Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification

Serum Apolipoproteins C-I and C-III Are Reduced in Stomach Cancer Patients: Results from MALDI-Based Peptidome and Immuno-Based Clinical Assays

Finding new peptide biomarkers for stomach cancer in human sera that can be implemented into a clinically practicable prediction method for monitoring of stomach cancer. We studied the serum peptidome from two different biorepositories. We first employed a C8-reverse phase liquid chromatography approach for sample purification, followed by mass-spectrometry analysis. These were applied onto serum samples from cancer-free controls and stomach cancer patients at various clinical stages. We then created a bioinformatics analysis pipeline and identified peptide signature discriminating stomach adenocarcinoma patients from cancer-free controls. Matrix Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) results from 103 samples revealed 9 signature peptides; with prediction accuracy of 89% in the training set and 88% in the validation set. Three of the discriminating peptides discovered were fragments of Apolipoproteins C-I and C-III (apoC-I and C-III); we further quantified their serum levels, as well as CA19-9 and CRP, employing quantitative commercial-clinical assays in 142 samples. ApoC-I and apoC-III quantitative results correlated with the MS results. We then employed apoB-100-normalized apoC-I and apoC-III, CA19-9 and CRP levels to generate rules set for stomach cancer prediction. For training, we used sera from one repository, and for validation, we used sera from the second repository. Prediction accuracies of 88.4% and 74.4% were obtained in the training and validation sets, respectively. Serum levels of apoC-I and apoC-III combined with other clinical parameters can serve as a basis for the formulation of a diagnostic score for stomach cancer patients

Modeling the Time Evolution of the Nanoparticle-Protein Corona in a Body Fluid

Background: Nanoparticles in contact with biological fluids interact with proteins and other biomolecules, thus forming a dynamic corona whose composition varies over time due to continuous protein association and dissociation events. Eventually equilibrium is reached, at which point the continued exchange will not affect the composition of the corona. Results: We developed a simple and effective dynamic model of the nanoparticle protein corona in a body fluid, namely human plasma. The model predicts the time evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants. An application to the interaction of human serum albumin, high density lipoprotein (HDL) and fibrinogen with 70 nm N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles is presented, including novel experimental data for HDL. Conclusions: The simple model presented here can easily be modified to mimic the interaction of the nanoparticle protein corona with a novel biological fluid or compartment once new data will be available, thus opening novel applications in nanotoxicity and nanomedicine

Application of multiple statistical tests to enhance mass spectrometry-based biomarker discovery

<p>Abstract</p> <p>Background</p> <p>Mass spectrometry-based biomarker discovery has long been hampered by the difficulty in reconciling lists of discriminatory peaks identified by different laboratories for the same diseases studied. We describe a multi-statistical analysis procedure that combines several independent computational methods. This approach capitalizes on the strengths of each to analyze the same high-resolution mass spectral data set to discover consensus differential mass peaks that should be robust biomarkers for distinguishing between disease states.</p> <p>Results</p> <p>The proposed methodology was applied to a pilot narcolepsy study using logistic regression, hierarchical clustering, t-test, and CART. Consensus, differential mass peaks with high predictive power were identified across three of the four statistical platforms. Based on the diagnostic accuracy measures investigated, the performance of the consensus-peak model was a compromise between logistic regression and CART, which produced better models than hierarchical clustering and t-test. However, consensus peaks confer a higher level of confidence in their ability to distinguish between disease states since they do not represent peaks that are a result of biases to a particular statistical algorithm. Instead, they were selected as differential across differing data distribution assumptions, demonstrating their true discriminatory potential.</p> <p>Conclusion</p> <p>The methodology described here is applicable to any high-resolution MALDI mass spectrometry-derived data set with minimal mass drift which is essential for peak-to-peak comparison studies. Four statistical approaches with differing data distribution assumptions were applied to the same raw data set to obtain consensus peaks that were found to be statistically differential between the two groups compared. These consensus peaks demonstrated high diagnostic accuracy when used to form a predictive model as evaluated by receiver operating characteristics curve analysis. They should demonstrate a higher discriminatory ability as they are not biased to a particular algorithm. Thus, they are prime candidates for downstream identification and validation efforts.</p

Tryptophan depletion in context of the inflammatory and general nutritional status of a low-income South African HIV-infected population

