Metabolomics in the fight against malaria

Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Using a single tablet daily to treat latent tuberculosis infection in Brazil: bioequivalence of two different isoniazid formulations (300 mg and 100 mg) demonstrated by a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry method in a randomised, crossover study

The recommended treatment for latent tuberculosis (TB) infection in adults is a daily dose of isoniazid (INH) 300 mg for six months. In Brazil, INH was formulated as 100 mg tablets. The treatment duration and the high pill burden compromised patient adherence to the treatment. The Brazilian National Programme for Tuberculosis requested a new 300 mg INH formulation. The aim of our study was to compare the bioavailability of the new INH 300 mg formulation and three 100 mg tablets of the reference formulation. We conducted a randomised, single dose, open label, two-phase crossover bioequivalence study in 28 healthy human volunteers. The 90% confidence interval for the INH maximum concentration of drug observed in plasma and area under the plasma concentration vs. time curve from time zero to the last measurable concentration “time t” was 89.61-115.92 and 94.82-119.44, respectively. The main limitation of our study was that neither adherence nor the safety profile of multiple doses was evaluated. To determine the level of INH in human plasma, we developed and validated a sensitive, simple and rapid high-performance liquid chromatography-tandem mass spectrometry method. Our results showed that the new formulation was bioequivalent to the 100 mg reference product. This finding supports the use of a single 300 mg tablet daily strategy to treat latent TB. This new formulation may increase patients’ adherence to the treatment and quality of life.

Determination of levodopa in human plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS): application to a bioequivalence study

A sensitive, accurate and simple method using HPLC-MS/MS was developed and validated for levodopa quantitation in human plasma. Analysis was achieved on a pursuit® C18 analytical column (5 µm; 150 x 4.6 mm i.d.) using a mobile phase (methanol and water , 90:10, v/v) containing formic acid 0.5% v/v, after extracting the samples using a simple protein plasma precipitation with perchloric acid. The developed method was validated in accordance with ANVISA guidelines and was successfully applied to a bioequivalence study in 60 healthy volunteers demonstrating the feasibility and reliability of the proposed method.

Mathematical equation correction to spectral and transport interferences in high-resolution continuum source flame atomic absorption spectrometry: determination of lead in phosphoric acid

In this work, a new mathematical equation correction approach for overcoming spectral and transport interferences was proposed. The proposal was applied to eliminate spectral interference caused by PO molecules at the 217.0005 nm Pb line, and the transport interference caused by variations in phosphoric acid concentrations. Correction may be necessary at 217.0005 nm to account for the contribution of PO, since Atotal217.0005 nm = A Pb217.0005 nm + A PO217.0005 nm. This may be easily done by measuring other PO wavelengths (e.g. 217.0458 nm) and calculating the relative contribution of PO absorbance (A PO) to the total absorbance (Atotal) at 217.0005 nm: A Pb217.0005 nm = Atotal217.0005 nm - A PO217.0005 nm = Atotal217.0005 nm - k (A PO217.0458 nm). The correction factor k is calculated from slopes of calibration curves built up for phosphorous (P) standard solutions measured at 217.0005 and 217.0458 nm, i.e. k = (slope217.0005 nm/slope217.0458 nm). For wavelength integrated absorbance of 3 pixels, sample aspiration rate of 5.0 ml min-1, analytical curves in the 0.1 - 1.0 mg L-1 Pb range with linearity better than 0.9990 were consistently obtained. Calibration curves for P at 217.0005 and 217.0458 nm with linearity better than 0.998 were obtained. Relative standard deviations (RSD) of measurements (n = 12) in the range of 1.4 - 4.3% and 2.0 - 6.0% without and with mathematical equation correction approach were obtained respectively. The limit of detection calculated to analytical line at 217.0005 nm was 10 µg L-1 Pb. Recoveries for Pb spikes were in the 97.5 - 100% and 105 - 230% intervals with and without mathematical equation correction approach, respectively.

Determination of copper at wide range concentrations using instrumental features of high-resolution continuum source flame atomic absorption spectrometry

This work describes a method to determine Cu at wide range concentrations in a single run without need of further dilutions employing high-resolution continuum source flame atomic absorption spectrometry. Different atomic lines for Cu at 324.754 nm, 327.396 nm, 222.570 nm, 249.215 nm and 224.426 nm were evaluated and main figures of merit established. Absorbance measurements at 324.754 nm, 249.215 nm and 224.426 nm allows the determination of Cu in the 0.07 - 5.0 mg L-1, 5.0 - 100 mg L-1 and 100 - 800 mg L-1 concentration intervals respectively with linear correlation coefficients better than 0.998. Limits of detection were 21 µg L-1, 310 µg L-1 and 1400 µg L-1 for 324.754 nm, 249.215 nm and 224.426 nm, respectively and relative standard deviations (n = 12) were £ 2.7%. The proposed method was applied to water samples spiked with Cu and the results were in agreement at a 95% of confidence level (paired t-test) with those obtained by line-source flame atomic absorption spectrometry.

