Carlos Fernando Odir Rodrigues Melo

A Lipidomics Approach in the Characterization of Zika-Infected Mosquito Cells: Potential Targets for Breaking the Transmission Cycle

Recent outbreaks of Zika virus in Oceania and Latin America, accompanied by unexpected clinical complications, made this infection a global public health concern. This virus has tropism to neural tissue, leading to microcephaly in newborns in a significant proportion of infected mothers. The clinical relevance of this infection, the difficulty to perform accurate diagnosis and the small amount of data in literature indicate the necessity of studies on Zika infection in order to characterize new biomarkers of this infection and to establish new targets for viral control in vertebrates and invertebrate vectors. Thus, this study aims at establishing a lipidomics profile of infected mosquito cells compared to a control group to define potential targets for viral control in mosquitoes. Thirteen lipids were elected as specific markers for Zika virus infection (Brazilian strain), which were identified as putatively linked to the intracellular mechanism of viral replication and/or cell recognition. Our findings bring biochemical information that may translate into useful targets for breaking the transmission cycle.

Serum Metabolic Alterations upon Zika Infection

Zika virus (ZIKV) infection has recently emerged as a major concern worldwide due to its strong association with nervous system malformation (microcephaly) of fetuses in pregnant women infected by the virus. Signs and symptoms of ZIKV infection are often mistaken with other common viral infections. Since transmission may occur through biological fluids exchange and coitus, in addition to mosquito bite, this condition is an important infectious disease. Thus, understanding the mechanism of viral infection has become an important research focus, as well as providing potential targets for assertive clinical diagnosis and quality screening for hemoderivatives. Within this context, the present work analyzed blood plasma from 79 subjects, divided as a control group and a ZIKV-infected group. Samples underwent direct-infusion mass spectrometry and statistical analysis, where eight markers related to the pathophysiological process of ZIKV infection were elected and characterized. Among these, Angiotensin (1-7) and Angiotensin I were upregulated under infection, showing an attempt to induce autophagy of the infected cells. However, this finding is concerning about hypertensive individuals under treatment with inhibitors of the Renin-Angiotensin System (RAS), which could reduce this response against the virus and exacerbate the symptoms of the infection. Moreover, one of the most abundant glycosphingolipids in the nervous tissue, Ganglioside GM2, was also elected in the present study as an infection biomarker. Considered an important pathogen receptor at membranes outer layer, this finding represents the importance of gangliosides for ZIKV infection and its association with brain tropism. Furthermore, a series of phosphatidylinositols were also identified as biomarkers, implying a significant role of the PI3K-AKT-mTOR Pathway in this mechanism. Finally, these pathways may also be understood as potential targets to be considered in pharmacological intervention studies on ZIKV infection management.

A Metabolomic Overview of Follicular Fluid in Cows

Follicular fluid (FF) protects the oocyte against proteolysis and extrusion during ovulation, providing an appropriate microenvironment that favors proper embryonic development; thereby, FF plays a key role in embryo quality. Being directly related to cattle breeding, studying FF is extremely important in livestock science to measure cattle fertility. This may eventually help to assess the quality of both meat and milk, products widely consumed worldwide. There is an important commercial interest in the evaluation and characterization of compounds present in the FF of livestock that present greater likelihood of pregnancy. Mass spectrometry is a great ally for this type of analysis and can provide quick and efficient screening for molecular markers in biological samples. The present study demonstrated the potential of high-resolution mass spectrometry in analyzing FF samples from two distinct groups of Nellore cows (Bos indicus): high and low fertility, as determined by the number of oocytes produced. We were able to delineate markers of interest for each group, which may ultimately be related to biochemical pathways that lead to higher or lower reproductive performance.

Outer Membrane Vesicles from Neisseria Meningitidis (Proteossome) Used for Nanostructured Zika Virus Vaccine Production

The increase of Zika virus (ZIKV) infections in Brazil in the last two years leaves a prophylactic measures on alert for this new and emerging pathogen. Concerning of our positive experience, we developed a new prototype using Neisseria meningitidis outer membrane vesicles (OMV) on ZIKV cell growth in a fusion of OMV in the envelope of virus particles. The fusion of nanoparticles resulting from outer membrane vesicles of N. meningitidis with infected C6/36 cells line were analyzed by Nano tracking analysis (NTA), zeta potential, differential light scattering (DLS), scan and scanning transmission eletronic microscopy (SEM and STEM) and high resolution mass spectometry (HRMS) for nanostructure characterization. Also, the vaccination effects were viewed by immune response in mice protocols immunization (ELISA and inflammatory chemokines) confirmed by Zika virus soroneutralization test. The results of immunizations in mice showed that antibody production had a titer greater than 1:160 as compared to unvaccinated mice. The immune response of the adjuvant and non-adjuvant formulation activated the cellular immune response TH1 and TH2. In addition, the serum neutralization was able to prevent infection of virus particles in the glial tumor cell model (M059J). This research shows efficient strategies without recombinant technology or DNA vaccines.

The role of lipids in the inception, maintenance and complications of dengue virus infection

Dengue fever is a viral condition that has become a recurrent issue for public health in tropical countries, common endemic areas. Although viral structure and composition have been widely studied, the infection phenotype in terms of small molecules remains poorly established. This contribution providing a comprehensive overview of the metabolic implications of the virus-host interaction using a lipidomic-based approach through direct-infusion high-resolution mass spectrometry. Our results provide further evidence that lipids are part of both the immune response upon Dengue virus infection and viral infection maintenance mechanism in the organism. Furthermore, the species described herein provide evidence that such lipids may be part of the mechanism that leads to blood-related complications such as hemorrhagic fever, the severe form of the disease.

