Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes

Background

Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes.

Methodology/Principal Findings

Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections.

Conclusions/Significance

These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity.     

Enzymes that hydrolyze adenine nucleotides of patients with hypercholesterolemia and inflammatory processes

The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in platelets from patients with increasing cholesterol levels. A possible association between cholesterol levels and inflammatory markers, such as oxidized low-density lipoprotein, highly sensitive C-reactive protein and oxidized low-density lipoprotein autoantibodies, was also investigated. Lipid peroxidation was estimated by measurement of thiobarbituric acid reactive substances in serum. The following groups were studied: group I, < 150 mg.dL(-1) cholesterol; group II, 151-200 mg.dL(-1) cholesterol; group III, 201-250 mg.dL(-1) cholesterol; and group IV, > 251 mg.dL(-1) cholesterol. The results demonstrated that both ATP hydrolysis and ADP hydrolysis were enhanced as a function of cholesterol level. Low-density lipoprotein levels increased concomitantly with total cholesterol levels. Triglyceride levels were increased in the groups with total cholesterol above 251 mg.dL(-1). Oxidized low-density lipoprotein levels were elevated in groups II, III, and IV. Highly sensitive C-reactive protein was elevated in the group with cholesterol levels higher than 251 mg.dL(-1). Oxidized low-density lipoprotein autoantibodies were elevated in groups III and IV. Thiobarbituric acid reactive substance content was enhanced as a function of cholesterol level. In summary, hypercholesterolemia is associated with enhancement of inflammatory response, oxidative stress, and ATP and ADP hydrolysis. The increased ATP and ADP hydrolysis in group IV was confirmed by an increase in CD39 expression on its surface. The increase in CD39 activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia.     

Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer

Background  

Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Pheno2Geno - High-throughput generation of genetic markers and maps from molecular phenotypes for crosses between inbred strains

Background

Genetic markers and maps are instrumental in quantitative trait locus (QTL) mapping in segregating populations. The resolution of QTL localization depends on the number of informative recombinations in the population and how well they are tagged by markers. Larger populations and denser marker maps are better for detecting and locating QTLs. Marker maps that are initially too sparse can be saturated or derived de novo from high-throughput omics data, (e.g. gene expression, protein or metabolite abundance). If these molecular phenotypes are affected by genetic variation due to a major QTL they will show a clear multimodal distribution. Using this information, phenotypes can be converted into genetic markers.

Results

The Pheno2Geno tool uses mixture modeling to select phenotypes and transform them into genetic markers suitable for construction and/or saturation of a genetic map. Pheno2Geno excludes candidate genetic markers that show evidence for multiple possibly epistatically interacting QTL and/or interaction with the environment, in order to provide a set of robust markers for follow-up QTL mapping.We demonstrate the use of Pheno2Geno on gene expression data of 370,000 probes in 148 A. thaliana recombinant inbred lines. Pheno2Geno is able to saturate the existing genetic map, decreasing the average distance between markers from 7.1 cM to 0.89 cM, close to the theoretical limit of 0.68 cM (with 148 individuals we expect a recombination every 100/148=0.68 cM); this pinpointed almost all of the informative recombinations in the population.

Conclusion

The Pheno2Geno package makes use of genome-wide molecular profiling and provides a tool for high-throughput de novo map construction and saturation of existing genetic maps. Processing of the showcase dataset takes less than 30 minutes on an average desktop PC. Pheno2Geno improves QTL mapping results at no additional laboratory cost and with minimum computational effort. Its results are formatted for direct use in R/qtl, the leading R package for QTL studies. Pheno2Geno is freely available on CRAN under “GNU GPL v3”. The Pheno2Geno package as well as the tutorial can also be found at: http://pheno2geno.nl.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0475-6) contains supplementary material, which is available to authorized users.     

Oxidative Stress in Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.     

The influence of some environmental factors on cytological and biometric parameters and chlorophyll content of Deschampsia antarctica Desv. in the maritime Antarctic

Under the environmental conditions of the Point Thomas Oasis (King George Island, the South Shetland Islands), we studied the influence of month-long artificial treatment with fresh water, salt water, and guano solution on the biometric characteristics, chlorophyll content, as well as the nuclear area of leaf parenchymal cells and nuclear DNA content, in a maritime Antarctic aboriginal plant Deschampsia antarctica. The modeled factors induced an increase in the generative shoot height and the length of the largest leaf, but did not influence the number of flowers. Treatment with guano caused an increase in the chlorophyll a and b contents, while fresh water treatment only led to some increase in chlorophyll a. Fluctuations of physiologically significant traits, such as the nuclear area and DNA content in the leaf parenchyma cells of D. antarctica, have been traced under the influence of the studied factors. Understanding of the hierarchy of influence of these factors as well as and sensitivity of plants of this species to external agents require further investigation.     