Measurement of serum estrogen and estrogen metabolites in pre- and postmenopausal women with osteoarthritis using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

Although 17β-estradiol (E2) deficiency has been linked to the development of osteoarthritis (OA) in middle-aged women, there are few studies relating other estrogens and estrogen metabolites (EMs) to this condition. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to measure the levels of six EMs (i.e., estrone, E2, estriol, 2-hydroxyestrone, 2-hydroxyestradiol, and 16a-hydroxyestrone) in healthy pre- and postmenopausal women and women with OA. This method had a precision ranging from 1.1 to 3.1% and a detection limit ranging from 10 to 15 pg. Compared to healthy women, serum-free E2 was lower in the luteal and postmenopausal phases in women with OA, and total serum E2 was lower in postmenopausal women with OA. Moreover, compared to healthy women, total serum 2-hydroxyestradiol was higher in postmenopausal women with OA and total serum 2-hydroxyestrone was lower in both the luteal and follicular phases in women with OA. In conclusion, our HPLC-ESI-MS/MS method allowed the measurement of multiple biochemical targets in a single assay, and, given its increased cost-effectiveness, simplicity, and speed relative to previous methods, this method is suitable for clinical studies.

Specificity and selectivity improvement in doping analysis using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry was used for the identification of forty doping agents. The improvement in the specificity was remarkable, allowing the resolution of analytes that could not be done by one-dimensional chromatographic systems. The sensitivity observed for different classes of prohibited substances was clearly below the value required by the World Anti-Doping Agency. In addition time-of-flight mass spectrometry gives full spectrum for all analytes without any interference from the matrix, resulting in selectivity improvements. These results could support the implementation of an exhaustive monitoring approach for hundreds of doping agents in a single injection.

Analysis of acylcarnitine profiles in umbilical cord blood and during the early neonatal period by electrospray ionization tandem mass spectrometry

Acylcarnitine profiling by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a potent tool for the diagnosis and screening of fatty acid oxidation and organic acid disorders. Few studies have analyzed free carnitine and acylcarnitines in dried blood spots (DBS) of umbilical cord blood (CB) and the postnatal changes in the concentrations of these analytes. We have investigated these metabolites in healthy exclusively breastfed neonates and examined possible effects of birth weight and gestational age. DBS of CB were collected from 162 adequate for gestational age neonates. Paired DBS of heel-prick blood were collected 4-8 days after birth from 106 of these neonates, the majority exclusively breastfed. Methanol extracts of DBS with deuterium-labeled internal standards were derivatized before analysis by ESI-MS/MS. Most of the analytes were measured using a full-scan method. The levels of the major long-chain acylcarnitines, palmitoylcarnitine, stearoylcarnitine, and oleoylcarnitine, increased by 27, 12, and 109%, respectively, in the first week of life. Free carnitine and acetylcarnitine had a modest increase: 8 and 11%, respectively. Propionylcarnitine presented a different behavior, decreasing 9% during the period. The correlations between birth weight or gestational age and the concentrations of the analytes in DBS were weak (r £ 0.20) or nonsignificant. Adaptation to breast milk as the sole source of nutrients can explain the increase of these metabolites along the early neonatal period. Acylcarnitine profiling in CB should have a role in the early detection of metabolic disorders in high-risk neonates.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

Carbohydrate-rich high-molecular-mass antigens are strongly recognized during experimental Histoplasma capsulatum infection

INTRODUCTION: During histoplasmosis, Histoplasma capsulatum soluble antigens (CFAg) can be naturally released by yeast cells. Because CFAg can be specifically targeted during infection, in the present study we investigated CFAg release in experimental murine histoplasmosis, and evaluated the host humoral immune response against high-molecular-mass antigens (hMMAg. >150 kDa), the more immunogenic CFAg fraction. METHODS: Mice were infected with 2.2x10(4) H. capsulatum IMT/HC128 yeast cells. The soluble CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg circulating immune complexes (CIC) levels were determined by enzymelinked immunosorbent assay, at days 0, 7, 14, and 28 post-infection. RESULTS: We observed a progressive increase in circulating levels of CFAg, IgG anti-CFAg, IgG anti-hMMAg, and IgG-hMMAg CIC after H. capsulatum infection. The hMMAg showed a high percentage of carbohydrates and at least two main immunogenic components. CONCLUSIONS: We verified for the first time that hMMAg from H. capsulatum IMT/HC128 strain induce humoral immune response and lead to CIC formation during experimental histoplasmosis.

