Gross, H.; Van Gemund, A.


    In development processes with high code production rates testing typically triggers fault diagnosis to localize the detected failures. However, current test prioritization algorithms are tuned for failure detection rate rather than diagnostic information. Consequently, unnecessary diagnostic effort

  7. Huntington Disease: Molecular Diagnostics Approach. (United States)

    Bastepe, Murat; Xin, Winnie


    Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in the first exon of the Huntingtin (HTT) gene. Molecular testing of Huntington disease for diagnostic confirmation and disease prediction requires detection of the CAG repeat expansion. There are three main types of HD genetic testing: (1) diagnostic testing to confirm or rule out disease, (2) presymptomatic testing to determine whether an at-risk individual inherited the expanded allele, and (3) prenatal testing to determine whether the fetus has inherited the expanded allele. This unit includes protocols that describe the complementary use of polymerase chain reactions (PCR) and Southern blot hybridization to accurately measure the CAG trinucleotide repeat size and interpret the test results. In addition, an indirect linkage analysis that does not reveal the unwanted parental HD status in a prenatal testing will also be discussed.

  8. Saliva as a diagnostic fluid. (United States)

    Samaranayake, Lakshman


    The use of saliva as a diagnostic fluid for various human ailments is gaining popularity as it offers distinct advantages over serum. These include the non-invasive nature of saliva collection compared with phlebotomy, simplicity of collection even for individuals with a modest training and the cost-effective applicability for screening large populations. Whole saliva is most frequently used for diagnosis of systemic diseases since it is readily collected and contains serum constituents while gland-specific saliva is useful for investigating pathology of major salivary glands. Broadly, saliva analysis is currently used for the diagnosis of infectious and malignant diseases, hereditary disorders, autoimmune diseases, and endocrine disorders, as well as for the assessment of therapeutic drug levels, particularly in monitoring drug abuse. This review addresses the current status of salivary diagnostics and their future potential.


    Directory of Open Access Journals (Sweden)

    Pezelj-Ribarić Sonja


    Full Text Available Saliva is a readily available oral fluid with many functions, from digestion, maintenance of oral tissues' integrity, to caries prevention. Changes regarding its secretion may be divided into qualitative and quantitative: both of them are a consequence of certain conditions/diseases (e.g. internal factors or nutrients/drugs ingested (e.g. external factors. During the last 15 years, technological advances gave a significant momentum to utilization of saliva as a diagnostic tool. Analysis of saliva, just like the blood analysis, has two main objectives: to identify the subjects suffering from a certain disorder, and to follow the development and progress of therapy. This paper provides an overview of possibilities for the use of saliva for diagnostic purposes and gives specific examples of some clinical investigations, with the final aim to stimulate the use of this noninvasive means for the health care promotion.

  10. Novette diagnostic support. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Cirigliano, R.; Franco, E.; Koppel, L.; Rodrigues, B.; Smith, J.


    The primary research areas were the following: (1) contribute x-ray diagnostic, experimental, and data reduction and analysis support for the Novette DANTE x-ray spectrometer experiments. This effort was expanded to improve the overall quality of the Novette database; (2) experimental and calculational characterization of the x-ray imaging properties of an ellipsoidal x-ray collection optic serving as a sensitivity enhancing component of the Transmission Grating Streak Spectrometer; (3) performance simulation of the x-ray dispersion properties of candidate x-ray laser cavity, normal incidence end-mirror optics; (4) contribute x-ray diagnostic, experimental, and data reduction and analysis support for the Novette Henway crystal spectrometer and the MCPIGS microchannel plate intensified grazing incident spectrometer experiments; and (5) perform a technical performance vs cost evaluation of commercially available hardware required to perform the NOVA neutron time-of-flight experiments.

  11. Advances in medical diagnostic technology

    CERN Document Server

    Lai, Khin Wee; Mohamad Salim, Maheza Irna; Ong, Sang-Bing; Utama, Nugraha Priya; Myint, Yin Mon; Mohd Noor, Norliza; Supriyanto, Eko


    This book provides the most recent findings and knowledge in advanced diagnostics technology, covering a wide spectrum including brain activity analysis, breast and lung cancer detection, echocardiography, computer aided skeletal assessment to mitochondrial biology imaging at the cellular level. The authors explored magneto acoustic approaches and tissue elasticity imaging for the purpose of breast cancer detection. Perspectives in fetal echocardiography from an image processing angle are included. Diagnostic imaging in the field of mitochondrial diseases as well as the use of Computer-Aided System (CAD) are also discussed in the book. This book will be useful for students, lecturers or professional researchers in the field of biomedical sciences and image processing.

  12. [Diagnostic workup of fragrance allergy]. (United States)

    Geier, J; Uter, W


    The diagnostic workup of contact allergy to fragrances must not be limited to patch testing with the two well-established fragrance mixes. False-positive reactions to these mixes occur in up to 50 % of the patch tested patients. For the diagnostic work-up of positive reactions, and in cases of suspected fragrance allergy, patch testing with the single mix components and additional fragrances is mandatory. Frequently sensitizing fragrance materials are the 14 components of the two fragrance mixes and tree moss (Evernia furfuracea), ylang ylang oil (I + II; Cananga odorata), lemongrass oil (Cymbopogon schoenanthus), sandalwood oil (Santalum album), jasmine absolute (Jasminum spp.), and, less frequently, clove oil (Eugenia caryophyllus), cedarwood oil (Cedrus atlantica/deodara, Juniperus virginiana), Neroli oil (Citrus aurantium amara flower oil), salicylaldehyde, narcissus absolute (Narcissus spp.), and patchouli oil (Pogostemon cablin).

  13. Diagnostics

    DEFF Research Database (Denmark)

    Donné, A.J.H.; Costley, A.E.; Barnsley, R.


    In order to support the operation of ITER and the planned experimental programme an extensive set of plasma and first wall measurements will be required. The number and type of required measurements will be similar to those made on the present-day large tokamaks while the specification of the mea...

  14. Laser Diagnostics for Spacecraft Propulsion (United States)


    AFTC/PA Clearance No. XXXX 8 Ion Engines & Hall Thrusters Operation Ion engines and Hall thrusters are electrostatic propulsion devices • Ion Engines... Hall thrusters are gridless electrostatic thrusters – Propellant ionized by electrons trapped in magnetic field – Ions accelerated by an electric field...Briefing Charts 3. DATES COVERED (From - To) 21 September 2015 – 13 October 2015 4. TITLE AND SUBTITLE Laser Diagnostics for Spacecraft Propulsion 5a

  15. [Diagnostic approach to infectious endocarditis]. (United States)

    Parize, Perrine; Mainardi, Jean-Luc


    Major advances in imaging and microbiological procedures have changed the diagnostic approach of infective endocarditis. New imaging tools have improved the search of local complications, systemic embolism and diagnosis of infection of cardiac devices. Moreover, microbiological diagnosis has been transformed by molecular techniques as long as these procedures have highlighted microorganisms thus far neglected by classical techniques. Despite these advances, endocarditis remained a clinical diagnosis which still depends on a high index of clinical suspicion.

  16. Gear Fatigue Diagnostics and Prognostics (United States)


    The first objective was to collect meaningful gear fault progression data starting from healthy NASA-designed spur test gears and ending with failed...and D. P. Townsend, "Analysis of the effects of surface pitting and wear on the vibration of a gear transmission system," Tribology International...Reporting Period: April 15, 2012 to September 27, 2012 Attached is the Gear Fatigue Diagnostics and Prognostics project progress report for the

  17. Molecular diagnostics and parasitic disease. (United States)

    Vasoo, Shawn; Pritt, Bobbi S


    Molecular parasitology represents an emerging field in microbiology diagnostics. Although most assays use nonstandardized, laboratory-developed methods, a few commercial systems have recently become available and are slowly being introduced into larger laboratories. In addition, a few methodologies show promise for use in field settings in which parasitic infections are endemic. This article reviews the available techniques and their applications to major parasitic diseases such as malaria, leishmaniasis, and trichomoniasis.

  18. Biomedical photonics handbook biomedical diagnostics

    CERN Document Server

    Vo-Dinh, Tuan


    Shaped by Quantum Theory, Technology, and the Genomics RevolutionThe integration of photonics, electronics, biomaterials, and nanotechnology holds great promise for the future of medicine. This topic has recently experienced an explosive growth due to the noninvasive or minimally invasive nature and the cost-effectiveness of photonic modalities in medical diagnostics and therapy. The second edition of the Biomedical Photonics Handbook presents fundamental developments as well as important applications of biomedical photonics of interest to scientists, engineers, manufacturers, teachers, studen

  19. Diagnostics for PLX-alpha (United States)

    Gilmore, Mark; Hsu, Scott


    The goal of the Plasma Liner eXperiment PLX-alpha at Los Alamos National Laboratory is to establish the viability of creating a spherically imploding plasma liner for MIF and HED applications, using a spherical array of supersonic plasma jets launched by innovative contoured-gap coaxial plasma guns. PLX- α experiments will focus in particular on establishing the ram pressure and uniformity scalings of partial and fully spherical plasma liners. In order to characterize these parameters experimentally, a suite of diagnostics is planned, including multi-camera fast imaging, a 16-channel visible interferometer (upgraded from 8 channels) with reconfigurable, fiber-coupled front end, and visible and VUV high-resolution and survey spectroscopy. Tomographic reconstruction and data fusion techniques will be used in conjunction with interferometry, imaging, and synthetic diagnostics from modeling to characterize liner uniformity in 3D. Diagnostic and data analysis design, implementation, and status will be presented. Supported by the Advanced Research Projects Agency - Energy - U.S. Department of Energy.

  20. Diagnostic guidlines for occupational epicondylitis

    Directory of Open Access Journals (Sweden)

    Patrycja Krawczyk-Szulc


    Full Text Available Making final decisions on the occupational etiology of musculoskeletal diseases is often difficult and problematic at every stage of the diagnostic procedure. Taking into account the need to facilitate decision-making about the causal relationship between the diagnosed disease entity and the working conditions guidelines for the recognition of work-related musculoskeletal diseases have been developed. This paper presents the guidelines for the diagnosis of occupational etiology of humeral epicondylitis, one of the most common occupational disease of the musculoskeletal system in Poland. The developed guidelines have been based on the literature data concerning occupational risk factors of humeral epicondylitis, workload classification, including repetitive movements, awkward postures, and force. Some criteria applied in ergonomic evaluation methods were also included. The presented diagnostic guidelines define approximate benchmarks for stating (after excluding non-occupational etiology that the identified humeral epicondylitis, is related to the way of working. Crucial work factors that should be analyzed include an operating time of movements overloading tendons connecting to the epicondyle, repetition and force used to perform occupational activities. The developed guidelines are aimed to facilitate occupational physicians diagnostic and certification procedures in case of humeral epicondylitis and determination whether there is a likelihood of its occupational etiology. Med Pr 2015;66(3:443–450

  1. [Diagnostic guidlines for occupational epicondylitis]. (United States)

    Krawczyk-Szulc, Patrycja; Wągrowska-Koski, Ewa; Puzder, Anna; Markowski, Przemysław; Walusiak-Skorupa, Jolanta


    Making final decisions on the occupational etiology of musculoskeletal diseases is often difficult and problematic at every stage of the diagnostic procedure. Taking into account the need to facilitate decision-making about the causal relationship between the diagnosed disease entity and the working conditions guidelines for the recognition of work-related musculoskeletal diseases have been developed. This paper presents the guidelines for the diagnosis of occupational etiology of humeral epicondylitis, one of the most common occupational disease of the musculoskeletal system in Poland. The developed guidelines have been based on the literature data concerning occupational risk factors of humeral epicondylitis, workload classification, including repetitive movements, awkward postures, and force. Some criteria applied in ergonomic evaluation methods were also included. The presented diagnostic guidelines define approximate benchmarks for stating (after excluding non-occupational etiology) that the identified humeral epicondylitis, is related to the way of working. Crucial work factors that should be analyzed include an operating time of movements overloading tendons connecting to the epicondyle, repetition and force used to perform occupational activities. The developed guidelines are aimed to facilitate occupational physicians diagnostic and certification procedures in case of humeral epicondylitis and determination whether there is a likelihood of its occupational etiology.

  2. Diagnostic interventions in nuclear medicine

    Energy Technology Data Exchange (ETDEWEB)

    Thrall, J.H.; Swanson, D.P.


    Diagnostic interventions in nuclear medicine may be defined as the coadministration of a nonradioactive drug or application of a physical stimulus or physiologic maneuver to enhance the diagnostic utility of a nuclear medicine test. The rationale for each interventional maneuver follows from the physiology or metabolism of the particular organ or organ system under evaluation. Diagnostic inference is drawn from the pattern of change in the biodistribution of the tracer in response to the intervention-induced change in metabolism or function. In current practice, the most commonly performed interventional maneuvers are aimed at studies of the heart, genitourinary system, hepatobiliary system, and gastrointestinal tract. The single most commonly performed interventional study in the United States is the stress Thallium-201 myocardial perfusion scan aimed at the diagnosis of coronary artery disease. The stress portion of the study is accomplished with dynamic leg exercise on a treadmill and is aimed at increasing myocardial oxygen demands. Areas of myocardium distal to hemodynamically significant lesions in the coronary arteries become ischemic at peak stress due to the inability of the stenotic vessel to respond to the oxygen demand/blood flow needs of the myocardium. Ischemic areas are readily recognized as photopenic defects on scans obtained immediately after exercise, with normalization upon delayed imaging. Diuresis renography is aimed at the differential diagnosis of hydroureteronephrosis. By challenging the urinary tract collecting structures with an augmented urine flow, dilated, unobstructed systems can be differentiated from systems with significant mechanical obstruction. 137 references.

  3. Fluorescence diagnostics in oncological gynecology (United States)

    Belyaeva, Ludmila A.; Adamyan, Leila V.; Kozachenko, Vladimir P.; Stratonnikov, Alexander A.; Stranadko, Eugene F.; Loschenov, Victor B.


    The method of fluorescent diagnostics (FD) of tumors is a promising tool that may allow to increase sensitivity of tumor detection especially at initial stages. One of the most promising photosensitizers today is 5-aminolevulinic acid (5-ALA) that, actually, is not photosensitizer itself but precursor of protoporphyrin IX (PpIX). This paper deals with cancer diagnostics in gynecology by means of ALA-induced Pp IX laser-fluorescence spectroscopy. The tissue fluorescence spectra in vivo were studied in patients with various pathologies of ovaries, uterine and vulva after 5-aminolevulinic acid administration. It was shown that different pathologies varies in accumulation of Pp IX. Coefficient of fluorescence kf for normal tissue is not high, but exceptions are endometrium and mucous membrane of uterine tubes. Benign tumors of uterus and ovary have low values of kf, but polyps of endometrium exhibit high kf. Optical express-biopsy is important for diagnosis of ovarian cancer and micrometastatic spread. Coefficients of diagnostic contrast were determined for cancer of endometrium, cervical cancer, vulvar cancer.

  4. Sequencing Needs for Viral Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S N; Lam, M; Mulakken, N J; Torres, C L; Smith, J R; Slezak, T


    We built a system to guide decisions regarding the amount of genomic sequencing required to develop diagnostic DNA signatures, which are short sequences that are sufficient to uniquely identify a viral species. We used our existing DNA diagnostic signature prediction pipeline, which selects regions of a target species genome that are conserved among strains of the target (for reliability, to prevent false negatives) and unique relative to other species (for specificity, to avoid false positives). We performed simulations, based on existing sequence data, to assess the number of genome sequences of a target species and of close phylogenetic relatives (''near neighbors'') that are required to predict diagnostic signature regions that are conserved among strains of the target species and unique relative to other bacterial and viral species. For DNA viruses such as variola (smallpox), three target genomes provide sufficient guidance for selecting species-wide signatures. Three near neighbor genomes are critical for species specificity. In contrast, most RNA viruses require four target genomes and no near neighbor genomes, since lack of conservation among strains is more limiting than uniqueness. SARS and Ebola Zaire are exceptional, as additional target genomes currently do not improve predictions, but near neighbor sequences are urgently needed. Our results also indicate that double stranded DNA viruses are more conserved among strains than are RNA viruses, since in most cases there was at least one conserved signature candidate for the DNA viruses and zero conserved signature candidates for the RNA viruses.

  5. DNA Microarray-Based Diagnostics. (United States)

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H


    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

  6. Portable Diagnostics and Rapid Germination

    Energy Technology Data Exchange (ETDEWEB)

    Dunn, Zachary Spencer [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)


    In the Bioenergy and Defense Department of Sandia National Laboratories, characterization of the BaDx (Bacillus anthracis diagnostic cartridge) was performed and rapid germination chemistry was investigated. BaDx was tested with complex sample matrixes inoculated with Bacillus anthracis, and the trials proved that BaDx will detect Bacillus anthracis in a variety of the medium, such as dirt, serum, blood, milk, and horse fluids. The dimensions of the device were altered to accommodate an E. coli or Listeria lateral flow immunoassay, and using a laser printer, BaDx devices were manufactured to identify E. coli and Listeria. Initial testing with E. coli versions of BaDx indicate that the device will be viable as a portable diagnostic cartridge. The device would be more effective with faster bacteria germination; hence studies were performed the use of rapid germination chemistry. Trials with calcium dipicolinic acid displayed increased cell germination, as shown by control studies using a microplate reader. Upon lyophilization the rapid germination chemistry failed to change growth patterns, indicating that the calcium dipicolinic acid was not solubilized under the conditions tested. Although incompatible with the portable diagnostic device, the experiments proved that the rapid germination chemistry was effective in increasing cell germination.