Diet with Diphenyl Diselenide Mitigates Quinclorac Toxicity in Silver Catfish (Rhamdia quelen)

In this study, the protective effects of diphenyl diselenide [(PhSe)₂] on quinclorac- induced toxicity were investigated in silver catfish (Rhamdia quelen). The fish were fed for 60 days with a diet in the absence or in the presence of 3.0 mg/Kg (PhSe)₂. Animals were further exposed to 1 mg/L quinclorac for 8 days. At the end of experimental period, fish were euthanized and biopsies from liver and gills, as well as blood samples, were collected. The cortisol and metabolic parameters were determined in plasma, and those enzyme activities related to osmoregulation were assayed in the gills. In liver, some important enzyme activities of the intermediary metabolism and oxidative stress-related parameters, such as thiobarbituric acid-reactive substance (TBARS), protein carbonyl, catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), nonprotein thiols (NPSH) and ascorbic acid contents were also evaluated. Compared to the control group, quinclorac exposure significantly decreased hepatosomatic index and increased cortisol and lactate values in plasma. Moreover, the activities of fructose biphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6Pase), glycogen phosphorilase (GPase) and aspartate aminotransferase (AST) were significantly increased in liver. Quinclorac also induced lipid peroxidation while the activity of SOD, NPSH and ascorbic acid levels decreased in the liver. However, dietary (PhSe)₂ reduced the herbicide-induced effects on the studied parameters. In conclusion, (PhSe)₂ has beneficial properties based on its ability to attenuate toxicity induced by quinclorac by regulating energy metabolism and oxidative stress-related parameters.     

Assessing landscape resistance to roe deer dispersal using fuzzy set theory and multicriteria analysis: a case study in Central Spain

The central Iberian Peninsula has one of the highest densities of roe deer populations in Spain. A new motorway is planned to pass through the middle of the distribution of roe deer, thus making it necessary to conduct a connectivity analysis. A map of resistance to roe deer dispersal movements was obtained based on the literature and expert judgment. Three factors were selected: land use (defined by the ability to hide movements, food source, and degree of naturalness), landforms, and influence due to proximity to elements that increase (such as roads and urban areas) or decrease (water resources and proximity to optimal habitat patches) resistance at the local level. Different combinations of factors derived using the analytical hierarchy and fuzzy logic processes were analysed and compared with the real distribution of the species. More realistic resistance (cost) values were obtained for gamma values close to 0.9. This highlights the greater predominance of the fuzzy sum over the fuzzy product in modelling the cost surface. Better results were obtained in scenarios where the predominant factors were either land use and landforms or land use and proximity to human-modified areas. This indicates that roe deer will readily range far from their optimal patches if the land use provides partial cover. These movements appear to be conditioned by steep terrain. Our case study offers a good example of building a cost resistance matrix to help locate areas where the expansion of the species may be curbed or encouraged.     

Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen)

Silver catfish (Rhamdia quelen; Teleostei) were exposed to commercial formulation Roundup, a glyphosate herbicide: 0 (control), 0.2 or 0.4 mg/L for 96 h. Fish exposed to glyphosate showed an increase in hepatic glycogen, but a reduction in muscle glycogen at both concentrations tested. Glucose decreased in liver and increased in muscle of fish at both herbicide concentrations. Glyphosate exposure increased lactate levels in liver and white muscle at both concentrations. Protein levels increased in liver and decreased in white muscle while levels of ammonia in both tissues increased in fish at both glyphosate concentrations. Specific AChE activity was reduced in brain after treatments, no changes were observed in muscle tissue. Catalase activity in liver did not change during of exposure. Fish exposed to glyphosate demonstrated increased TBARS production in muscle tissue at both concentrations tested. For both glyphosate concentrations tested brain showed a reduction of TBARS after 96 h of exposure. The present results showed that in 96 h, glyphosate changed AChE activity, metabolic parameters and TBARS production. The parameters measured can be used as herbicide toxicity indicators considering environmentally relevant concentration.