Persistent asthma in adults: comparison of high resolution computed tomography of the lungs after one year of follow-up

OBJECTIVE: The aims of this study were to evaluate the role of high resolution computed tomography of the torax in detecting abnormalities in chronic asthmatic patients and to determine the behavior of these lesions after at least one year. METHOD: Fourteen persistent asthmatic patients with a mean forced expiratory volume in 1-second that was 63% of predicted and a mean forced expiratory volume in 1-second /forced vital capacity of 60% had two high resolution computed tomographys separated by an interval of at least one year. RESULTS: All 14 patients had abnormalities on both scans. The most common abnormality was bronchial wall thickening, which was present in all patients on both computed tomographys. Bronchiectasis was suggested on the first computed tomography in 5 of the 14 (36%) patients, but on follow-up, the bronchial dilatation had disappeared in 2 and diminished in a third. Only one patient had any emphysematous changes; a minimal persistent area of paraseptal emphysema was present on both scans. In 3 patients, a "mosaic" appearance was observed on the first scan, and this persisted on the follow-up computed tomography. Two patients had persistent areas of mucoid impaction. In a third patient, mucus plugging was detected only on the second computed tomography. CONCLUSIONS: We conclude that there are many abnormalities on the high resolution computed tomography of patients with persistent asthma. Changes suggestive of bronchiectasis, namely bronchial dilatation, frequently resolve spontaneously. Therefore, the diagnosis of bronchiectasis by high resolution computed tomography in asthmatic patients must be made with caution, since bronchial dilatation can be reversible or can represent false dilatation. Nonsmoking chronic asthmatic subjects in this study had no evidence of centrilobular or panacinar emphysema.

Brazil nuts: determination of natural elements and aflatoxin

A study was carried out to evaluate the association of levels of radioactivity, selenium and aflatoxin in shelled Brazil nuts, which were classified in different sizes, for export. The selenium determinations were performed by inductively coupled plasma optical emission spectrometry (LOQ = 3.0 µg g-1), and aflatoxins were detected by Liquid chromatography-mass spectrometry (LOQ = 0.85 µg kg-1), recovery rates were between 92 and 100%. Radioactivity was measured by high-resolution gamma spectrometry. The selenium mean concentration was (22.7 ± 7.4) µg g-1. (n = 30). Mean activities determined for the following radium isotopes were: 15.77 Bq kg-1 for 224Ra, 104.8 Bq kg-1 for 226Ra and 99.48 Bq kg-1 for 228Ra. For 226Ra, the levels did not vary significantly with nut sizes, although such differences were observed for 224Ra and 228Ra. There was no statistically significant association between the level of selenium and the activity of radionuclides, however, there was correlation between the radionuclides. Aflatoxins above the quantification limit were not found.

Evaluation of geostatistical techniques to estimate the spatial distribution of aboveground biomass in the Amazon rainforest using high-resolution remote sensing data

ABSTRACT The spatial distribution of forest biomass in the Amazon is heterogeneous with a temporal and spatial variation, especially in relation to the different vegetation types of this biome. Biomass estimated in this region varies significantly depending on the applied approach and the data set used for modeling it. In this context, this study aimed to evaluate three different geostatistical techniques to estimate the spatial distribution of aboveground biomass (AGB). The selected techniques were: 1) ordinary least-squares regression (OLS), 2) geographically weighted regression (GWR) and, 3) geographically weighted regression - kriging (GWR-K). These techniques were applied to the same field dataset, using the same environmental variables derived from cartographic information and high-resolution remote sensing data (RapidEye). This study was developed in the Amazon rainforest from Sucumbíos - Ecuador. The results of this study showed that the GWR-K, a hybrid technique, provided statistically satisfactory estimates with the lowest prediction error compared to the other two techniques. Furthermore, we observed that 75% of the AGB was explained by the combination of remote sensing data and environmental variables, where the forest types are the most important variable for estimating AGB. It should be noted that while the use of high-resolution images significantly improves the estimation of the spatial distribution of AGB, the processing of this information requires high computational demand.

Sensitivity of the Amazon biome to high resolution climate change projections

ABSTRACT: Despite the reduction in deforestation rate in recent years, the impact of global warming by itself can cause changes in vegetation cover. The objective of this work was to investigate the possible changes on the major Brazilian biome, the Amazon Rainforest, under different climate change scenarios. The dynamic vegetation models may simulate changes in vegetation distribution and the biogeochemical processes due to climate change. Initially, the Inland dynamic vegetation model was forced with initial and boundary conditions provided by CFSR and the Eta regional climate model driven by the historical simulation of HadGEM2-ES. These simulations were validated using the Santarém tower data. In the second part, we assess the impact of a future climate change on the Amazon biome by applying the Inland model forced with regional climate change projections. The projections show that some areas of rainforest in the Amazon region are replaced by deciduous forest type and grassland in RCP4.5 scenario and only by grassland in RCP8.5 scenario at the end of this century. The model indicates a reduction of approximately 9% in the area of tropical forest in RCP4.5 scenario and a further reduction in the RCP8.5 scenario of about 50% in the eastern region of Amazon. Although the increase of CO2 atmospheric concentration may favour the growth of trees, the projections of Eta-HadGEM2-ES show increase of temperature and reduction of rainfall in the Amazon region, which caused the forest degradation in these simulations.