  7. Human African trypanosomiasis. (United States)

    Lejon, Veerle; Bentivoglio, Marina; Franco, José Ramon


    Human African trypanosomiasis or sleeping sickness is a neglected tropical disease that affects populations in sub-Saharan Africa. The disease is caused by infection with the gambiense and rhodesiense subspecies of the extracellular parasite Trypanosoma brucei, and is transmitted to humans by bites of infected tsetse flies. The disease evolves in two stages, the hemolymphatic and meningoencephalitic stages, the latter being defined by central nervous system infection after trypanosomal traversal of the blood-brain barrier. African trypanosomiasis, which leads to severe neuroinflammation, is fatal without treatment, but the available drugs are toxic and complicated to administer. The choice of medication is determined by the infecting parasite subspecies and disease stage. Clinical features include a constellation of nonspecific symptoms and signs with evolving neurological and psychiatric alterations and characteristic sleep-wake disturbances. Because of the clinical profile variability and insidiously progressive central nervous system involvement, disease staging is currently based on cerebrospinal fluid examination, which is usually performed after the finding of trypanosomes in blood or other body fluids. No vaccine being available, control of human African trypanosomiasis relies on diagnosis and treatment of infected patients, assisted by vector control. Better diagnostic tools and safer, easy to use drugs are needed to facilitate elimination of the disease.

  8. Diagnostics of Pupils' Attitude to Education (United States)

    Eminli, Tovuz


    The paper is devoted to the investigation of the questions connected with the pedagogical diagnostics, in particular, the diagnostics of pupils' attitude to education. It is considered reasonable to apply the practice of development of an individual pedagogical and psychological map for productive implementation of the pedagogical diagnostics and…

  9. Diagnostic accuracy of the care dependency scale

    NARCIS (Netherlands)

    Dijkstra, Ate; Tiesinga, LJ; Plantinga, L; Dassen, TWN; Veltman, G.


    Aim. This paper reports an investigation of the diagnostic accuracy of the Care Dependency Scale (CDS). Background. Assessment tools can be described in terms of diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy can be determin

  10. Relating faults in diagnostic reasoning with diagnostic errors and patient harm.

    NARCIS (Netherlands)

    Zwaan, L.; Thijs, A.; Wagner, C.; Wal, G. van der; Timmermans, D.R.M.


    Purpose: The relationship between faults in diagnostic reasoning, diagnostic errors, and patient harm has hardly been studied. This study examined suboptimal cognitive acts (SCAs; i.e., faults in diagnostic reasoning), related them to the occurrence of diagnostic errors and patient harm, and studied

  11. Inferences of clinical diagnostic reasoning and diagnostic error. (United States)

    Lawson, Anton E; Daniel, Erno S


    This paper discusses clinical diagnostic reasoning in terms of a pattern of If/then/Therefore reasoning driven by data gathering and the inference of abduction, as defined in the present paper, and the inferences of retroduction, deduction, and induction as defined by philosopher Charles Sanders Peirce. The complex inferential reasoning driving clinical diagnosis often takes place subconsciously and so rapidly that its nature remains largely hidden from the diagnostician. Nevertheless, we propose that raising such reasoning to the conscious level reveals not its basic pattern and basic inferences, it also reveals where errors can and do occur and how such errors might be reduced or even eliminated.

  12. PLS-Prediction and Confirmation of Hydrojuglone Glucoside as the Antitrypanosomal Constituent of Juglans Spp. (United States)

    Ellendorff, Therese; Brun, Reto; Kaiser, Marcel; Sendker, Jandirk; Schmidt, Thomas J


    Naphthoquinones (NQs) occur naturally in a large variety of plants. Several NQs are highly active against protozoans, amongst them the causative pathogens of neglected tropical diseases such as human African trypanosomiasis (sleeping sickness), Chagas disease and leishmaniasis. Prominent NQ-producing plants can be found among Juglans spp. (Juglandaceae) with juglone derivatives as known constituents. In this study, 36 highly variable extracts were prepared from different plant parts of J. regia, J. cinerea and J. nigra. For all extracts, antiprotozoal activity was determined against the protozoans Trypanosoma cruzi, T. brucei rhodesiense and Leishmania donovani. In addition, an LC-MS fingerprint was recorded for each extract. With each extract's fingerprint and the data on in vitro growth inhibitory activity against T. brucei rhodesiense a Partial Least Squares (PLS) regression model was calculated in order to obtain an indication of compounds responsible for the differences in bioactivity between the 36 extracts. By means of PLS, hydrojuglone glucoside was predicted as an active compound against T. brucei and consequently isolated and tested in vitro. In fact, the pure compound showed activity against T. brucei at a significantly lower cytotoxicity towards mammalian cells than established antiprotozoal NQs such as lapachol.

  13. PLS-Prediction and Confirmation of Hydrojuglone Glucoside as the Antitrypanosomal Constituent of Juglans Spp.

    Directory of Open Access Journals (Sweden)

    Therese Ellendorff


    Full Text Available Naphthoquinones (NQs occur naturally in a large variety of plants. Several NQs are highly active against protozoans, amongst them the causative pathogens of neglected tropical diseases such as human African trypanosomiasis (sleeping sickness, Chagas disease and leishmaniasis. Prominent NQ-producing plants can be found among Juglans spp. (Juglandaceae with juglone derivatives as known constituents. In this study, 36 highly variable extracts were prepared from different plant parts of J. regia, J. cinerea and J. nigra. For all extracts, antiprotozoal activity was determined against the protozoans Trypanosoma cruzi, T. brucei rhodesiense and Leishmania donovani. In addition, an LC-MS fingerprint was recorded for each extract. With each extract’s fingerprint and the data on in vitro growth inhibitory activity against T. brucei rhodesiense a Partial Least Squares (PLS regression model was calculated in order to obtain an indication of compounds responsible for the differences in bioactivity between the 36 extracts. By means of PLS, hydrojuglone glucoside was predicted as an active compound against T. brucei and consequently isolated and tested in vitro. In fact, the pure compound showed activity against T. brucei at a significantly lower cytotoxicity towards mammalian cells than established antiprotozoal NQs such as lapachol.

  14. Overview of the microfluidic diagnostics commercial landscape. (United States)

    Kim, Lily


    Since its birth in the late 1980s, the field of microfluidics has continued to mature, with a growing number of companies pursuing diagnostic applications. In 2009 the worldwide in vitro diagnostics market was estimated at >$40 billion USD, and microfluidic diagnostics are poised to reap a significant part of this market across a range of areas including laboratory diagnostics, point-of-care diagnostics, cancer diagnostics, and others. The potential economic advantages of microfluidics are numerous and compelling: lower reagent and/or sample volumes, lower equipment costs, improved portability, increased automation, and increased measurement speed. All of these factors may help put more information in the hands of doctors and patients sooner, enabling earlier disease detection and more tailored, effective treatments. This chapter reviews the microfluidic diagnostics commercial landscape and discusses potential commercialization challenges and opportunities.

  15. DEMO diagnostics and burn control

    Energy Technology Data Exchange (ETDEWEB)

    Biel, Wolfgang, E-mail: [Institute of Energy and Climate Research, Forschungszentrum Jülich GmbH, Jülich (Germany); Department of Applied Physics, Ghent University (Belgium); Baar, Marco de [FOM-Institute DIFFER, Nieuwegein (Netherlands); Eindhoven University of Technology (Netherlands); Dinklage, Andreas [Max-Planck-Institut für Plasmaphysik, Greifswald (Germany); Felici, Federico [Eindhoven University of Technology (Netherlands); König, Ralf [Max-Planck-Institut für Plasmaphysik, Greifswald (Germany); Meister, Hans; Treutterer, Wolfgang [Max-Planck-Institut für Plasmaphysik, Garching (Germany); Wenninger, Ronald [Max-Planck-Institut für Plasmaphysik, Garching (Germany); EFDA Power Plant Physics and Technology, Garching (Germany)


    Highlights: • An initial concept for the DEMO diagnostic and control system is presented. • A preliminary list of control functions and candidate diagnostics is developed. • Challenges regarding disruptions, power exhaust and radiation control are highlighted. • The need for introducing realistic control margins is emphasized. • On outline of the future R&D plan is presented. - Abstract: The development of the control system for a tokamak demonstration fusion reactor (DEMO) faces unprecedented challenges. First, the requirements for control reliability and accuracy are more stringent than on existing fusion devices: any loss of plasma control on DEMO may result in a disruption which could damage the inner wall of the machine, while operating the device with larger margins against the operational limits would lead to a reduction of the electrical output power. Second, the performance of DEMO control is limited by space restrictions for the implementation of components (optimization of the tritium breeding rate), by lifetime issues for the front-end parts (neutron and gamma radiation, erosion and deposition acting on all components) and by slow, weak and indirect action of the available actuators (plasma shaping, heating and fuelling). The European DEMO conceptual design studies include the development of a reliable control system, since the details of the achievable plasma scenario and the machine design may depend on the actual performance of the control system. In the first phase of development, an initial understanding of the prime choices of diagnostic methods applicable to DEMO, implementation and performance issues, the interrelation with the plasma scenario definition, and the planning of necessary future R&D have been obtained.

  16. PDR diagnostics study with CLOUDY

    Institute of Scientific and Technical Information of China (English)

    Rui Xue; Maohai Huang


    A series of plane-parallel photodissociation region (PDR) models are calculated using the spectral synthesis code CLOUDY. These models span a wide range of physical conditions, with gas densities of n = 102 - 106 cm-3 and incident far-ultraviolet (FUV) fields of G0 = 100 - 106 (where Go is the FUV flux in units of the local interstellar value), which are comparable with various astrophysical environments from interstellar diffuse clouds to the dense neutral gas around galactic compact HII regions. Based on the calculated results, we study the thermal balance of PDR gas and the emissions of [ CII ], [ CI ] and [ OI ] fine-structure lines under different physical conditions. The intensities and strength ratios of the studied lines, which are frequently used as PDR diagnostics, are presented using contour diagrams as functions of n and Go. We compare the calculated PDR surface gas temperatures T8 with those from Kaufman et al. and find that Ts from our models are systematically higher over most of the adopted n-G0 parameter space. The predicated line intensities and ratios from our work and those from Kaufman et al. can be different by a factor greater than 10, and such large differences usually occur near the border of our parameter space. The different methods of treating the dust grain physics, the change of H2 formation and dissociation rates, and the improvement in the radiation transfer of line emissions in our CLOUDY models are likely to be the major reasons for the divergences. Our models represent an up-to-date treatment of PDR diagnostic calcula- tions and can be used to interpret observational data. Meanwhile, the uncertainties in the treatment of microphysics and chemical processes in PDR models have significant effects on PDR diagnostics.

  17. Diagnostic tools for neuropathic pain

    Directory of Open Access Journals (Sweden)

    CHEN Xiang-jun


    Full Text Available Neuropathic pain (NP is a kind of chronic, severe and persistent pain syndrome. Due to the underlying mechanisms, the treatment for NP differs from that of nociceptive pain. An accurate diagnosis of NP is very important. However, the present diagnostic process which mainly depends on clinical and neurophysiological assessments is quite time-consuming and low efficient. In recent years, various screening tools and drug efficacy assessments for NP have been developed and validated. They become very useful in the diagnosis and treatment of NP, as well as in epidemiological study. These tools are also very useful in elucidating the underlying mechanism of NP.

  18. Diagnostic management of renal colic. (United States)

    Nicolau, C; Salvador, R; Artigas, J M


    Renal colic is a common reason for presentation to emergency departments, and imaging has become fundamental for the diagnosis and clinical management of this condition. Ultrasonography and particularly noncontrast computed tomography have good diagnostic performance in diagnosing renal colic. Radiologic management will depend on the tools available at the center and on the characteristics of the patient. It is essential to use computed tomography techniques that minimize radiation and to use alternatives like ultrasonography in pregnant patients and children. In this article, we review the epidemiology, clinical and radiologic presentations, and clinical management of ureteral lithiasis.

  19. Optoelectronic tweezers for medical diagnostics (United States)

    Kremer, Clemens; Neale, Steven; Menachery, Anoop; Barrett, Mike; Cooper, Jonathan M.


    Optoelectronic tweezers (OET) allows the spatial patterning of electric fields through selected illumination of a photoconductive surface. This enables the manipulation of micro particles and cells by creating non-uniform electrical fields that then produce dielectrophoretic (DEP) forces. The DEP responses of cells differ and can produce negative or positive (repelled or attracted to areas of high electric field) forces. Therefore OET can be used to manipulate individual cells and separate different cell types from each other. Thus OET has many applications for medical diagnostics, demonstrated here with work towards diagnosing Human African Trypanosomiasis, also known as sleeping sickness.

  20. Vibration diagnostics instrumentation for ILC

    Energy Technology Data Exchange (ETDEWEB)

    Bertolini, A.


    The future e{sup -}e{sup +} 500 GeV International Linear Collider will rely on unprecedented nanometer scale particle beam size at the interaction point, in order to achieve the design luminosity. Tight tolerances on static and dynamic alignment of the accelerator cavities and optical components are demanded to transport and focus the high energy electron and positron beams with reasonable position jitter and low emittance. A brief review of techniques and devices evaluated and developed so far for the vibration diagnostics of the machine is presented in this paper. (orig.)

  1. Chordoid meningioma: A diagnostic dilemma

    Directory of Open Access Journals (Sweden)

    Fouzia Siraj


    Full Text Available Chordoid meningioma (CM, classified as Grade II/atypical meningioma according to the World Health Organization classification, is a rare subtype, which represents only 0.5% of all meningiomas. Morphologically, it can mimic other chondroid and myxoid tumors within the brain and its vicinity thus posing a diagnostic challenge. Accurate diagnosis, therefore, assumes importance as these tumors have an aggressive clinical course and propensity to recur compared to classical meningiomas. Furthermore, the prognosis and treatment strategies vary when compared to tumors with morphological overlap. We present a case of CM in a 14-year-old girl and discuss its clinicopathological and immunohistochemical features.

  2. Diagnostic Ophthalmic Ultrasound for Radiologists. (United States)

    Kendall, Cynthia J; Prager, Thomas C; Cheng, Han; Gombos, Dan; Tang, Rosa A; Schiffman, Jade S


    Ophthalmic ultrasound is an invaluable tool that provides quick and noninvasive evaluation of the eye and the orbit. It not only allows the clinicians to view structures that may not be visible with routine ophthalmic equipment or neuroimaging techniques but also provides unique diagnostic information in various ophthalmic conditions. In this article, the basic principles of ophthalmic ultrasound and examination techniques are discussed. Its clinical application is illustrated through a variety of ocular pathologic abnormalities (eg, narrow angles, ciliary body tumor, detached retina, choroidal melanoma, and papilledema).

  3. Tuberculosis diagnostics: Challenges and opportunities

    Directory of Open Access Journals (Sweden)

    Vijay Nema


    Full Text Available Tuberculosis (TB has been a disease affecting almost all parts of the world since ages. Lot many efforts came in the past for improving diagnosis and treatment. Also, an effective vaccine has been sought after for long. With the emergence of resistant strains of Mycobacterium tuberculosis, the causal organisms of tuberculosis, and complexities emerging due to other associated infections and disease conditions, there is a desperate need for further research input in the field. Be it the better medication and care or better resistance management, proper diagnostics holds the key to success. It has been observed that a high burden of the disease was accompanied by resource limitations and poor research set-up. The scenario remained like this for several decades. With the refreshed vision of resourceful countries and funding agencies, funding is being provided in many areas of research in tuberculosis diagnosis and treatment. This review has been written with an aim to bring forth the limitations of available methods in the field of diagnostics and making researchers aware about the changing scenario with better funding opportunities and support. The author visualizes an enthusiasm from all over the world for the development of better modalities and urges scientists to join the struggle at this very perfect time to take the challenge and come forward with innovations in this field.

  4. Patient dosimetry in diagnostic radiology

    Directory of Open Access Journals (Sweden)

    Ciraj-Bjelac Olivera F.


    Full Text Available The objective of this work is to assess patient organ doses, effective doses and entrance surface doses in conventional diagnostic radiology procedures for standard adult patient. The survey consists of measurements of doses delivered to 239 patients in nine types of X-ray examinations. Three types of data were collected: X-ray machine data, patient data, and output measurements. Entrance surface dose was assessed based on the survey data and subsequently, using conversion coefficients, the organ doses and effective doses were calculated. Values of the entrance surface dose and the effective dose were estimated to be 0.4 to 5.8 mGy and 0.03 to 3.00 mSv for different examinations. Derived doses were compared with recommended general diagnostic reference levels. The impact of examination parameters on dose values was discussed. Except for posterior-anterior chest examination, all estimated doses are lower than stated reference levels. Survey data are aimed at helping development of national quality control and radiation protection programmed for medical exposures.