MV was the project leader. PB developed and validated the GC-MS method for the analysis of tryptophan and performed the biochemical and immunological analyses. MV and PB were responsible for the project design, analyses of the results and writing of the manuscript. PL was involved in the sourcing of patients and the clinical examination of all patients.The authors wish to thank the participants and staff of the Immunology Clinic at Kalafong Hospital and the South African National Blood Service at the Pretoria West satellite site.BACKGROUND : The essential amino acid tryptophan cannot be synthesised in the body and must be acquired through dietary intake. Oxidation of tryptophan, due to immune induction of the enzyme indoleamine 2,3- dioxygenase (IDO), is considered to be the main cause of tryptophan depletion in HIV infection and AIDS. We examined plasma tryptophan levels in a low-income sub-Saharan HIV-infected population and compared it to that of developed countries. Tryptophan levels were further examined in context of the general nutritional and inflammatory status. METHODS : This cross-sectional study included 105 HIV-positive patients recruited from the Kalafong Hospital in Pretoria, South Africa, and 60 HIV-negative controls. RESULTS : Patient tryptophan levels were in general markedly lower than those reported for developed countries. In contrast to reports from developed countries that showed tryptophan levels on average to be 18.8 % lower than their control values, tryptophan levels in our study were 44.1 % lower than our controls (24.4 ± 4.1 vs. 43.6 ± 11.9 μmol/l; p < 0.001). Tryptophan levels correlated with both CD4 counts (r = 0.341; p = 0.004) and with proinflammatory activity as indicated by neopterin levels (r = −0.399; p = 0.0001). Nutritional indicators such as albumin and haemoglobin correlated positively with tryptophan and negatively with the pro-inflammatory indicators neopterin, interleukin 6 and C-reactive protein. The most probable causes of the lower tryptophan levels seen in our population are food insecurity and higher levels of inflammatory activity. CONCLUSIONS : We contend that inflammation-induced tryptophan depletion forms part of a much wider effect of pro-inflammatory activity on the nutritional profile of HIV-infected patients.This research was supported by grant funding received from the Medical Research Council of South Africa and the South African Sugar Association (SASA Project 213).http://www.jhpn.net/index.php/jhpnam2016Internal MedicinePhysiologyPsychiatr

The kynurenine pathway activities in a sub-Saharan HIV/AIDS population

BACKGROUND : Tryptophan is an essential amino acid for the synthesis of proteins and important metabolites such as serotonin, melatonin, tryptamine and niacin. After protein synthesis, more than 90 % of tryptophan catabolism occurs along the kynurenine pathway. The inflammation-inducible enzyme indoleamine 2,3 dioxygenase (IDO) is responsible for the first rate-limiting step in the kynurenine pathway, i.e., oxidation of tryptophan to kynurenine. Excessive IDO activity in conditions such as HIV/AIDS may lead to tryptophan depletion and accumulation of metabolites downstream from kynurenine. Little is known about the kynurenine pathway of HIV/AIDS patients in sub-Saharan regions. This study, in a low income sub-Saharan HIV/AIDS population, examined the effects of activities in the kynurenine pathway on plasma levels of tryptophan, kynurenine and the neurotoxin quinolinic acid, and on de novo synthesis of nicotinamide. METHODS : Plasma samples were obtained from a cohort of 105 HIV patients and 60 controls. Kynurenine pathway metabolites were analysed using gas chromatography – mass spectrometry. ELISA and flow cytometry were used to assess plasma inflammatory markers. RESULTS : IDO activity, depletion of tryptophan, as well as accumulation of kynurenine and the neurotoxin quinolinic acid, were not only significantly greater in the patients than in the controls, but also markedly greater than in HIV/AIDS patients from developed countries. Tryptophan levels were 12.3 % higher, kynurenine levels 16.2 % lower, quinolinic acid levels 43.2 % lower and nicotinamide levels 27,2 % lower in patients on antiretroviral treatment than in antiretroviral-naïve patients. Patients’ kynurenine pathway metabolites correlated with the levels of inflammatory markers, including that of the major IDO-inducer, interferon-gamma. Indications are that the rate of de novo synthesis of nicotinamide in the kynurenine pathway correlates with increases in quinolinic acid levels up to a point where saturation of the enzyme quinolinate phosphoribosyl transferase occurs. CONCLUSIONS : Higher levels of inflammatory activity in this low income sub-Saharan HIV/AIDS population than in patients from developed countries lead to greater tryptophan depletion and greater accumulation of metabolites downstream from tryptophan with quinolinic acid levels often reaching levels associated with the development of HIV/AIDS-associated neurocognitive dysfunction. De novo synthesis of nicotinamide from quinolinic acid contributes to the maintenance of nicotinamide, and by implication NAD levels, in HIV/AIDS patients from low income populations. Antiretroviral treatment partially corrects disturbances in the kynurenine pathway.Medical Research Council of South Africa and the South African Sugar Association (SASA Project 213).http://www.biomedcentral.com/bmcinfectdis/hb201