  5. [Vasculitis - diagnostic and therapeutic advances]. (United States)

    De Albuquerque, R; Machado, Filipa


    Vasculitis is characterized by inflammation and necrosis of blood vessels walls. It represents a heterogeneous group of conditions, whose etiopathogenic mechanisms remain unclear. Although uncommon, with an annual incidence of 40-54 cases per 1.000.000 persons, this is an important cause of multiorganic dysfunction and premature mortality. Depending on the affected vessels, it can cause diverse clinical presentations, which makes difficult its recognition. It is therefore a challenge for any clinician. This paper reviews the diagnostic and therapeutic advances of the most common forms of vasculitis, in order to optimize the approach and management of this clinical entity. We have conducted a search in Medline database on articles written in English, published for the last 10 years using the keywords: vasculitis, epidemiology, classification, diagnosis and treatment. To minimize the impact of vasculitis it is essential an early diagnosis, allowing a timely institution of the appropriate treatment. The diagnosis depends on the integration of clinical, laboratory, imaging and histopathologic data. According to the clinical condition, it may be indicated the removal of the offending antigen, the treatment of the underlying disease or specific treatment of the primary vasculitis. The introduction of immunosuppressive therapy with glucocorticoids and cyclophosphamide has revolutionized the prognosis of these patients but, despite its efficacy, it is associated with frequent relapses and significant toxicity. The study of the pathogenesis has been providing more effective and safer diagnostic and therapeutic options, for example B-cell depleting agents, but additional studies are needed to confirm the potential of these alternatives.

  6. Diagnostic validity of basic symptoms. (United States)

    Klosterkötter, J; Ebel, H; Schultze-Lutter, F; Steinmeyer, E M


    Although the Bonn Scale for the Assessment of Basic Symptoms (BSABS) [13] has come into use in several European countries, its diagnostic validity has not yet been sufficiently examined. That is why we have assessed BSABS items on a sample of 243 consecutive admissions to the Department of Psychiatry at the RWTH University, Aachen, and 79 psychologically healthy persons. Then, a cluster analysis was calculated to identify the empirical item-grouping. Five well-interpretable BSABS subsyndromes were found. In addition, uni- and multivariate analyses were computed to evaluate the diagnostic validity of these subsyndromes. We were able to show that every BSABS subsyndrome separates at least schizophrenic, organic mental and affective disorders from personality, neurotic and substance-induced disorders, as well as from psychological health. Furthermore, the subsyndrome "information processing disturbances" differentiates between schizophrenic and organic mental disorders, on the one hand, and affective disorders, on the other, and additionally, the subsyndrome "interpersonal irritation" between schizophrenics and all other persons examined.

  7. Ultrasound diagnostics of thyroid diseases

    Energy Technology Data Exchange (ETDEWEB)

    Kharchenko, Vladimir P. [Russian Radiology Research Center, Moscow (Russian Federation); Kotlyarov, Peter M. [Russian Center of Roentgenradiology, Moscow (Russian Federation); Mogutov, Mikhail S.; Sencha, Alexander N.; Patrunov, Yury N.; Belyaev, Denis V. [Yaroslavl Railway Clinic (Russian Federation); Alexandrov, Yury K. [State Medical Academy, Yaroslavl (Russian Federation)


    This book is based on the authors' extensive practical experience in the use of modern ultrasound, and other radiological methods, in the diagnosis of thyroid diseases. The authors have analyzed more than 100,000 ultrasound examinations performed between 1995 and 2008 in patients with thyroid and parathyroid disease, as well as many thousands of diagnostic and therapeutic ultrasound-guided minimally invasive procedures. The opening chapters include discussion of current ultrasound techniques, pitfalls, and the specifics of ultrasound examination of the thyroid in children. Detailed attention is then devoted to findings in the normal thyroid and in the presence of diffuse and focal changes. Further chapters focus on such topics as ultrasound examination after thyroid surgery and ultrasound diagnosis of parathyroid disease, recurrent goiter, and neck masses. Ultrasound-guided minimally invasive techniques, such as fine-needle aspiration biopsy, percutaneous laser ablation, and ethanol and glucocorticoid injections, are considered in depth. This up-to-date and richly illustrated book will interest and assist specialists in ultrasound diagnostics, radiologists, endocrinologists, and neck surgeons. (orig.)

  8. New tuberculosis diagnostics and rollout

    Directory of Open Access Journals (Sweden)

    Ruth McNerney


    Full Text Available Early detection and effective treatment are crucial for tuberculosis control, but global case detection rates remain low. The diagnosis of paediatric and extrapulmonary disease is problematic and there are, as yet, no rapid screening tests to assist active case finding in the community. Progress has been made in clinic-based detection tools with the introduction of Xpert MTB/RIF, a nucleic acid amplification test that combines sample processing and analysis in a single instrument to provide a diagnostic result and detection of resistance to rifampicin in under 2 h. Enthusiasm for Xpert MTB/RIF has been high and global rollout has been facilitated by donor agencies. However, concerns remain about access and sustainability due to the high cost and infrastructure requirements. Although more sensitive than smear microscopy, early studies suggest the impact of the new test on case detection rates and patient survival has been limited. Alternative technologies are being developed, including non-sputum-based tests to assist the detection of extrapulmonary disease. Evaluation studies are needed to provide evidence of the impact of the new technologies on patient outcomes. This will enable appropriate placement of new diagnostic products in the healthcare system to support the control and eventual eradication of tuberculosis disease.

  9. Implementation of Rapid Molecular Infectious Disease Diagnostics: the Role of Diagnostic and Antimicrobial Stewardship. (United States)

    Messacar, Kevin; Parker, Sarah K; Todd, James K; Dominguez, Samuel R


    New rapid molecular diagnostic technologies for infectious diseases enable expedited accurate microbiological diagnoses. However, diagnostic stewardship and antimicrobial stewardship are necessary to ensure that these technologies conserve, rather than consume, additional health care resources and optimally affect patient care. Diagnostic stewardship is needed to implement appropriate tests for the clinical setting and to direct testing toward appropriate patients. Antimicrobial stewardship is needed to ensure prompt appropriate clinical action to translate faster diagnostic test results in the laboratory into improved outcomes at the bedside. This minireview outlines the roles of diagnostic stewardship and antimicrobial stewardship in the implementation of rapid molecular infectious disease diagnostics.

  10. A GPA diagnostic system for aeroengine applications

    Institute of Scientific and Technical Information of China (English)

    Yi-Guang Li


    In this research,a GPA(Gas Path Analysis)diagnostic system enhanced with GPA Index is described for gas path sensor and component fault diagnosis.A method of measurement correction is used in order that the measurement data obtained at un-standard ambient and operating conditions can be used for diagnostic analysis.The developed diagnostic system has been implemented into a Cranfield University gas turbine performance and diagnostic analysis software PYTHIA for gas turbine performance degradation analysis.The developed method and software have been applied to a model aero gas turbine engine to test the effectiveness of the system.The analysis shows that the developed diagnostic system can diagnose degraded sensor and components effectively using performance deviation measured at un-standard ambient and operational conditions.Theoretically,the idea of the diagnostic approach can be applied to different gas turbine engines.

  11. 42 CFR 415.180 - Teaching setting requirements for the interpretation of diagnostic radiology and other diagnostic... (United States)


    ... interpretation of diagnostic radiology and other diagnostic tests. 415.180 Section 415.180 Public Health CENTERS... for the interpretation of diagnostic radiology and other diagnostic tests. (a) General rule. Physician fee schedule payment is made for the interpretation of diagnostic radiology and other diagnostic...

  12. Unmet Diagnostic Needs in Infectious Disease (United States)

    Blaschke, Anne J.; Hersh, Adam L.; Beekmann, Susan E.; Ince, Dilek; Polgreen, Philip M.; Hanson, Kimberly E.


    Accurate diagnosis is critical to providing appropriate care in infectious diseases. New technologies for infectious disease diagnostics are emerging, but gaps remain in test development and availability. The Emerging Infections Network surveyed Infectious Diseases physicians to assess unmet diagnostic needs. Responses reflected the urgent need to identify drug-resistant infections and highlighted the potential for early diagnosis to improve antibiotic stewardship. Information gained from this survey can help inform recommendations for new diagnostic test development in the future. PMID:25456043

  13. Motional Stark effect diagnostic on TEXTOR (United States)

    Jakubowska, K.; De Bock, M.; Jaspers, R.; von Hellermann, M.; Shmaenok, L.


    A motional Stark effect diagnostic at the tokamak TEXTOR has been constructed and brought recently into operation. In contrast to diagnostics used on other tokamaks, this diagnostic reveals the direction of the magnetic field from the intensity ratio of the π and σ components of the emitted Balmer-α and not from a polarization measurement of a single line. Moreover, the complete spectrum is measured which allows determining the radial position of the measurement, and in principle the radial electric field.

  14. The Beam Diagnostics for SESAME

    CERN Document Server

    Varnasseri, S


    SESAME (Synchrotron-light for Experimental Science and Applications in the Middle East) is an Independent Intergovernmental Organization developed and officially established under the auspices of UNESCO. SESAME will become a major international research center in the Middle East, located in Allan, Jordan. The machine design is based on a 2.5 GeV 3rd generation Light Source with an emittance of 26 nm*rad and 12 straights for insertion devices. The conceptual design of the accelerator complex has been frozen and the engineering design is started. The completion of the accelerators complex construction is scheduled for the end of 2009. In the following an overview of the electron beam diagnostic system is presented, with special emphasis on the beam position monitoring system and the synchrotron light monitor.

  15. Improving tuberculosis diagnostics with biomarkers

    Directory of Open Access Journals (Sweden)

    Shu CC


    Full Text Available Chin-Chung Shu,1,2 Jann-Yuan Wang,2 Li-Na Lee,2,3 Chong-Jen Yu,2 Kwen-Tay Luh3 1Department of Traumatology, 2Department of Internal Medicine, 3Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan Abstract: Although many laboratory methods have been developed to expedite the diagnosis of active tuberculosis (TB and Mycobacterium tuberculosis (Mtb infection, delays in diagnosis remain a major problem in clinical practice. Biomarkers may contribute favorably or unfavorably to TB diagnosis in a clinical suspect TB case with inconclusive diagnostic findings. A good understanding of the effectiveness and practical limitations of these biomarkers is important to improve diagnosis. This review summarizes currently used biomarkers, mainly as validation, and focuses on latent TB infection, active pulmonary TB, and tuberculous pleural effusion. Keywords: tuberculosis, biomarker, diagnosis, latent tuberculosis infection, pleural effusion 

  16. AI-Based Diagnostic Shell

    Directory of Open Access Journals (Sweden)

    R. L. Verma


    Full Text Available This paper datails the design and implementation of an AI-based diagnostic shell. The shell has a user-interface which takes in the complaint and aids the user throughout the consultation. The 'expert knowledge' is acquired and encoded in the form of 'IF-THEN' rules, The control mechanism routes through the rules chaining first backwards to identify a fault and then forwards to confirm it.Explanation facilities have been provided to enable the user query the reason for any question asked, a facility to go back and re-answer any previous question, and a trace and explanation of the path of reasoning.This shell was developed and first used for the diagnosis of a digital exchange. It was then applied for the fault-finding of the moving target indicator used in the radar.

  17. The sleepy teenager - diagnostic challenges

    Directory of Open Access Journals (Sweden)

    Anne-Marie eLandtblom


    Full Text Available The sleepy teenager is a diagnostic challenge because the problems may be physiological or pathological, with behavioural, social and pychological expressions. It is of great importance that health staff that encounter young people with sleep disturbance have good knowledge about the diseases that must be excluded. Narcolepsy, periodic hypersomnia like Kleine Levin syndrome, delayed sleep phase syndrome and obstructive sleep apnoea syndrome, depression and substance use as well as fatigue from chronic disease like multiple sclerosis should be investigated. Clinical assessment, neurophysiological and laboratory investigations constitute important support in these investigations. Functional methods, for example fMRI, are being developed. The role of computer gaming and use of social media in the night is discussed in relation to these diseases. Cognitive dysfunction may develop with several of the conditions. There is need for increased awareness of how to investigate sleep disturbance in children and young people.

  18. [Molecular diagnostics of lung cancer]. (United States)

    Ryska, A; Dziadziuszko, R; Olszewski, W; Berzinec, P; Öz, B; Gottfried, M; Cufer, T; Samarzija, M; Plank, L; Ostoros, Gy; Tímár, J


    Development of the target therapies of lung cancer was a rapid process which fundamentally changed the pathological diagnosis as well. Furthermore, molecular pathology became essential part of the routine diagnostics of lung cancer. These changes generated several practical problems and in underdeveloped countries or in those with reimbursement problems have been combined with further challenges. The central and eastern region of Europe are characterized by similar problems in this respect which promoted the foundation of NSCLC Working Group to provide up to date protocols or guidelines. This present paper is a summary of the molecular pathology and target therapy guidelines written with the notion that it has to be upgraded continuously according to the development of the field.

  19. [Acute rhinosinusitis: diagnostics and treatment]. (United States)

    Lazarevich, I A; Kozlov, V S


    Acute rhinosinusitis is a challenging clinical problem due to its high prevalence. The overwhelming majority of the cases of acute rhinosinusitis (ARS) have viral etiology. Clinical manifestations of viral and bacterial rhinosinusitis can be very similar. Similar expert communities have proposed their recommendations on diagnostics and treatment of acute rhinosinusitis in the recent decade. These recommendations are underlain by the principles of evidence-based medicine, take into consideration the result of reliable investigations, and reflect the opinions of leading specialists in otorhinolaryngology, allergology and immunology. The present review contains the analysis of consensus documents and recommendations. The results of ongoing research provide convincing evidence of the effectiveness of intranasal application of corticosteroids in the patients presenting with acute rhinosinusitis. Antibacterial therapy of acute rhinosinusitis is indicated only in the case of severe or complicated clinical course of the disease.

  20. Interstitial lung disease: Diagnostic approach

    Directory of Open Access Journals (Sweden)

    Kaushik Saha


    Full Text Available Interstitial lung disease (ILD is a final common pathway of a broad heterogeneous group of parenchymal lung disorders. It is characterized by progressive fibrosis of the lung leading to restriction and diminished oxygen transfer. Clinically, the presenting symptoms of ILD are non-specific (cough and progressive dyspnea on exertion and are often attributed to other diseases, thus delaying diagnosis and timely therapy. Clues from the medical history along with the clinical context and radiologic findings provide the initial basis for prioritizing diagnostic possibilities for a patient with ILD. An accurate prognosis and optimal treatment strategy for patients with ILDs can only be after an accurate diagnosis. This review will assist pulmonary physicians and medicine specialist in recognition of ILD. Extensive literature search has been made through PubMed and also Book References has been used for writing this review.

  1. Necrotizing sialometaplasia: A diagnostic dilemma!

    Directory of Open Access Journals (Sweden)

    Samir A Joshi


    Full Text Available Necrotizing sialometaplasia (NS is a benign, self-limiting inflammatory reaction of salivary gland tissue which may mimic squamous cell carcinoma or mucoepidermoid carcinoma, both clinically and histologically, that creates diagnostic dilemma leading to unwarranted aggressive surgery. Most commonly affected site is the minor salivary glands of the palate. The pathogenesis is unknown but is believed to be due to ischemia of vasculature supplying the salivary gland lobules. A simple incisional biopsy is required to confirm the histological diagnosis and to rule out more serious disease processes. It is a self-limiting disease process and requires no treatment. It will be prudent to do repeat biopsy in case if the lesion does not heal within 3 months.

  2. Diagnostic Ultrasound in Colorectal Cancer

    DEFF Research Database (Denmark)

    Rafaelsen, Søren Rafael


    SUMMARYBackground and purpose Colorectal cancer is a common disease in Denmark with considerable morbidity and mortality. Although survival in recent years has improved, Denmark still has the lowest 5-year survival compared to the other Nordic countries. The treatment of patients depends on local...... the potential to contribute to the staging of colorectal cancer. The purpose of these studies was to determine the usefulness of ultrasound diagnostics in patients with colorectal cancer.The purpose of the TRUS studies was to compare staging of rectal carcinomas using digital rectal exploration...... with the resulting pathological examination in relation to differentiating benign from malignant polyps and determining tumour stage and lymph node status. In this context we also performed an observer comparison using both TRUS and MRI. Consistency of tumour outgrowth of rectal cancer rated by TRUS and MRI...

  3. Molecular diagnostics of foodborne pathogens

    DEFF Research Database (Denmark)

    Hansen, Trine

    for screening of unknown bacteria in bottled water without prior cultivation. B. cereusartificially inoculated in bottled water was used as a model. The results revealed that the method was able to detect B. cereusat levels of 105-106 CFU/L, a detection level low enough for detection in outbreaks situations...... behavior in pork processing environments, and detection of B. cereusin food, feed and water samples without prior cultivation. The persistence of Salmonellain food production chains has been suggested to be a result of bacterial attachment and surface colonization. It was found that the physiological state...... or accidental contamination of food, feed and water supplies pose a threat to human health worldwide and the need for generic detection methods that can screen for many pathogens at the time are highly desirable. A metagenomics based direct 16S rDNA sequencing approach was evaluated as a diagnostic tool...

  4. Beam diagnostics in the CIRFEL

    Energy Technology Data Exchange (ETDEWEB)

    Krishnaswamy, J.; Lehrman, I.S.; Hartley, R. [Northrop Grumman Advanced Technology and Development Center, Princeton, NJ (United States)] [and others


    The CIRFEL system has been operating with electron energies in the range of 11 to 12 MeV and RF pulse length of 3 to 4 {mu}secs. The electrons produced by a Magnesium photocathode illuminated by a 261nm mode locked laser are accelerated in the RF gun, and further boosted in energy by a booster section downstream of the RIF gun. The electrons are energy selected in the bending section before insertion into a permanent magnet wiggler. We describe several recent diagnostic measurements carried out on the CIRFEL system: emittance measurements in two different sections of the beam line, energy and energy spread measurements, and jitter characteristics of the photo cathode drive laser as well as the electron beam energy.

  5. Brucellosis: Epizootiologic and diagnostic challenge

    Directory of Open Access Journals (Sweden)

    Radojičić Sonja


    Full Text Available Although it has been known as a separate etiological entity for almost 150 years, brucellosis is, on a global scale, one of the most frequent diseases that is transferred from animals to humans. It is present as an endemic disease in almost all countries of the Mediterranean Sea, which indicates that a large number of aspects in connection with the control and epizootiology of this disease still remain unexplained. Three of the six types of brucella have been officially confirmed in our country as well. They are Brucella melitensis biotip 3, Brucella suis biotip 2 and Brucella canis. Brucellosis is endemically present in Kosovo and Metohija province and in southern Serbia proper; over the past few years, the spread of brucellosis in sheep and goats as primary hosts for B. melitensis to new territories, mostly in Vojvodina province, has shown that risk analysis is one of the main factors in selecting and implementing control programmes. A correctly selected set of diagnostic tests yields reliable data in most cases, but interpretations of results are prone to result in subjective assessments as well. A special problem in the serological diagnosis of brucellosis is the cross reactivity of brucellas and some other bacteria, often a weak immunological response of the animal, or that the type of brucella that causes the infection determines the sensitivity and specificity of the applied tests, most often screenings tests. Due to the big economic losses resulting from disease control and eradication, and the serious risk to human health, brucellosis still poses an epizootiological, and, in particular, a diagnostic challenge.

  6. Diagnostic randomized controlled trials: the final frontier. (United States)

    Rodger, Marc; Ramsay, Tim; Fergusson, Dean


    Clinicians, patients, governments, third-party payers, and the public take for granted that diagnostic tests are accurate, safe and effective. However, we may be seriously misled if we are relying on robust study design to ensure accurate, safe, and effective diagnostic tests. Properly conducted, randomized controlled trials are the gold standard for assessing the effectiveness and safety of interventions, yet are rarely conducted in the assessment of diagnostic tests. Instead, diagnostic cohort studies are commonly performed to assess the characteristics of a diagnostic test including sensitivity and specificity. While diagnostic cohort studies can inform us about the relative accuracy of an experimental diagnostic intervention compared to a reference standard, they do not inform us about whether the differences in accuracy are clinically important, or the degree of clinical importance (in other words, the impact on patient outcomes). In this commentary we provide the advantages of the diagnostic randomized controlled trial and suggest a greater awareness and uptake in their conduct. Doing so will better ensure that patients are offered diagnostic procedures that will make a clinical difference.

  7. Recent Advancements in Microwave Imaging Plasma Diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    H. Park; C.C. Chang; B.H. Deng; C.W. Domier; A.J.H. Donni; K. Kawahata; C. Liang; X.P. Liang; H.J. Lu; N.C. Luhmann, Jr.; A. Mase; H. Matsuura; E. Mazzucato; A. Miura; K. Mizuno; T. Munsat; K. and Y. Nagayama; M.J. van de Pol; J. Wang; Z.G. Xia; W-K. Zhang


    Significant advances in microwave and millimeter wave technology over the past decade have enabled the development of a new generation of imaging diagnostics for current and envisioned magnetic fusion devices. Prominent among these are revolutionary microwave electron cyclotron emission imaging (ECEI), microwave phase imaging interferometers, imaging microwave scattering and microwave imaging reflectometer (MIR) systems for imaging electron temperature and electron density fluctuations (both turbulent and coherent) and profiles (including transport barriers) on toroidal devices such as tokamaks, spherical tori, and stellarators. The diagnostic technology is reviewed, and typical diagnostic systems are analyzed. Representative experimental results obtained with these novel diagnostic systems are also presented.

  8. Combustion & Laser Diagnostics Research Complex (CLDRC) (United States)

    Federal Laboratory Consortium — Description: The Combustion and Laser Diagnostics Research Complex (CLRDC) supports the experimental and computational study of fundamental combustion phenomena to...

  9. Constraints to estimating the prevalence of trypanosome infections in East African zebu cattle

    Directory of Open Access Journals (Sweden)

    Cox Andrew P


    Full Text Available Abstract Background In East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations. Results The Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%, T. congolense (42.9% and T. vivax (22.9%. Conclusions These results show that, despite the convenience of Whatman FTA cards and specific PCR based

  10. Diagnostic accuracy of consensus diagnostic criteria for frontotemporal dementia in a memory clinic population

    NARCIS (Netherlands)

    Y. Pijnenburg (Yolande); J.L. Mulder (Jacqueline); J.C. van Swieten (John); B.M. Uitdehaag (Bernard); M. Stevens (Martijn); P. Scheltens (Philip); C. Jonker (Cees)


    textabstractBackground/Aims: The goal of the present study was to evaluate the diagnostic accuracy of the core diagnostic criteria for frontotemporal dementia (FTD) [Neary D, et al: Neurology 1998;51:1546-1554] within a memory clinic population. Methods: The 5 core diagnostic criteria for FTD were o

  11. [Ultrasound diagnostics in ophthalmology (standardized echography): part 2: diseases of the orbit - ultrasound biomicroscopy diagnostics]. (United States)

    Hasenfratz, G; Mardin, C


    Ultrasound diagnostics have been one of the most important noninvasive supplementary diagnostic procedures in ophthalmology for many decades and are indispensable for many intraocular and orbital diseases. When the echography examination and analysis of the echograms obtained are correctly carried out, ultrasound diagnostics are characterized by a high measure of specificity and sensitivity.

  12. Tuberculosis diagnostics: innovating to make an impact. (United States)

    Ghanashyam, Bharathi


    The 'International Symposium on TB Diagnostics: Innovating to Make an Impact' was organized by the International Centre for Genetic Engineering & Biotechnology, New Delhi, India, on December 16-17, 2010, with sponsorship support from the Bill & Melinda Gates Foundation, Foundation for Innovative New Diagnostics and AERAS Global TB Vaccine Foundation. This highly successful symposium attracted more than 300 participants from India and several other countries and covered several aspects of TB diagnostics, including recent scientific advances in TB diagnostics, progress made in expanding the TB diagnostics pipeline including a portfolio of WHO-endorsed, validated new tools and improved technologies, the successful development of newer molecular assays that have the potential to be used at the point of treatment and the growing contributions of emerging economies such as India. In addition to highlighting the positive aspects of TB diagnostics, the symposium speakers also highlighted the need to focus on worrisome aspects of TB diagnosis, including widespread abuse of inappropriate tests that can prevent the use of good diagnostics, lack of quality assurance in laboratories, lack of adequate regulation of diagnostics and how these can pose a major challenge for roll-out and implementation of new tools. The symposium ended with a very stimulating discussion on how India can become a global leader in TB innovations.

  13. [The diagnostic algorithm in twin pregnancy]. (United States)

    Ropacka-Lesiak, Mariola; Szaflik, Krzysztof; Breborowicz, Grzegorz H


    This paper presents the diagnostic algorithm in twin pregnancy. The most important sonographic parameters in the assessment of twins have been discussed. Moreover, the most significant complications of twin pregnancy as well as diagnostic possibilities and management, have been also presented and defined.

  14. Diagnostic Research : improvements in design and analysis

    NARCIS (Netherlands)

    Biesheuvel, C.J.


    In the era of evidence-based medicine, diagnostic procedures also need to undergo critical evaluations. In contrast to guidelines for randomised trials and observational etiologic studies, principles and methods for diagnostic evaluations are still incomplete. The research described in this thesis w

  15. Novel insights into esophageal diagnostic procedures

    DEFF Research Database (Denmark)

    Savarino, Edoardo; Ottonello, Andrea; Tolone, Salvatore;


    The 21st century offers new advances in diagnostic procedures and protocols in the management of esophageal diseases. This review highlights the most recent advances in esophageal diagnostic technologies, including clinical applications of novel endoscopic devices, such as ultrathin endoscopy and...

  16. Developing Score Reports for Cognitive Diagnostic Assessments (United States)

    Roberts, Mary Roduta; Gierl, Mark J.


    This paper presents a framework to provide a structured approach for developing score reports for cognitive diagnostic assessments ("CDAs"). Guidelines for reporting and presenting diagnostic scores are based on a review of current educational test score reporting practices and literature from the area of information design. A sample diagnostic…

  17. Modeling Diagnostic Assessments with Bayesian Networks (United States)

    Almond, Russell G.; DiBello, Louis V.; Moulder, Brad; Zapata-Rivera, Juan-Diego


    This paper defines Bayesian network models and examines their applications to IRT-based cognitive diagnostic modeling. These models are especially suited to building inference engines designed to be synchronous with the finer grained student models that arise in skills diagnostic assessment. Aspects of the theory and use of Bayesian network models…

  18. Comparison of Diagnostic Methods for Asperger Syndrome (United States)

    Kopra, Kristiina; von Wendt, Lennart; Nieminen-von Wendt, Taina; Paavonen, E. Julia


    Several different diagnostic sets of criteria exist for Asperger syndrome (AS), but there is no agreement on a gold standard. The aim of this study was to compare four diagnostic sets of criteria for AS: the ICD-10, the DSM-IV, the Gillberg & Gillberg, and the Szatmari criteria. The series consists of 36 children who had been referred to two…

  19. Quality of reporting of diagnostic accuracy studies

    NARCIS (Netherlands)

    Smidt, N.; Rutjes, A.W.; Windt - Mens, van der D.A.W.M.; Ostelo, R.W.J.G.; Reitsma, J.B.; Bouter, L.M.; Vet, de H.C.W.


    PURPOSE: To evaluate quality of reporting in diagnostic accuracy articles published in 2000 in journals with impact factor of at least 4 by using items of Standards for Reporting of Diagnostic Accuracy (STARD) statement published later in 2003. MATERIALS AND METHODS: English-language articles on pri

  20. Diagnostic Inflation Causes and a Suggested Cure

    NARCIS (Netherlands)

    Batstra, Laura; Frances, Allen


    There have been a striking diagnostic inflation and a corresponding increase in the use of psychotropic drugs during the past 30 years. DSM-5, scheduled to appear in May 2013, proposes another grand expansion of mental illness. In this article, we will review the causes of diagnostic exuberance and

  1. [Klatskin tumors: rational diagnostics and staging]. (United States)

    Denzer, U W; Rösch, T


    Klatskin tumors continue to be a challenge for diagnostic assessment and staging due to their longitudinal tumor growth along the perihilar bile ducts. Therefore the rate of non-resectable tumors remains relatively stable despite modern imaging and endoscopic techniques. This article reviews the current diagnostic methods for preoperative staging and the significance for predicting resectability.

  2. Integrated diagnostic technique for nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Gofuku, Akio [Graduate School of Natural Science and Technology, Okayama University, Okayama (Japan)


    It is very important to detect and identify small anomalies and component failures for the safe operation of complex and large-scale artifacts such as nuclear power plants. Each diagnostic technique has its own advantages and limitations. These facts inspire us not only to enhance the capability of diagnostic techniques but also to integrate the results of diagnostic subsystems in order to obtain more accurate diagnostic results. The article describes the outline of four diagnostic techniques developed for the condition monitoring of the fast breeder reactor 'Monju'. The techniques are (1) estimation technique of important state variables based on a physical model of the component, (2) a state identification technique by non-linear discrimination function applying SVM (Support Vector Machine), (3) a diagnostic technique applying WT (Wavelet Transformation) to detect changes in the characteristics of measurement signals, and (4) a state identification technique effectively using past cases. In addition, a hybrid diagnostic system in which a final diagnostic result is given by integrating the results from subsystems is introduced, where two sets of values called confidence values and trust values are used. A technique to determine the trust value is investigated under the condition that the confidence value is determined by each subsystem.

  3. How to appraise a diagnostic test. (United States)

    Manikandan, Ramanitharan; Dorairajan, Lalgudi N


    Urologists frequently encounter problems in making a clinical diagnosis whose resolution requires the use of diagnostic tests. With an ever increasing choice of investigations being available, the urologist often has to decide which diagnostic test(s) will best resolve the patient's diagnostic problem. In this article, we aim to help the urologist understand how to critically appraise studies on diagnostic tests and make a rational choice. This article presents the guiding principles in scientifically assessing studies on diagnostic tests by proposing a clinical scenario. The authors describe a standardized protocol to assess the validity of the test and its relevance to the clinical problem that can help the urologist in decision making. The three important issues to be considered when evaluating the validity of the study are to identify how the study population was chosen, how the test was performed and whether there is a comparison to the gold standard test so as to confirm or refute the diagnosis. Then, the urologist would need to know the probability of the test in providing the correct diagnosis in an individual patient in order to decide about its utility in solving the diagnostic dilemma. By performing the steps described in this article, the urologist would be able to critically appraise diagnostic studies and draw meaningful conclusions about the investigations in terms of validity, results and its applicability to the patient's problem. This would provide a scientific basis for using diagnostic tests for improving patient care.

  4. Timely Diagnostic Feedback for Database Concept Learning (United States)

    Lin, Jian-Wei; Lai, Yuan-Cheng; Chuang, Yuh-Shy


    To efficiently learn database concepts, this work adopts association rules to provide diagnostic feedback for drawing an Entity-Relationship Diagram (ERD). Using association rules and Asynchronous JavaScript and XML (AJAX) techniques, this work implements a novel Web-based Timely Diagnosis System (WTDS), which provides timely diagnostic feedback…

  5. Diagnostics of solar flare reconnection

    Directory of Open Access Journals (Sweden)

    M. Karlický


    Full Text Available We present new diagnostics of the solar flare reconnection, mainly based on the plasma radio emission. We propose that the high-frequency (600-2000 MHz slowly drifting pulsating structures map the flare magnetic field reconnection. These structures correspond to the radio emission from plasmoids which are formed in the extended current sheet due to tearing and coalescence processes. An increase of the frequency drift of the drifting structures is interpreted as an increase of the reconnection rate. Using this model, time scales of slowly drifting pulsating structure observed during the 12 April 2001 flare by the Trieste radiopolarimeter with high time resolution (1 ms are interpreted as a radio manifestation of electron beams accelerated in the multi-scale reconnection process. For short periods Fourier spectra of the observed structure have a power-law form with power-law indices in the 1.3-1.6 range. For comparison the 2-D MHD numerical modeling of the multi-scale reconnection is made and it is shown that Fourier spectrum of the reconnection dissipation power has also a power-law form, but with power-law index 2. Furthermore, we compute a time evolution of plasma parameters (density, magnetic field etc in the 2-D MHD model of the reconnection. Then assuming a plasma radio emission from locations, where the 'double-resonance' instability generates the upper-hybrid waves due to unstable distribution function of suprathermal electrons, we model radio spectra. Effects of the MHD turbulence are included. The resulting spectra are compared with those observed. It is found, that depending on model parameters the lace bursts and the decimetric spikes can be reproduced. Thus, it is shown that the model can be used for diagnostics of the flare reconnection process. We also point out possible radio signatures of reconnection outflow termination shocks. They are detected as type II-like herringbone structures in the 200-700 MHz frequency range. Finally

  6. Rapid Diagnostics of Onboard Sequences (United States)

    Starbird, Thomas W.; Morris, John R.; Shams, Khawaja S.; Maimone, Mark W.


    Keeping track of sequences onboard a spacecraft is challenging. When reviewing Event Verification Records (EVRs) of sequence executions on the Mars Exploration Rover (MER), operators often found themselves wondering which version of a named sequence the EVR corresponded to. The lack of this information drastically impacts the operators diagnostic capabilities as well as their situational awareness with respect to the commands the spacecraft has executed, since the EVRs do not provide argument values or explanatory comments. Having this information immediately available can be instrumental in diagnosing critical events and can significantly enhance the overall safety of the spacecraft. This software provides auditing capability that can eliminate that uncertainty while diagnosing critical conditions. Furthermore, the Restful interface provides a simple way for sequencing tools to automatically retrieve binary compiled sequence SCMFs (Space Command Message Files) on demand. It also enables developers to change the underlying database, while maintaining the same interface to the existing applications. The logging capabilities are also beneficial to operators when they are trying to recall how they solved a similar problem many days ago: this software enables automatic recovery of SCMF and RML (Robot Markup Language) sequence files directly from the command EVRs, eliminating the need for people to find and validate the corresponding sequences. To address the lack of auditing capability for sequences onboard a spacecraft during earlier missions, extensive logging support was added on the Mars Science Laboratory (MSL) sequencing server. This server is responsible for generating all MSL binary SCMFs from RML input sequences. The sequencing server logs every SCMF it generates into a MySQL database, as well as the high-level RML file and dictionary name inputs used to create the SCMF. The SCMF is then indexed by a hash value that is automatically included in all command

  7. Current possibilities of chorioretinites diagnostics

    Directory of Open Access Journals (Sweden)

    O. V. Chudinova


    Full Text Available Purpose: to study the morphometric changes in retina and the state of regional hemodynamics for chorioretinites of different etiology, to draw parallels between these methods of study with evaluation of their diagnostical significance.Methods: Clinical and instrumental examination was performed in 15 patients (15 eyes — group 1 — with the verified diagnosis of toxoplasmous chorioretinitis and in 13 patients (13 eyes — group 2 — with the diagnosis of tuberculous chorioretinitis. Control (group 3 consisted of 20 subjects (40 eyes, 9 males, 11 females, without any pathology of organ of vision. Complex ophthalmologic examination was performed in all the patients; the examination included the following procedures: determination of visual acuity with correction, computer perimetry, biomicroscopy of eye fundus, inspection of eye fundus using Goldman lens, optic coherent tomogra- phy (OCt, ultrasound Dopplerography (USDG of eye vessels.Results: the following was determined by OCt data: subclinical serous retinal detachment, isolated cells of cyst-like edema, cyst- like edema in macular zone, unevenness of hyperreflective band of pigment epithelium, thinning of neurosensory retina in the area of scarry focus, hyperreflectivity of the zone of the fibrosis being formed, architectonics disorder of NE layers in foveolar zone and para- foveally at the expense of the presence of small hyperreflective parts. In the presence of proliferative process in the vascular coat the reliable decrease of blood flow maximal and minimal velocities in the posterior short ciliary arteries, maximal and minimal velocities of blood flow in the posterior long ciliary arteries in comparison with the values of patients from control group. the data obtained are supposed that proliferative processes in the vascular coat are accompanied by marked local hemodynamic disorders, which should be taken into consideration when complex therapy is prescribed.Conclusion: Dynamic

  8. Diagnostic criteria for autoimmune chronic pancreatitis revisited

    Institute of Scientific and Technical Information of China (English)

    Kyu-Pyo Kim; Myung-Hwan Kim; Jong Cheol Kim; Sang Soo Lee; Dong Wan Seo; Sung Koo Lee


    Autoimmune chronic pancreatitis (AIP) is increasingly being recognized worldwidely, as knowledge of this entity builds up. Above all, AIP is a very attractive disease to clinicians in terms of its dramatic response to the oral steroid therapy in contrast to ordinary chronic pancreatitis. Although many characteristic findings of AIP have been described, definite diagnostic criteria have not been fully established. In the year 2002, the Japan Pancreas Society published the diagnostic criteria of AIP and many clinicians around the world use these criteria for the diagnosis of AIP. The diagnostic criteria proposed by the Japan Pancreas Society, however, are not completely satisfactory and some groups use their own criteria in reporting AIP. This review discusses several potential limitations of current diagnostic criteria for this increasingly recognized condition. The manuscript is organized to emphasize the need for convening a consensus to develop improved diagnostic criteria.

  9. Small business development for molecular diagnostics. (United States)

    Anagostou, Anthanasia; Liotta, Lance A


    Molecular profiling, which is the application of molecular diagnostics technology to tissue and blood -specimens, is an integral element in the new era of molecular medicine and individualized therapy. Molecular diagnostics is a fertile ground for small business development because it can generate products that meet immediate demands in the health-care sector: (a) Detection of disease risk, or early-stage disease, with a higher specificity and sensitivity compared to previous testing methods, and (b) "Companion diagnostics" for stratifying patients to receive a treatment choice optimized to their individual disease. This chapter reviews the promise and challenges of business development in this field. Guidelines are provided for the creation of a business model and the generation of a marketing plan around a candidate molecular diagnostic product. Steps to commercialization are outlined using existing molecular diagnostics companies as learning examples.

  10. Electrochemical Biosensors for Early Stage Zika Diagnostics. (United States)

    Kaushik, Ajeet; Tiwari, Sneham; Jayant, Rahul D; Vashist, Arti; Nikkhah-Moshaie, Roozbeh; El-Hage, Nazira; Nair, Madhavan


    Health agencies have declared the recent Zika virus (ZIKV) infection an epidemic and a public health emergency of global concern due to its association with microcephaly and serious neurological disorders. The unavailability of effective drugs, vaccines, and diagnostic tools increases the demand for efficient analytical devices to detect ZIKV infection. However, high costs, longer diagnostic times, and stringent expertise requirements limit the utility of reverse transcriptase-PCR methods for rapid diagnostics. Therefore, developing portable, sensitive, selective, and cost-effective sensing systems to detect ZIKV at picomolar concentrations in biofluids would be a breakthrough in diagnostics and therapeutics. This paper highlights the advancements in developing smart sensing strategies to monitor ZIKV progression, with rapid point-of-care diagnostics as the ultimate aim.

  11. Diagnostic Categories in Autobiographical Accounts of Illness. (United States)

    Kelly, Michael P


    Working within frameworks drawn from the writings of Immanuel Kant, Alfred Schutz, and Kenneth Burke, this article examines the role that diagnostic categories play in autobiographical accounts of illness, with a special focus on chronic disease. Four lay diagnostic categories, each with different connections to formal medical diagnostic categories, serve as typifications to make sense of the way the lifeworld changes over the course of chronic illness. These diagnostic categories are used in conjunction with another set of typifications: lay epidemiologies, lay etiologies, lay prognostics, and lay therapeutics. Together these serve to construct and reconstruct the self at the center of the lifeworld. Embedded within the lay diagnostic categories are narratives of progression, regression, or stability, forms of typification derived from literary and storytelling genres. These narratives are developed by the self in autobiographical accounts of illness.

  12. Diagnostic interval and mortality in colorectal cancer

    DEFF Research Database (Denmark)

    Tørring, Marie Louise; Frydenberg, Morten; Hamilton, William;


    Objective To test the theory of a U-shaped association between time from the first presentation of symptoms in primary care to the diagnosis (the diagnostic interval) and mortality after diagnosis of colorectal cancer (CRC). Study Design and Setting Three population-based studies in Denmark...... presentation, the association between the length of the diagnostic interval and 5-year mortality rate after the diagnosis of CRC was the same for all three types of data: displaying a U-shaped association with decreasing and subsequently increasing mortality with longer diagnostic intervals. Conclusion Unknown...... confounding and in particular confounding by indication is likely to explain the counterintuitive findings of higher mortality among patients with very short diagnostic intervals, but cannot explain the increasing mortality with longer diagnostic intervals. The results support the theory that longer...

  13. International Workshop on Diagnostics for ITER

    CERN Document Server

    Gorini, Giuseppe; Sindoni, Elio


    This book of proceedings collects the papers presented at the Workshop on Diagnostics for ITER, held at Villa Monastero, Varenna (Italy), from August 28 to September 1, 1995. The Workshop was organised by the International School of Plasma Physics "Piero Caldirola. " Established in 1971, the ISPP has organised over fifty advanced courses and workshops on topics mainly related to plasma physics. In particular, courses and workshops on plasma diagnostics (previously held in 1975, 1978, 1982, 1986, and 1991) can be considered milestones in the history of this institution. Looking back at the proceedings of the previous meetings in Varenna, one can appreciate the rapid progress in the field of plasma diagnostics over the past 20 years. The 1995 workshop was co-organised by the Istituto di Fisica del Plasma of the National Research Council (CNR). In contrast to previous Varenna meetings on diagnostics, which have covered diagnostics in present-day tokamaks and which have had a substantial tutorial component, the 1...

  14. Tuberculosis diagnostic methods in buffaloes

    Directory of Open Access Journals (Sweden)

    Gabriela Capriogli Oliveira


    Full Text Available The low productivity of buffalo herds and condemnation of carcasses in slaughterhouses due to tuberculosis lesions have resulted in increasing economic losses because these animals cannot be treated and must be destroyed by sanitary slaughter. Tuberculosis is a widely distributed zoonosis that affects the beef supply chain of the Brazilian agribusiness economically and socially. Like cattle, buffaloes are sensitive to Mycobacterium bovis, which is the main causative agent of zoonotic tuberculosis. Tuberculosis in buffaloes has been reported in several countries, including Brazil. In order to control and eradicate this disease among cattle and buffaloes in Brazil, the Ministry of Agriculture, Livestock, and Supply created the National Program for the Control and Eradication of Brucellosis and Tuberculosis with the main objective of finding a significant number of disease-free herds throughout the national territory using reliable methods. This review summarizes the main data on the history of occurrence of M. bovis in Brazilian herds and the diagnostic methods for the disease in buffaloes. Little information is available on buffalo tuberculosis. Due to the increasing population of buffaloes and their economic importance, more studies investigating the occurrence and identification of tuberculosis in this species are clearly needed.

  15. Antiphospholipid syndrome: A diagnostic challenge. (United States)

    Mallhi, R S; Kushwaha, Neerja; Chatterjee, T; Philip, J


    The antiphospholipid syndrome (APS) is an acquired autoimmune thrombophilic disorder that is characterized by thrombosis (venous, arterial and microvascular) and obstetric morbidity due to a diverse family of antibodies against phospholipid-binding proteins present in plasma. The term antiphospholipid antibody is actually a misnomer as the antibodies are not against the phospholipid per se, but target the plasma protein co-factors, which bind to anionic PLs. The exact etiology has not been elucidated and is multifactorial. The initial guidelines for the diagnosis of APS were laid down in Sapporo, 1999, which were subsequently revised as the Sydney Consensus Conference criteria in 2006. Major changes were the inclusion of β2GPI as independent laboratory criteria, addition of ischemic stroke and transient cerebral ischemia as established clinical criteria and the requirement of repeating the test after 12 weeks. The laboratory tests recommended are coagulation assays, which study the effect of lupus anticoagulant on the clotting time and immunological assays, mostly ELISAs to detect IgG and IgM antibodies against cardiolipin and/or β2 glycoprotein I. For the diagnosis of APS, at least one clinical criterion and one laboratory criterion should be present. Limitations pertaining to the standardization, reproducibility and robustness of the currently recommended diagnostic tests still remain. Despite elaborate guidelines and syndrome defining criteria, the diagnosis of APS still remains a challenge. A greater interaction between the clinicians and the laboratory professionals is necessary for arriving at the correct diagnosis as a misdiagnosis of APS can have grave consequences.

  16. Diagnostic strategies in nasal congestion

    Directory of Open Access Journals (Sweden)

    John Krouse


    Full Text Available John Krouse1, Valerie Lund2, Wytske Fokkens3, Eli O Meltzer41Temple University School of Medicine, Philadelphia, PA, USA; 2Ear Institute, University College London, UK; 3Department of Otorhinolaryngology, Amsterdam Medical Centre, Netherlands; 4Allergy and Asthma Medical Group and Research Center, San Diego, CA and Department of Pediatrics, University of California, San Diego, USAAbstract: Nasal congestion is a major symptom of upper respiratory tract disorders, and its characterization an important part of the diagnosis of these illnesses. Patient history and assessment of nasal symptoms are essential components of diagnosis, providing an initial evaluation that may be adequate to rule out serious conditions. However, current congestion medications are not always fully effective. Thus, if symptoms do not respond adequately to therapy, or symptoms suggestive of more serious conditions are present, specialized assessments may be needed. Various techniques are available for diagnosing patients, including those used chiefly by primary care clinicians and those requiring the expertise of otolaryngologists, allergists, and other specialists. Endoscopy remains a mainstay for evaluating nasal blockage and its causes, while modalities such as peak nasal inspiratory flow and acoustic rhinometry are evolving to provide easy-to-use, noninvasive procedures that are sensitive enough to measure small but clinically important abnormalities and therapeutic changes. Several imaging modalities are available to the specialist for severe or unusual cases, as are specialized diagnostic procedures that measure adjunctive features of congestion, such as impaired mucociliary function.Keywords: allergic rhinitis, congestion, diagnosis, obstruction, rhinosinusitis

  17. Nanotechnology in Disease Diagnostic Techniques. (United States)

    Savaliya, Reema; Shah, Darshini; Singh, Ragini; Kumar, Ashutosh; Shankar, Rishi; Dhawan, Alok; Singh, Sanjay


    Currently the major research highlights of bioengineering and medical technology are directed towards development of improved diagnostic techniques to screen complex diseases. Screening requirements are for the identification of the cause of illnesses, monitoring the improvement or progression of the state of diseases such as cancer, cardiovascular or neurodegenerative diseases. Nanotechnology enables the manipulation of materials at nanoscale and has shown potential to enhance sensitivity, selectivity and lower the cost of a diagnosis. The causative biomolecules (DNA, proteins) can be detected by red-shifted absorbance of gold nanoparticles or alteration in the conductance of a nanowire or nanotubes, and deflection of a micro or nano-cantilever. Several types of nanomaterials such as metals, metal-oxides and quantum dots have shown ample advantages over traditional diagnosis, intracellular labeling and visualization of target cells/tissues. Nanotechnology has also opened several avenues which could be further developed to enable enhanced visualization of tissues, cells, DNA and proteins over a point-of-care device. Protein or gene chips created using nanomaterials could be further be integrated into a convenient nano-fluidic device for better disease diagnosis.

  18. Communication skills in diagnostic pathology. (United States)

    Lehr, Hans-Anton; Bosman, Fred T


    Communication is an essential element of good medical practice also in pathology. In contrast to technical or diagnostic skills, communication skills are not easy to define, teach, or assess. Rules almost do not exist. In this paper, which has a rather personal character and cannot be taken as a set of guidelines, important aspects of communication in pathology are explored. This includes what should be communicated to the pathologist on the pathology request form, communication between pathologists during internal (interpathologist) consultation, communication around frozen section diagnoses, modalities of communication of a final diagnosis, with whom and how critical and unexpected findings should be communicated, (in-)adequate routes of communication for pathology diagnoses, who will (or might) receive pathology reports, and what should be communicated and how in case of an error or a technical problem. An earlier more formal description of what the responsibilities are of a pathologist as communicator and as collaborator in a medical team is added in separate tables. The intention of the paper is to stimulate reflection and discussion rather than to formulate strict rules.


    Directory of Open Access Journals (Sweden)

    Vinicius Orso


    Full Text Available Objective : To analyze aspects related to the diagnostic difficulty in patients with bacterial spondylodiscitis. Methods : Cross-sectional observational study with retrospective data collected in the period from March 2004 to January 2014.Twenty-one patients diagnosed with bacterial spondylodiscitis were analyzed. Results : Women were the most affected, as well as older individuals. Pain in the affected region was the initial symptom in 52% of patients, and 45.5% of the patients had low back pain, and those with dorsal discitis had back pain as the main complaint; the patients with thoracolumbar discitis had pain in that region, and only one patient had sacroiliac discitis. The average time between onset of symptoms and treatment was five months. The lumbar segment was the most affected with 11 cases (52%, followed by thoracolumbar in 24%, dorsal in 19% of cases and a case in the sacroiliac segment. Only seven patients had fever. Pain in the affected level was coincidentally the most common symptom. Conclusions : Early diagnosis of bacterial spondylodiscitis remains a challenge due to the nonspecific signs and symptoms reported by the patient and the wide variability of laboratory results and imaging. The basis for early diagnosis remains the clinical suspicion at the time of initial treatment.

  20. Microbiological diagnostics of fungal infections

    Directory of Open Access Journals (Sweden)

    Corrado Girmenia


    Full Text Available Laboratory tests for the detection of fungal infections are easy to perform. The main obstacle to a correct diagnosis is the correlation between the laboratory findings and the clinical diagnosis. Among pediatric patients, the most common fungal pathogen is Candida. The detection of fungal colonization may be performed through the use of chromogenic culture media, which allows also the identification of Candida subspecies, from which pathogenicity depends. In neonatology, thistest often drives the decision to begin a empiric therapy; in this regard, a close cooperation between microbiologists and clinicians is highly recommended. Blood culture, if positive, is a strong confirmation of fungal infection; however, its low sensitivity results in a high percentage of false negatives, thus decreasing its reliability. Molecular diagnostics is still under evaluation, whereas the detection of some fungal antigens, such as β-D-glucan, galactomannan, mannoprotein, and cryptococcal antigen in the serum is used for adults, but still under evaluations for pediatric patients.

  1. Tibial hyperostosis: A diagnostic approach

    Energy Technology Data Exchange (ETDEWEB)

    Touraine, Sébastien, E-mail: [Radiologie ostéo-articulaire, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris Cedex 10 (France); Parlier-Cuau, Caroline, E-mail: [Radiologie ostéo-articulaire, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris Cedex 10 (France); Bousson, Valérie, E-mail: [Radiologie ostéo-articulaire, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris Cedex 10 (France); Sverzut, Jean-Michel, E-mail: [Radiologie ostéo-articulaire, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris Cedex 10 (France); Centre d’imagerie du centre cardiologique du Nord, 32-36 rue des Moulins Gémeaux, 93200 Saint-Denis (France); Genah, Idan, E-mail: [Radiologie ostéo-articulaire, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris Cedex 10 (France); and others


    Tibial hyperostosis may be encountered in musculoskeletal imaging, incidentally or during the investigation of a leg pain. Hyperostosis involves the exuberant production of osseous tissue and results in cortical, periosteal and/or endosteal thickening of the bone. As a long bone with thick cortices, the tibia has a significant probability of being affected by ubiquitous bone diseases. As a tubular long bone, the tibia is likely to be involved in extensive infectious conditions such as osteomyelitis. As a bone of the lower limb, the tibia undergoes high stresses and may be affected by decrease in bone strength or repetitive submaximal stress. The tibia is also particularly involved in some bone sclerosing dysplasias and Paget's disease. In this work, we aim at highlighting the main conditions leading to tibial hyperostosis and try to provide key elements to narrow down the several diagnostic possibilities. Osteoid osteomas, fatigue or insufficiency fractures, infectious conditions, vascular lesions, sclerosing bone dysplasias and Paget's disease represent the main challenging diagnoses to discuss.

  2. Companion diagnostics: a regulatory perspective from the last 5 years of molecular companion diagnostic approvals. (United States)

    Roscoe, Donna M; Hu, Yun-Fu; Philip, Reena


    Companion diagnostics are essential for the safe and effective use of the corresponding therapeutic products. The US FDA has approved a number of companion diagnostics used to select cancer patients for treatment with contemporaneously approved novel therapeutics. The processes of co-development and co-approval of a therapeutic product and its companion diagnostic have been a learning experience that continues to evolve. Using several companion diagnostics as examples, this article describes the challenges associated with the scientific, clinical and regulatory hurdles faced by FDA and industry alike. Taken together, this discussion is intended to assist manufacturers toward a successful companion diagnostics development plan.

  3. A Framework to Debug Diagnostic Matrices (United States)

    Kodal, Anuradha; Robinson, Peter; Patterson-Hine, Ann


    Diagnostics is an important concept in system health and monitoring of space operations. Many of the existing diagnostic algorithms utilize system knowledge in the form of diagnostic matrix (D-matrix, also popularly known as diagnostic dictionary, fault signature matrix or reachability matrix) gleaned from physical models. But, sometimes, this may not be coherent to obtain high diagnostic performance. In such a case, it is important to modify this D-matrix based on knowledge obtained from other sources such as time-series data stream (simulated or maintenance data) within the context of a framework that includes the diagnostic/inference algorithm. A systematic and sequential update procedure, diagnostic modeling evaluator (DME) is proposed to modify D-matrix and wrapper logic considering least expensive solution first. This iterative procedure includes conditions ranging from modifying 0s and 1s in the matrix, or adding/removing the rows (failure sources) columns (tests). We will experiment this framework on datasets from DX challenge 2009.

  4. Diagnostic challenges in celiac disease

    Directory of Open Access Journals (Sweden)

    M Haghighat


    Full Text Available   1-The most important challenge in diagnosis of celiac disease is not- performing the diagnostic tests in suspected persons. Because of multi-organ damage and multiple manifestations of disease, diagnosis of celiac disease may be delayed. It seems general physicians should be awared about uncommon presentations of disease and indications of celiac tests 2-The second most important challenge is in patients with suspected disease but negative serologic tests. In these cases evaluating of HLA can be useful. 3- The third challenge is in cases with positive serologic tests but negative histopathological findings. There may be false positive serologic response or consumption of gluten before testing. We recommend introduction of gluten for at least 3 mo and re- endoscopy and if diagnosis is equivocal HLA-typing  for DQ8 and  DQ2 should be done. 4-The forth challenge is about performing endoscopy. Based on guideline from ESPGHAN if there are typical clinical manifestations of celiac disease, Anti-TTG more than ten times UPN , positive Anti-EMA and HLA DQ2, performing endoscopy may not be necessary, but many physicians don’t agree with this idea. 5-In people who are genetically predisposed to celiac disease antibody levels may be fluctuating thus endoscopy with biopsy should be done in these patients. 6-In children lower than 2years, Anti- TTG and Anti –EMA have low sensitivity. we recommend Anti-TTG and Anti-DGP in these patients. 7-Resolution of symptoms after gluten free diet is not necessarily a feature of celiac disease. This condition may be seen in patients with IBS or non-celiac gluten sensitivity.  

  5. Diagnostic Challenges in Celiac Disease

    Directory of Open Access Journals (Sweden)

    Mahmood Haghighat


    Full Text Available 1. The most important challenge in diagnosis of celiac disease is not-performing the diagnostic tests in suspected persons. Because of multi-organ damage and multiple manifestations of disease, diagnosis of celiac disease may be delayed. It seems general physicians should be aware about uncommon presentations of disease and indications of celiac tests. 2. The second most important challenge is in patients with suspected disease but negative serologic tests. In these cases evaluating of HLA can be useful. 3. The third challenge is in cases with positive serologic tests but negative histopathological findings. There may be false positive serologic response or consumption of gluten before testing. We recommend introduction of gluten for at least 3 mo and re- endoscopy and if diagnosis is equivocal HLA-typing for DQ8 and DQ2 should be done. 4. The forth challenge is about performing endoscopy. Based on guideline from ESPGHAN if there are typical clinical manifestations of celiac disease, Anti-TTG more than ten times UPN, positive Anti-EMA and HLA DQ2, performing endoscopy may not be necessary, but many physicians don’t agree with this idea. 5. In people who are genetically predisposed to celiac disease antibody levels may be fluctuating thus endoscopy with biopsy should be done in these patients. 6. In children lower than 2years, Anti- TTG and Anti –EMA have low sensitivity. we recommend Anti-TTG and Anti-DGP in these patients. 7. Resolution of symptoms after gluten free diet is not necessarily a feature of celiac disease. This condition may be seen in patients with IBS or non-celiac gluten sensitivity.

  6. Searching for the elusive typhoid diagnostic

    Directory of Open Access Journals (Sweden)

    Baker Stephen


    Full Text Available Abstract Typhoid (enteric fever is still a common disease in many developing countries but current diagnostic tests are inadequate. Studies on pathogenesis and genomics have provided new insight into the organisms that cause enteric fever. Better understanding of the microorganisms explains, in part, why our current typhoid methodologies are limited in their diagnostic information and why developing new strategies may be a considerable challenge. Here we discuss the current position of typhoid diagnostics, highlight the need for technological improvements and suggest potential ways of advancing this area.

  7. Collected abstracts on particle beam diagnostic systems

    Energy Technology Data Exchange (ETDEWEB)

    Hickok, R.L.


    This report contains a compilation of abstracts on work related to particle beam diagnostics for high temperature plasmas. The abstracts were gathered in early 1978 and represent the status of the various programs as of that date. It is not suggested that this is a comprehensive list of all the work that is going on in the development of particle beam diagnostics, but it does provide a representative view of the work in this field. For example, no abstracts were received from the U.S.S.R. even though they have considerable activity in particle beam diagnostics.

  8. Chronic Meningitis: Simplifying a Diagnostic Challenge. (United States)

    Baldwin, Kelly; Whiting, Chris


    Chronic meningitis can be a diagnostic dilemma for even the most experienced clinician. Many times, the differential diagnosis is broad and encompasses autoimmune, neoplastic, and infectious etiologies. This review will focus on a general approach to chronic meningitis to simplify the diagnostic challenges many clinicians face. The article will also review the most common etiologies of chronic meningitis in some detail including clinical presentation, diagnostic testing, treatment, and outcomes. By using a case-based approach, we will focus on the key elements of clinical presentation and laboratory analysis that will yield the most rapid and accurate diagnosis in these complicated cases.

  9. Polymeric micelles as carriers of diagnostic agents. (United States)



    This review deals with diagnostic applications of polymeric micelles composed of amphiphilic block-copolymers. In aqueous solutions these polymers spontaneously form particles with diameter 20-100 nm. A variety of diagnostic moieties can be incorporated covalently or non-covalently into the particulates with high loads. Resulting particles can be used as particulate agents for diagnostic imaging using three major imaging modalities: gamma-scintigraphy, magnetic resonance imaging and computed tomography. The use of polyethyleneoxide-diacyllipid micelles loaded with chelated (111)In/Gd(3+) as well as iodine-containing amphiphilic copolymer in percutaneous lymphography and blood pool/liver imaging are discussed as specific examples.

  10. National NIF Diagnostic Program Interim Management Plan

    Energy Technology Data Exchange (ETDEWEB)

    Warner, B


    The National Ignition Facility (NIF) has the mission of supporting Stockpile Stewardship and Basic Science research in high-energy-density plasmas. To execute those missions, the facility must provide diagnostic instrumentation capable of observing and resolving in time events and radiation emissions characteristic of the plasmas of interest. The diagnostic instrumentation must conform to high standards of operability and reliability within the NIF environment. These exacting standards, together with the facility mission of supporting a diverse user base, has led to the need for a central organization charged with delivering diagnostic capability to the NIF. The National NIF Diagnostics Program (NNDP) has been set up under the aegis of the NIF Director to provide that organization authority and accountability to the wide user community for NIF. The funds necessary to perform the work of developing diagnostics for NIF will be allocated from the National NIF Diagnostics Program to the participating laboratories and organizations. The participating laboratories and organizations will design, build, and commission the diagnostics for NIF. Restricted availability of funding has had an adverse impact, unforeseen at the time of the original decision to projectize NIF Core Diagnostics Systems and Cryogenic Target Handing Systems, on the planning and initiation of these efforts. The purpose of this document is to provide an interim project management plan describing the organizational structure and management processes currently in place for NIF Core Diagnostics Systems. Preparation of a Program Execution Plan for NIF Core Diagnostics Systems has been initiated and a current draft is provided as Attachment 1 to this document. The National NIF Diagnostics Program Interim Management Plan provides a summary of primary design criteria and functional requirements, current organizational structure, tracking and reporting procedures, and current planning estimates of project scope

  11. Diagnostic criteria of autoimmune hepatitis. (United States)

    Liberal, Rodrigo; Grant, Charlotte R; Longhi, Maria Serena; Mieli-Vergani, Giorgina; Vergani, Diego


    Autoimmune hepatitis (AIH) is a chronic immune-mediated liver disorder characterised by female preponderance, elevated transaminase and immunoglobulin G levels, seropositivity for autoantibodies and interface hepatitis. Presentation is highly variable, therefore AIH should be considered during the diagnostic workup of any increase in liver enzyme levels. A set of inclusion and exclusion criteria for the diagnosis of AIH have been established by the International Autoimmune Hepatitis Group (IAIHG). There are two main types of AIH: type 1, positive for anti-nuclear (ANA) and/or anti-smooth muscle antibodies (SMAs) and type 2, defined by the presence of anti-liver kidney microsomal antibody type 1 (LKM-1) and/or anti-liver cytosol type 1 (LC-1) autoantibodies. The central role of autoantibodies in the diagnosis of AIH has led the IAIHG to produce a consensus statement detailing appropriate and effective methods for their detection. Autoantibodies should be tested by indirect immunofluorescence at an initial dilution of 1/40 in adults and 1/10 in children on a freshly prepared rodent substrate that includes kidney, liver and stomach sections to allow for the simultaneous detection of all reactivities relevant to AIH. Anti-LKM-1 is often confused with anti-mitochondrial antibody (AMA) if rodent kidney is used as the sole immunofluorescence substrate. The identification of the molecular targets of anti-LKM-1 and AMA has led to the establishment of immuno-assays based on the use of the recombinant or purified autoantigens. Perinuclear anti-nuclear neutrophil antibody (p-ANNA) is an additional marker of AIH-1; anti soluble liver antigen (SLA) antibodies are specific for autoimmune liver disease, can be present in AIH-1 and AIH-2 and are associated with a more severe clinical course. Anti-SLA are detectable by ELISA or radio-immuno-assays, but not by immunofluorescence. AIH is exquisitely responsive to immunosuppressive treatment, which should be instituted promptly to

  12. Insulation Degradation Diagnostic Technology to XLPE Cable (United States)

    Uchida, Katsumi; Okamoto, Tatsuki

    XLPE power cables have been in use for long time now and some of them are approaching 30 years of their designed lifetime. However, under the present severe economic situation, it is necessary to use the existing apparatus as long as possible. So, degradation diagnostic methods with high reliability are needed earnestly. This paper explains the insulation degradation mechanism of XLPE power cables and the newest diagnostic methods on each voltage class. In 3.3-77kV classes, fairly high extents of the cable accidents depend on the water-tree degradation. For 3.3-6.6kV class cables, on-line diagnostic methods with high reliability to detect bridged water-trees have been developed and put in practical use. And, for 22-77kV classes, two promising off-line diagnostic methods to detect un-bridged water-trees have begun to carry out in on-site tests.

  13. Vibration diagnostics of weak base embankments

    Institute of Scientific and Technical Information of China (English)

    Evgenij Ashpiz; Vladimir Kapustin; Svetlana Klepikova; Maxim Shirobokov


    In this paper the theoretical background was analyzed for vibration diagnostics method and experience in its application for weak base embankments. General schemes of survey and recommendations on hardware systems and further prospective development are outlined.

  14. Painful Shoulder in Swimmers: A Diagnostic Challenge. (United States)

    McMaster, William C.


    This article discusses the incidence, diagnosis, and treatment of painful shoulder in swimmers, including: regional problems that can cause shoulder pain; physical, clinical, and laboratory tests for diagnostic use; and approaches to management of the problem. (Author/CB)

  15. Fusion Enhanced Vehicle Level Diagnostic System Project (United States)

    National Aeronautics and Space Administration — Global Technology Connection, Inc. in conjunction with its partner, Vanderbilt University, is proposing to build a Fusion-enhanced Vehicle Diagnostics System (FVDS)...

  16. Revised Diagnostic Diagrams for Planetary Nebulae

    CERN Document Server

    Riesgo, H


    Diagnostic diagrams of electron density - excitation for a sample of 613 planetary nebulae are presented. The present extensive sample allows the definition of new statistical limits for the distribution of planetary nebulae in the log [Ha/[SII

  17. Selected Diagnostics for Microgravity Combustion Science (United States)

    Greenberg, Paul S.; Weiland, Karen J.; Griffin, DeVon W.; Yanis, William


    The evolution of our understanding of combustion phenomena occurring under reduced gravity conditions poses a continuing demand for diagnostic tools of increased sophistication. Existing methods have become insufficient to keep pace with emerging refinements in the underlying theories and predictive models. The coupling of physical mechanisms inherent in combustion systems mandates the simultaneous determination of numerous thermophysical quantities, principally temperature, velocity, and species concentrations. Observed differences attributable to variations in experimental configuration, fuels and diluents, and initial conditions enhance the difficulty of developing diagnostic methods suitable for the acquisition of the required data over the desired range of experimental parameters. Efforts to provide for ongoing diagnostic development supporting microgravity combustion science experiments are conducted within the Microgravity Science Division at the NASA-Lewis Research Center. The following describes recent results from several diagnostic development efforts conducted under this project.

  18. Systematic reviews of diagnostic test accuracy. (United States)

    Leeflang, Mariska M G; Deeks, Jonathan J; Gatsonis, Constantine; Bossuyt, Patrick M M


    More and more systematic reviews of diagnostic test accuracy studies are being published, but they can be methodologically challenging. In this paper, the authors present some of the recent developments in the methodology for conducting systematic reviews of diagnostic test accuracy studies. Restrictive electronic search filters are discouraged, as is the use of summary quality scores. Methods for meta-analysis should take into account the paired nature of the estimates and their dependence on threshold. Authors of these reviews are advised to use the hierarchical summary receiver-operating characteristic or the bivariate model for the data analysis. Challenges that remain are the poor reporting of original diagnostic test accuracy studies and difficulties with the interpretation of the results of diagnostic test accuracy research.

  19. Ares I-X Ground Diagnostic Prototype (United States)

    National Aeronautics and Space Administration — The automation of pre-launch diagnostics for launch vehicles offers three potential benefits: improving safety, reducing cost, and reducing launch delays. The Ares...

  20. Diagnostic and Prognostic Biomarkers in Melanoma (United States)

    Leininger, Jennifer; Hamby, Carl; Safai, Bijan


    Melanoma is a lethal melanocytic neoplasm. Unfortunately, the histological diagnosis can be difficult at times. Distinguishing ambiguous melanocytic neoplasms that are benign nevi from those that represent true melanoma is important both for treatment and prognosis. Diagnostic biomarkers currently used to assist in the diagnosis of melanoma are usually specific only for melanocytic neoplasms and not necessarily for their ability to metastasize. Traditional prognostic biomarkers include depth of invasion and mitotic count. Newer diagnostic and prognostic biomarkers utilize immunohistochemical staining as well as ribonucleic acid, micro-ribonucleic acid, and deoxyribonucleic acid assays and fluorescence in situ hybridization. Improved diagnostic and prognostic biomarkers are of increasing importance in the treatment of melanoma with the development of newer and more targeted therapies. Herein, the authors review many of the common as well as newer diagnostic and prognostic biomarkers used in melanoma. PMID:25013535

  1. Noninvasive Medical Diagnostics & Treatment Using Ultrasonics (United States)

    Bar-Cohen, Y.; Siegel, R.; Grandia, W.


    In parallel to the industrial application of NDE to flaw detection and material property determination, the medical community has succesfully adapted such methods to the noninvasaive diagnostics and treatment of many conditions and disorders of the human body.

  2. Fuel-motion diagnostics and cineradiography

    Energy Technology Data Exchange (ETDEWEB)

    DeVolpi, A.


    Nuclear and non-nuclear applications of cineradiography are reviewed, with emphasis on diagnostic instrumentation for in-pile transient-reactor safety testing of nuclear fuel motion. The primary instrument for this purpose has been the fast-neutron hodoscope, which has achieved quantitative monitoring of time, location, mass, and velocity of fuel movement under the difficult conditions associated with transient-reactor experiments. Alternative diagnostic devices that have been developed have not matched the capabilities of the hodoscope. Other applications for the fuel-motion diagnostic apparatus are also evolving, including time-integrated radiography and direct time- and space-resolved fuel-pin power monitoring. Although only two reactors are now actively equipped with high-resolution fuel-motion diagnostic systems, studies and tests have been carried out in and for many other reactors.

  3. Sensitive Diagnostics for Chemically Reacting Flows

    KAUST Repository

    Farooq, Aamir


    This talk will feature latest diagnostic developments for sensitive detection of gas temperature and important combustion species. Advanced optical strategies, such as intrapulse chirping, wavelength modulation, and cavity ringdown are employed.

  4. Practical Diagnostics for Evaluating Residential Commissioning Metrics

    Energy Technology Data Exchange (ETDEWEB)

    Wray, Craig; Walker, Iain; Siegel, Jeff; Sherman, Max


    In this report, we identify and describe 24 practical diagnostics that are ready now to evaluate residential commissioning metrics, and that we expect to include in the commissioning guide. Our discussion in the main body of this report is limited to existing diagnostics in areas of particular concern with significant interactions: envelope and HVAC systems. These areas include insulation quality, windows, airtightness, envelope moisture, fan and duct system airflows, duct leakage, cooling equipment charge, and combustion appliance backdrafting with spillage. Appendix C describes the 83 other diagnostics that we have examined in the course of this project, but that are not ready or are inappropriate for residential commissioning. Combined with Appendix B, Table 1 in the main body of the report summarizes the advantages and disadvantages of all 107 diagnostics. We first describe what residential commissioning is, its characteristic elements, and how one might structure its process. Our intent in this discussion is to formulate and clarify these issues, but is largely preliminary because such a practice does not yet exist. Subsequent sections of the report describe metrics one can use in residential commissioning, along with the consolidated set of 24 practical diagnostics that the building industry can use now to evaluate them. Where possible, we also discuss the accuracy and usability of diagnostics, based on recent laboratory work and field studies by LBNL staff and others in more than 100 houses. These studies concentrate on evaluating diagnostics in the following four areas: the DeltaQ duct leakage test, air-handler airflow tests, supply and return grille airflow tests, and refrigerant charge tests. Appendix A describes those efforts in detail. In addition, where possible, we identify the costs to purchase diagnostic equipment and the amount of time required to conduct the diagnostics. Table 1 summarizes these data. Individual equipment costs for the 24

  5. Intelligent systems in technical and medical diagnostics

    CERN Document Server

    Korbicz, Jozef


    For many years technical and medical diagnostics has been the area of intensive scientific research. It covers well-established topics as well as emerging developments in control engineering, artificial intelligence, applied mathematics, pattern recognition and statistics. At the same time, a growing number of applications of different fault diagnosis methods, especially in electrical, mechanical, chemical and medical engineering, is being observed. This monograph contains a collection of 44 carefully selected papers contributed by experts in technical and medical diagnostics, and constitutes

  6. Survey of Diagnostic Techniques for Dynamic Components (United States)


    sensitive to torque fluctuations (19). • NP4 deteriorates as the severity of the damage increases on multiple gear teeth (25). • Crest factor indicates...diagnostic techniques that have been developed for dynamic components such as bearings and gears . There has been a tremendous amount of research... Gears 13  3.1  Diagnostic or Signal Enhancement Techniques ............................................................14  3.2  Time Synchronous Average

  7. [Spigelian hernia: clinical, diagnostic and therapeutical aspects]. (United States)

    Versaci, A; Rossitto, M; Centorrino, T; Barbera, A; Fonti, M T; Broccio, M; Ciccolo, A


    The Authors describing a case of Spigelian hernia observed point out clinical, diagnostic and therapeutic considerations about this rare pathology of abdominal wall. They specify the anatomic characteristics of the region and underline as any diagnostic difficulties are by passed by use of USG and TC imaging for formulation of correct preoperative diagnosis. They confirm as surgical treatment by a correct access isn't different by a normal hernioplasty and guarantee the long term surgical outcome.

  8. Respiratory Disease: Diagnostic Approaches in the Horse. (United States)

    Hewson, Joanne; Arroyo, Luis G


    Evaluation of the upper and lower respiratory tract of horses requires strategic selection of possible diagnostic tests based on location of suspected pathologic lesions and purpose of testing and must also include consideration of patient status. This article discusses the various diagnostic modalities that may be applied to the respiratory system of horses under field conditions, indications for use, and aspects of sample collection, handling, and laboratory processing that can impact test results and ultimately a successful diagnosis in cases of respiratory disease.

  9. [Recent development of microfluidic diagnostic technologies]. (United States)

    Li, Haifang; Zhang, Qianyun; Lin, Jin-Ming


    Microfluidic devices exhibit a great promising development in clinical diagnosis and disease screening due to their advantages of precise controlling of fluid flow, requirement of miniamount sample, rapid reaction speed and convenient integration. In this paper, the improvements of microfluidic diagnostic technologies in recent years are reviewed. The applications and developments of on-chip disease marker detection, microfluidic cell selection and cell drug metabolism, and diagnostic micro-devices are discussed.

  10. Reliability and diagnostic of modular systems

    Directory of Open Access Journals (Sweden)

    J. Kohlas


    Full Text Available Reliability and diagnostic are in general two problems discussed separately. Yet the two problems are in fact closely related to each other. Here, this relation is considered in the simple case of modular systems. We show, how the computation of reliability and diagnostic can efficiently be done within the same Bayesian network induced by the modularity of the structure function of the system.

  11. Observations and diagnostics in high brightness beams

    Energy Technology Data Exchange (ETDEWEB)

    Cianchi, A., E-mail: [University of Rome Tor Vergata and INFN-Roma Tor Vergata, Via della Ricerca Scientifica 1, 00133 Rome (Italy); Anania, M.P.; Bisesto, F.; Castellano, M.; Chiadroni, E.; Pompili, R.; Shpakov, V. [INFN-LNF, Via Enrico Fermi 40, 00044 Frascati (Italy)


    The brightness is a figure of merit largely used in the light sources, like FEL (Free Electron Lasers), but it is also fundamental in several other applications, as for instance Compton backscattering sources, beam driven plasma accelerators and THz sources. Advanced diagnostics are essential tools in the development of high brightness beams. 6D electron beam diagnostics will be reviewed with emphasis on emittance measurement.

  12. Human Vision Pathology Diagnostics by Photogrammetrics Means (United States)

    Murynin, A.; Knyaz, V.; Mateev, I.


    One of the reasons of such vision pathology as human stereoscopic vision capability dysfunction is an asymmetry of a human face. As a rule, such dysfunctions occur as early as in the babyhood, when diagnostic methods applied for adults are ineffective. Early diagnostics and prophylaxis could help in treatment of such pathology and face 3D modeling is one of the promising ways to solve this problem.

  13. Applying Diagnostics to Enhance Cable System Reliability (Cable Diagnostic Focused Initiative, Phase II)

    Energy Technology Data Exchange (ETDEWEB)

    Hartlein, Rick [Georgia Tech Research Corporation (GTRC), Atlanta, GA (United States). National Electric Energy Testing, Research and Applications Center (NEETRAC); Hampton, Nigel [Georgia Tech Research Corporation (GTRC), Atlanta, GA (United States). National Electric Energy Testing, Research and Applications Center (NEETRAC); Perkel, Josh [Georgia Tech Research Corporation (GTRC), Atlanta, GA (United States). National Electric Energy Testing, Research and Applications Center (NEETRAC); Hernandez, JC [Univ. de Los Andes, Merida (Venezuela); Elledge, Stacy [Georgia Tech Research Corporation (GTRC), Atlanta, GA (United States). National Electric Energy Testing, Research and Applications Center (NEETRAC); del Valle, Yamille [Georgia Tech Research Corporation (GTRC), Atlanta, GA (United States). National Electric Energy Testing, Research and Applications Center (NEETRAC); Grimaldo, Jose [Georgia Inst. of Technology, Atlanta, GA (United States). School of Electrical and Computer Engineering; Deku, Kodzo [Georgia Inst. of Technology, Atlanta, GA (United States). George W. Woodruff School of Mechanical Engineering


    The Cable Diagnostic Focused Initiative (CDFI) played a significant and powerful role in clarifying the concerns and understanding the benefits of performing diagnostic tests on underground power cable systems. This project focused on the medium and high voltage cable systems used in utility transmission and distribution (T&D) systems. While many of the analysis techniques and interpretations are applicable to diagnostics and cable systems outside of T&D, areas such as generating stations (nuclear, coal, wind, etc.) and other industrial environments were not the focus. Many large utilities in North America now deploy diagnostics or have changed their diagnostic testing approach as a result of this project. Previous to the CDFI, different diagnostic technology providers individually promoted their approach as the “the best” or “the only” means of detecting cable system defects.

  14. IR thermography diagnostics for the WEST project

    Energy Technology Data Exchange (ETDEWEB)

    Courtois, X., E-mail: [CEA, IRFM, F-13108 Saint-Paul-Lez-Durance (France); Aumeunier, M.H. [OPTIS, ZE de La Farlède, F-83078 Toulon Cedex 9 (France); Joanny, M.; Roche, H.; Micolon, F.; Salasca, S.; Balorin, C.; Jouve, M. [CEA, IRFM, F-13108 Saint-Paul-Lez-Durance (France)


    Highlights: • The WEST project requires a set of three infrared diagnostics. • The tungsten divertor will be monitored by the existing diagnostic renewed. • The antennas monitoring require the development of an innovative diagnostic. • A fiber bundle will be used as image transport for the antennas monitoring. • A wide angle tangential view of the upper divertor and the first wall is studied. - Abstract: To operate long plasma discharge in tokamak equipped with actively cooled plasma facing components (PFC), infrared (IR) thermography is a key diagnostic. Indeed IR data are used for both PFC safety monitoring, to avoid material degradation and water leak, and various physics studies on plasma-wall interaction. The IR monitoring is becoming even more crucial with today metallic PFCs. This is the case for the WEST project (Tungsten (W) Environment for Steady State Tokamak), which aims at installing a W divertor in Tore Supra (TS), in order to operate the 1st tokamak with a full W actively cooled divertor in long plasma discharges. The IR thermography system for the WEST project described in this paper will consist of a set of 3 different diagnostics: (1) Six cameras located in upper ports viewing the full W divertor, which reuse a part of the existing diagnostic of TS. (2) Five novel views located behind the inner protection panels for the antennas monitoring, based on an innovative imaging fibers bundle technology. (3) A tangential wide angle view located in a median port, for the upper divertor and first wall monitoring.

  15. Nanotechnology based diagnostics for neurological disorders

    Energy Technology Data Exchange (ETDEWEB)

    Kurek, Nicholas S.; Chandra, Sathees B., E-mail: [Department of Biological, Chemical and Physical Sciences, Roosevelt University, Chicago, IL (United States)


    Nanotechnology involves probing and manipulating matter at the molecular level. Nanotechnology based molecular diagnostics have the potential to alleviate the suffering caused by many diseases, including neurological disorders, due to the unique properties of nanomaterials. Most neurological illnesses are multifactorial conditions and many of these are also classified as neurobehavioral disorders. Alzheimer's disease, Parkinson's disease, Huntington disease, cerebral ischemia, epilepsy, schizophrenia and autism spectrum disorders like Rett syndrome are some examples of neurological disorders that could be better treated, diagnosed, prevented and possibly cured using nanotechnology. In order to improve the quality of life for disease afflicted people, a wide range of nanomaterials that include gold and silica nanoparticles, quantum dots and DNA along with countless other forms of nanotechnology have been investigated regarding their usefulness in advancing molecular diagnostics. Other small scaled materials like viruses and proteins also have potential for use as molecular diagnostic tools. Information obtained from nanotechnology based diagnostics can be stored and manipulated using bioinformatics software. More advanced nanotechnology based diagnostic procedures for the acquisition of even greater proteomic and genomic knowledge can then be developed along with better ways to fight various diseases. Nanotechnology also has numerous applications besides those related to biotechnology and medicine. In this article, we will discuss and analyze many novel nanotechnology based diagnostic techniques at our disposal today. (author)

  16. Marketing diagnostics in consumer cooperatives trade enterprises

    Directory of Open Access Journals (Sweden)

    O.S. Krivoruchko


    Full Text Available The aim of the article. The article highlights main demands and levels of realization of consumer cooperatives trade enterprises` diagnostics.We demonstrate the chain of marketing diagnostics; we offer the model of diagnostics process of marketing problems (opportunities of cooperative trade enterprises.The results of the analysis. Marketing diagnostics is one of the marketing researches directions, which is matching of the researched object characteristics with comparison base for definition of objects quality condition (its diagnosis. In the context of our research marketing diagnostics should be considered as separate technological module which enables to form development backgrounds of competitive marketing strategies of consumer cooperatives trade enterprises according to conditions of inner and outer environment.We consider the following demands of marketing diagnostics conduction of consumer cooperatives trade enterprises: authenticity, objectivity, accuracy, resultativity, systematicness, sequence, scientific foundation, flexibility, timeliness, effectiveness, validity.Strategic diagnostics is the direction of researches that is responsible for receiving the information, necessary for further functioning of the enterprise. We should refer to comparative researches of strategic state of enterprise economy portfolio, enterprise competitiveness estimation, and enterprise activity threats and possibilities definition.Tactical diagnostics is the researches direction which forms information for plans programs development. Economy interest matching of marketing activity participants in this situation gains special meaning. Due to this meaning the procedures of their sequence can be developed. We consider the mentioned type of diagnostics to be insufficiently researched nowadays, though it has significant importance in the process of managerial decisions supporting and making.The main task of operative diagnostics is defining deviation borders

  17. Diagnostics for FIRE: A status report (United States)

    Young, Kenneth M.


    The mission for the proposed fusion ignition research experiment (FIRE) device is to "attain, explore, understand and optimize fusion-dominated plasmas." Operation at Q⩾5, for 20 s with a fusion power output of ˜150 MW is the major goal. Attaining this mission sets demands for plasma measurement which are at least as comprehensive as on present tokamaks, with the additional capabilities needed for control of the plasma and for understanding the effects of the alpha-particles. Because of the planned operation in advanced tokamak scenarios, with steep transport barriers, the diagnostic instrumentation must be able to provide fine spatial and temporal resolution. It must also be able to withstand the impact of the intense neutron and gamma irradiation. There are practical engineering issues of minimizing radiation streaming while providing essential diagnostic access to the plasma. Many components will operate close to the first wall, e.g., ceramics and mineral insulated cable for magnetic diagnostics and mirrors for optical diagnostics; these components must be selected and mounted so that they will operate and survive in fluxes which require special material selection. The measurement requirements have been assessed so that the diagnostics for the FIRE device can be defined. Clearly, a better set of diagnostics of alpha-particles than that available for TFTR is essential, since the alpha-particles provide the dominant sources of heating and of instability-drive in the plasma.

  18. Clinically based diagnostic wax-up for optimal esthetics: the diagnostic mock-up. (United States)

    Simon, Harel; Magne, Pascal


    A diagnostic wax-up can enhance the predictability of treatment by modeling the desired result in wax prior to treatment. It is critical to correlate the wax-up to the patient to avoid a result that appears optimal on the casts but does not correspond to the patient's smile. This article reviews the applications and techniques for clinically based diagnostic wax-up, and focuses on the diagnostic mock-up philosophy as a means to obtain predictable esthetics and function.

  19. Structure-activity study of pentamidine analogues as antiprotozoal agents. (United States)

    Bakunova, Svetlana M; Bakunov, Stanislav A; Patrick, Donald A; Kumar, E V K Suresh; Ohemeng, Kwasi A; Bridges, Arlene S; Wenzler, Tanja; Barszcz, Todd; Jones, Susan Kilgore; Werbovetz, Karl A; Brun, Reto; Tidwell, Richard R


    Diamidine 1 (pentamidine) and 65 analogues (2-66) have been tested for in vitro antiprotozoal activities against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani, and for cytotoxicity against mammalian cells. Dications 32, 64, and 66 exhibited antitrypanosomal potencies equal or greater than melarsoprol (IC(50) = 4 nM). Nine congeners (2-4, 12, 27, 30, and 64-66) were more active against P. falciparum than artemisinin (IC(50) = 6 nM). Eight compounds (12, 32, 33, 44, 59, 62, 64, and 66) exhibited equal or better antileishmanial activities than 1 (IC(50) = 1.8 microM). Several congeners were more active than 1 in vivo, curing at least 2/4 infected animals in the acute mouse model of trypanosomiasis. The diimidazoline 66 was the most promising compound in the series, showing excellent in vitro activities and high selectivities against T. b. rhodesiense, P. falciparum, and L. donovani combined with high antitrypanosomal efficacy in vivo.

  20. Fusion neutron diagnostics on ITER tokamak (United States)

    Bertalot, L.; Barnsley, R.; Direz, M. F.; Drevon, J. M.; Encheva, A.; Jakhar, S.; Kashchuk, Y.; Patel, K. M.; Arumugam, A. P.; Udintsev, V.; Walker, C.; Walsh, M.


    ITER is an experimental nuclear reactor, aiming to demonstrate the feasibility of nuclear fusion realization in order to use it as a new source of energy. ITER is a plasma device (tokamak type) which will be equipped with a set of plasma diagnostic tools to satisfy three key requirements: machine protection, plasma control and physics studies by measuring about 100 different parameters. ITER diagnostic equipment is integrated in several ports at upper, equatorial and divertor levels as well internally in many vacuum vessel locations. The Diagnostic Systems will be procured from ITER Members (Japan, Russia, India, United States, Japan, Korea and European Union) mainly with the supporting structures in the ports. The various diagnostics will be challenged by high nuclear radiation and electromagnetic fields as well by severe environmental conditions (ultra high vacuum, high thermal loads). Several neutron systems with different sensitivities are foreseen to measure ITER expected neutron emission from 1014 up to almost 1021 n/s. The measurement of total neutron emissivity is performed by means of Neutron Flux Monitors (NFM) installed in diagnostic ports and by Divertor Neutron Flux Monitors (DNFM) plus MicroFission Chambers (MFC) located inside the vacuum vessel. The neutron emission profile is measured with radial and vertical neutron cameras. Spectroscopy is accomplished with spectrometers looking particularly at 2.5 and 14 MeV neutron energy. Neutron Activation System (NAS), with irradiation ends inside the vacuum vessel, provide neutron yield data. A calibration strategy of the neutron diagnostics has been developed foreseeing in situ and cross calibration campaigns. An overview of ITER neutron diagnostic systems and of the associated challenging engineering and integration issues will be reported.

  1. Microwave diagnostics of atmospheric plasmas (United States)

    Scott, David

    Plasma treatment of biological tissues has tremendous potential due to the wide range of applications. Most plasmas have gas temperatures which greatly exceed room temperature. These are often utilized in electro-surgery for cutting and coagulating tissue. Another type of plasma, referred to as cold atmospheric plasma, or CAP, is characterized by heavy particle temperatures which are at or near room temperature. Due to this lack of thermal effect, CAP may provide less invasive medical procedures. Additionally, CAP have been demonstrated to be effective at targeting cancer cells while minimizing damage to the surrounding tissue. A recently fabricated Microwave Electron Density Device (MEDD) utilizes microwave scattering on small atmospheric plasmas to determine the electron plasma density. The MEDD can be utilized on plasmas which range from a fraction of a millimeter to several centimeters at atmospheric pressure when traditional methods cannot be applied. Microwave interferometry fails due to the small size of the plasma relative to the microwave wavelength which leads to diffraction and negligible phase change; electrostatic probes introduce very strong perturbation and are associated with difficulties of application in strongly-collisional atmospheric conditions; and laser Thomson scattering is not sensitive enough to measure plasma densities less than 1012 cm-3. The first part of this dissertation provides an overview of two types of small atmospheric plasma objects namely CAPs and plasmas utilized in the electro-surgery. It then goes on to describe the fabrication, testing and calibration of the MEDD facility. The second part of this dissertation is focused on the application of the MEDD and other diagnostic techniques to both plasma objects. A series of plasma images that illustrate the temporal evolution of a discharge created by an argon electrosurgical device operating in the coagulation mode and its behavior was analyzed. The discharge of the argon

  2. Diagnostic criteria for headache attributed to temporomandibular disorders

    DEFF Research Database (Denmark)

    Schiffman, Eric; Ohrbach, Richard; List, Thomas;


    We assessed and compared the diagnostic accuracy of two sets of diagnostic criteria for headache secondary to temporomandibular disorders (TMD).......We assessed and compared the diagnostic accuracy of two sets of diagnostic criteria for headache secondary to temporomandibular disorders (TMD)....

  3. Diagnostic devices for osteoporosis in the general population

    DEFF Research Database (Denmark)

    Høiberg, M P; Rubin, Katrine Hass; Hermann, Pernille


    INTRODUCTION: A diagnostic gap exists in the current dual photon X-ray absorptiometry (DXA) based diagnostic approach to osteoporosis. Other diagnostic devices have been developed, but no comprehensive review concerning the applicability of these diagnostic devices for population-based screening...

  4. TPX diagnostics for tokamak operation, plasma control and machine protection

    Energy Technology Data Exchange (ETDEWEB)

    Edmonds, P.H. [Texas Univ., Austin, TX (United States). Fusion Research Center; Medley, S.S.; Young, K.M. [Princeton Univ., NJ (United States). Plasma Physics Lab.] [and others


    The diagnostics for TPX are at an early design phase, with emphasis on the diagnostic access interface with the major tokamak components. Account has to be taken of the very severe environment for diagnostic components located inside the vacuum vessel. The placement of subcontracts for the design and fabrication of the diagnostic systems is in process.

  5. Transcriptome marker diagnostics using big data. (United States)

    Han, Henry; Liu, Ying


    The big omics data are challenging translational bioinformatics in an unprecedented way for its complexities and volumes. How to employ big omics data to achieve a rivalling-clinical, reproducible disease diagnosis from a systems approach is an urgent problem to be solved in translational bioinformatics and machine learning. In this study, the authors propose a novel transcriptome marker diagnosis to tackle this problem using big RNA-seq data by viewing whole transcriptome as a profile marker systematically. The systems diagnosis not only avoids the reproducibility issue of the existing gene-/network-marker-based diagnostic methods, but also achieves rivalling-clinical diagnostic results by extracting true signals from big RNA-seq data. Their method demonstrates a better fit for personalised diagnostics by attaining exceptional diagnostic performance via using systems information than its competitive methods and prepares itself as a good candidate for clinical usage. To the best of their knowledge, it is the first study on this topic and will inspire the more investigations in big omics data diagnostics.

  6. ISTTOK plasma control with the tomography diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, H.; Caralho, P.J.; Duarte, P.; Pereira, T.; Coelho, R.; Silva, C. [Association Euratom/IST, Institute of Plasmas and Nuclear Fusion, Technology Graduate Institute, P-1049-001 Lisbon (Portugal)


    A real-time plasma position control system is mandatory to achieve long duration (up to 250 ms), Alternating Current (AC) discharges on the ISTTOK tokamak. Such a system has been used for some time supported only on magnetic field diagnostic data. However, this system does not function accurately when the plasma current is low, rendering it inoperative during the plasma current reversal. A tomography diagnostic with 3 pinhole cameras and 8 silicone photodiode channels per camera was installed and customized to supply alternative plasma position to be used for plasma position control. As no filtering is applied, most of the radiation detected is in the visible/near-UV range. This system (i) executes a tomographic reconstruction, (ii) determines the average emissivity position from it, (iii) calculates the shift from the required position and (iv) supplies the vertical field power supply unit with the desired current value, all in less than 100 {mu}s. The horizontal magnetic field power supply unit is expected to be included in the system and will have no impact in the process time. This paper presents the tomography diagnostic architecture together with results of its scientific exploitation in ISTTOK AC discharges, where it has proven to be capable of supplying an accurate plasma position during the current reversal. The use of the tomography diagnostic for plasma position overcomes some limitations of the magnetic diagnostics, but poses challenges of its own such as blindness to plasma current direction. (authors)

  7. Diagnostics of the Enterprise Export Potential

    Directory of Open Access Journals (Sweden)

    Skrynkovskyy Ruslan M.


    Full Text Available The aim of the article is to form and develop theoretical and methodological foundations for diagnostics of the enterprise export potential, which should be understood as a process of identification, analysis and assessment of the level of potential possibility and readiness of the enterprise to implement export activities taking into account the impact of interrelated internal variables (goals, technology, structure, tasks, educational and professional potential of the staff and factors of the enterprise external environment (customers, competitors, suppliers, intermediaries, contact audience, etc. in order to ensure a qualitatively new level of its development and formation of its prospects. It is determined that the key business indicators of the system for diagnostics of enterprise export potential are: the level of enterprises competitiveness in the international (global market; the level of competitiveness of enterprise products; the level of competitiveness of enterprise export products; the level of enterprise resource opportunities; the level of enterprise export performance. Prospects for further research in this direction are to develop a classification (specifically detailed list of objectives of the enterprise diagnostics by the level of detail (element, partial, complex, taking into account diagnostics of export potential of the enterprise as part of diagnostic objective.

  8. Toward theory-based diagnostic categories. (United States)

    Gordon, M


    As a social institution, nursing has a responsibility to society for the development of knowledge in the areas described by nursing diagnoses. This article focuses on the need for developing the theoretical basis of each diagnostic category. Diagnoses are viewed as summarizations of underlying conceptual models for interpreting observations, and as such, they provide a perspective for understanding and thinking about a set of clinical observations. At present many diagnostic concepts do not meet this standard and suggest primitive, pretheoretical ideas with a minimal knowledge base. The importance of having valid and reliable diagnostic categories for use in making clinical judgements and as a focus for care planning is discussed. A cycle of development is outlined in three phases: diagnostic concept identification, concept analysis-model development, and construction/reconstruction of diagnostic categories. It is suggested that a useful category captures the conceptual understanding and state of knowledge development about a phenomena in its (a) name, (b) definition, and (c) cluster of defining characteristics.


    Directory of Open Access Journals (Sweden)

    A. Yu. Galkin


    Full Text Available In article we analyzed national and international regulations concerning the quality and safety of medical devices for in vitro diagnostics. We discussed the possibility of a partial application of the recommendations of the State Pharmacopoeia of Ukraine to this type of product. The main guiding regulatory documents establishing requirements for quality and safety tools for the serological diagnosis products are The technical regulation on medical devices for the diagnosis in vitro, DSTU ISO 13485 “Medical devices. Quality management system. Regulatory requirements”, and DSTU ISO/IEC 17025 “General requirements for the competence of testing and calibration laboratories”. Similar requirements of the State Pharmacopoeia of Ukraine which are used for drug standardization can not be directly applied to the medical devises for in vitro diagnostics due to a number of features, namely, the serological diagnosis products pre-designed to determine the unknown concentration of a particular analyte in a biological material, the diagnostic kits has to include the control samples (internal standard systems that need to be calibrated. It was determined following parameters of bioanalytical standardization and validation characterization for of qualitative (semi quantitative test-kits for serological diagnosis: precision (convergence, intralaboratory precision and reproducibility, diagnostic and analytical specificity, diagnostic sensitivity. It’s necessary to inspect additional parameters for quantitative test-kits such as accuracy (precision, linearity, analytical sensitivity and range.

  10. Long distance transmission of diagnostic cardiovascular information. (United States)

    Caldwell, M A; Miles, R; Barrington, W


    Availability of advanced cardiac diagnostic procedures has proven to decrease the morbidity and mortality associated with common cardiovascular diseases. It was requested that the University of Nebraska Medical Center (UNMC) provide a rural hospital with advanced cardiology services. The services included monitoring of inpatients and rapid interpretation of cardiac diagnostic tests. UNMC proposed to solve this problem by building a computer-based system to transmit diagnostic cardiac information from a rural hospital to UNMC for interpretation by a cardiologist. The information transmitted to UNMC was grouped into two categories: 1) cardiovascular ultrasound, 2) ambulatory and 12-lead electrocardiograms. The information consisted of digital and analog waveform information, static images, and 30 fps video. The system provided rapid data transmission to UNMC over a T-1 line. The system utilized a compression technology which did not degrade the interpretation quality of the data. This system has increased the availability of advanced cardiac diagnostic services to general medical practitioners in a rural hospital. In addition, the system has significantly reduced the time and cost to transmit vital cardiac diagnostic information, thus improving the quality of care received by rural patients.

  11. Elementary School-Age Children's Capacity To Choose Positive Diagnostic and Negative Diagnostic Tests. (United States)

    Samuels, Mark C.; McDonald, John


    Two experiments compared 10-year-olds' and adults' ability to choose positive and negative diagnostic tests over positive and negative nondiagnostic tests. Findings indicated that both age groups were more likely to prefer positive diagnostic tests over positive nondiagnostic tests, although only adults showed a significant preference for negative…

  12. Defining Characteristics of Diagnostic Classification Models and the Problem of Retrofitting in Cognitive Diagnostic Assessment (United States)

    Gierl, Mark J.; Cui, Ying


    One promising application of diagnostic classification models (DCM) is in the area of cognitive diagnostic assessment in education. However, the successful application of DCM in educational testing will likely come with a price--and this price may be in the form of new test development procedures and practices required to yield data that satisfy…

  13. A diagnostic methodology for refrigerating systems; Methodologie de diagnostic des installations frigorifiques

    Energy Technology Data Exchange (ETDEWEB)

    Vrinat, G. [Association Francaise du Froid (AFF), 75 - Paris (France)


    A diagnostic methodology for refrigerating machines, equipment and plants has been defined and evaluated for EDF, the French national power utility and ADEME, the French Agency for Energy Conservation, in the framework of energy conservation objectives: the diagnostic method should enable to identify malfunctions, assess the cost efficiency of the equipment, identify limiting factors, and consider corrective measures

  14. In-vitro diagnostic devices introduction to current point-of-care diagnostic devices

    CERN Document Server

    Cheng, Chao-Min; Chen, Chien-Fu


    Addressing the origin, current status, and future development of point-of-care diagnostics, and serving to integrate knowledge and tools from Analytical Chemistry, Bioengineering, Biomaterials, and Nanotechnology, this book focusses on addressing the collective and combined needs of industry and academia (including medical schools) to effectively conduct interdisciplinary research. In addition to summarizing and detailing developed diagnostic devices, this book will attempt to point out the possible future trends of development for point-of-care diagnostics using both scientifically based research and practical engineering needs with the aim to help novices comprehensively understand the development of point-of-care diagnostics. This includes demonstrating several common but critical principles and mechanisms used in point-of-care diagnostics that address practical needs (e.g., disease or healthcare monitoring) using two well-developed examples so far: 1) blood glucose meters (via electrochemistry); and, 2) p...

  15. Trends in Laboratory Diagnostic Methods in Periodontology

    Directory of Open Access Journals (Sweden)

    Beáta Bolerázska


    Full Text Available This work presents a summary of current knowledge on the laboratory diagnosis of periodontitis. It focuses on the theoretical foundations and is supplemented with new knowledge. It subsequently describes specifically the laboratory diagnosis methods of periodontitis: the protein expression of inflammation, oral microbiology and molecular diagnostics. Periodontitis is a serious disease worldwide and its confirmed association with systemic diseases means its severity is increasing. Its laboratory diagnosis has the potential to rise to the level of clinical and diagnostic imaging. The transfer of diagnostic methods from laboratory to clinical use is increasingly used in the prevention and monitoring of the exacerbation and treatment of periodontal disease, as well as of its impact on systemic disease.

  16. Multiple diagnostic approaches to palpable breast mass

    Energy Technology Data Exchange (ETDEWEB)

    Chin, Soo Yil; Kim, Kie Hwan; Moon, Nan Mo; Kim, Yong Kyu; Jang, Ja June [Korea Cancer Center Hospital, Seoul (Korea, Republic of)


    The combination of the various diagnostic methods of palpable breast mass has improved the diagnostic accuracy. From September 1983 to August 1985 pathologically proven 85 patients with palpable breast masses examined with x-ray mammography, ultrasonography, penumomammography and aspiration cytology at Korea Cancer Center Hospital were analyzed. The diagnostic accuracies of each methods were 77.6% of mammogram, 74.1% of ultrasonogram, 90.5% of penumomammogram and 92.4% of aspiration cytology. Pneumomammograms was accomplished without difficulty or complication and depicted more clearly delineated mass with various pathognomonic findings; air-ductal pattern in fibroadenoma (90.4%) and cystosarcoma phylloides (100%), air-halo in fibrocystic disease (14.2%), fibroadenoma (100%), cystosarcoma phylloides (100%), air-cystogram in cystic type of fibrocystic disease (100%) and vaculoar pattern or irregular air collection without retained peripheral gas in carcinoma.

  17. Technical Report: Rayleigh Scattering Combustion Diagnostic

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Wyatt [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Hecht, Ethan [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)


    A laser Rayleigh scattering (LRS) temperature diagnostic was developed over 8 weeks with the goal of studying oxy-combustion of pulverized coal char in high temperature reaction environments with high concentrations of carbon dioxide. Algorithms were developed to analyze data collected from the optical diagnostic system and convert the information to temperature measurements. When completed, the diagnostic will allow for the kinetic gasification rates of the oxy-combustion reaction to be obtained, which was previously not possible since the high concentrations of high temperature CO2 consumed thermocouples that were used to measure flame temperatures inside the flow reactor where the combustion and gasification reactions occur. These kinetic rates are important for studying oxycombustion processes suitable for application as sustainable energy solutions.

  18. Remote online machine fault diagnostic system (United States)

    Pan, Min-Chun; Li, Po-Ching


    The study aims at implementing a remote online machine fault diagnostic system built up in the architecture of both the BCB software-developing environment and Internet transmission communication. Variant signal-processing computation schemes for signal analysis and pattern recognition purposes are implemented in the BCB graphical user interface. Hence, machine fault diagnostic capability can be extended by using the socket application program interface as the TCP/IP protocol. In the study, the effectiveness of the developed remote diagnostic system is validated by monitoring a transmission-element test rig. A complete monitoring cycle includes data acquisition, signal processing, feature extraction, pattern recognition through the ANNs, and online video monitoring, is demonstrated.

  19. Methodology, models and algorithms in thermographic diagnostics

    CERN Document Server

    Živčák, Jozef; Madarász, Ladislav; Rudas, Imre J


    This book presents  the methodology and techniques of  thermographic applications with focus primarily on medical thermography implemented for parametrizing the diagnostics of the human body. The first part of the book describes the basics of infrared thermography, the possibilities of thermographic diagnostics and the physical nature of thermography. The second half includes tools of intelligent engineering applied for the solving of selected applications and projects. Thermographic diagnostics was applied to problematics of paraplegia and tetraplegia and carpal tunnel syndrome (CTS). The results of the research activities were created with the cooperation of the four projects within the Ministry of Education, Science, Research and Sport of the Slovak Republic entitled Digital control of complex systems with two degrees of freedom, Progressive methods of education in the area of control and modeling of complex object oriented systems on aircraft turbocompressor engines, Center for research of control of te...

  20. Molecular Diagnostic Applications in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Laura Huth


    Full Text Available Colorectal cancer, a clinically diverse disease, is a leading cause of cancer-related death worldwide. Application of novel molecular diagnostic tests, which are summarized in this article, may lead to an improved survival of colorectal cancer patients.  Distinction of these applications is based on the different molecular principles found in colorectal cancer (CRC. Strategies for molecular analysis of single genes (as KRAS or TP53 as well as microarray based techniques are discussed. Moreover, in addition to the fecal occult blood testing (FOBT and colonoscopy some novel assays offer approaches for early detection of colorectal cancer like the multitarget stool DNA test or the blood-based Septin 9 DNA methylation test. Liquid biopsy analysis may also exhibit great diagnostic potential in CRC for monitoring developing resistance to treatment. These new diagnostic tools and the definition of molecular biomarkers in CRC will improve early detection and targeted therapy of colorectal cancer.