A quantitative study on growth variability and production of ochratoxin A and its derivatives by A. carbonarius and A. niger in grape-based medium

Aspergillus carbonarius and Aspergillus niger are the main responsible fungi for the accumulation of ochratoxin A (OTA) in wine grapes. Some strains are able to convert the parent mycotoxin into other compounds by means of hydrolysis and/or conjugation reactions through their defense mechanisms and enzymatic activity, leading to the formation of a modified mycotoxin. Thus, the variability of growth and metabolite production are inherent to the strain, occurring distinctively even when submitted to similar conditions. In this sense, this contribution aimed at determining the variability in multiplication and production of OTA by strains of A. carbonarius and A. niger isolated from grapes, as well as investigating the formation of modified mycotoxins. Strains were incubated in grape-based medium, and the diameter of the colonies measured daily. The determination of OTA was performed by high-performance liquid chromatography and the identification of modified mycotoxins was carried out using high-resolution mass spectrometry. Variabilities in terms of growth and OTA production were assessed across five different strains. Peak production of OTA was detected on day 15, and a decline on day 21 was observed, indicating that the observed reduction may be associated with the degradation or modification of the OTA over time by the fungus. Ethylamide ochratoxin A, a modified mycotoxin identified in this study, provides evidence that there may be underreporting of total mycotoxin levels in food, increasing uncertainty concerning health risks to the population.

Skin Biomarkers for Cystic Fibrosis: A Potential Non-Invasive Approach for Patient Screening

Background Cystic fibrosis (CF) is a disabling genetic disease with an increased prevalence in European heritage populations. Currently, the most used technique for collection of CF samples and diagnosis is provided through uncomfortable tests, with uncertain results, mostly based on chloride concentration in sweat. Since CF mutation induces many metabolic changes in patients, exploring these alterations might be an alternative to visualize potential biomarkers that could be used as interesting tools for further diagnostic upgrade, prioritizing simplicity, low cost, and quickness. Methods This contribution describes an accurate strategy to provide potential biomarkers related to CF, which may be understood as a potential tool for new diagnostic approaches and/or for monitoring disease evolution. Therefore, the present proposal consists of using skin imprints on silica plates as a way of sample collection, followed by direct-infusion high-resolution mass spectrometry and multivariate data analysis, intending to identify metabolic changes in skin composition of CF patients. Results Metabolomics analysis allowed identifying chemical markers that can be traced back to CF in patients’ skin imprints, differently from control subjects. Seven chemical markers from several molecular classes were elected, represented by bile acids, a glutaric acid derivative, thyrotropin-releasing hormone, an inflammatory mediator, a phosphatidic acid, and diacylglycerol isomers, all reflecting metabolic disturbances that occur due to of CF. Conclusion The comfortable method of sample collection combined with the identified set of biomarkers represent potential tools that open the range of possibilities to manage CF and follow the disease evolution. This exploratory approach points to new perspectives about the development of diagnostic assay using biomarkers and the management CF.

New Approach of QuEChERS and GC-MS Triple-Quadrupole for the Determination of Ethyl Carbamate Content in Brazilian cachaças

Cachaça is a popular spirit produced in Brazil, obtained by distillation of fermented sugar cane. Among the contaminants arising from production, ethyl carbamate is a carcinogenic compound that occurs naturally in fermented foods and beverages; in Brazil, the maximum limit established by current legislation is 150 µg L−1. Quality control is usually performed using gas chromatography; however, robustness and reproducibility of quantitative results may be severely impaired, as the addition of 6–30 g L−1 of sucrose is a common procedure for taste standardization, directly interfering in the results. This work describes the development of a novel method to improve ethyl carbamate quantification in cachaças using a new approach of QuEChERS extraction based on salting-out phenomenon, to effectively separate ethanol from sugar-containing water. Eighteen different brands of cachaça were analyzed. The proposed methodology was able to eliminate components that contaminate the sample flow path in the gas chromatography system, while improving precision and accuracy by using a triple-quadrupole approach, in comparison with the methodology usually employed: direct analysis of cachaça samples with no sample prep. Results indicate that this approach is more effective due to the removal of sugar content, with no impact in costs per analysis.

A Machine Learning Application Based in Random Forest for Integrating Mass Spectrometry-Based Metabolomic Data: A Simple Screening Method for Patients With Zika Virus

Recent Zika outbreaks in South America, accompanied by unexpectedly severe clinical complications have brought much interest in fast and reliable screening methods for ZIKV (Zika virus) identification. Reverse-transcriptase polymerase chain reaction (RT-PCR) is currently the method of choice to detect ZIKV in biological samples. This approach, nonetheless, demands a considerable amount of time and resources such as kits and reagents that, in endemic areas, may result in a substantial financial burden over affected individuals and health services veering away from RT-PCR analysis. This study presents a powerful combination of high-resolution mass spectrometry and a machine-learning prediction model for data analysis to assess the existence of ZIKV infection across a series of patients that bear similar symptomatic conditions, but not necessarily are infected with the disease. By using mass spectrometric data that are inputted with the developed decision-making algorithm, we were able to provide a set of features that work as a “fingerprint” for this specific pathophysiological condition, even after the acute phase of infection. Since both mass spectrometry and machine learning approaches are well-established and have largely utilized tools within their respective fields, this combination of methods emerges as a distinct alternative for clinical applications, providing a diagnostic screening—faster and more accurate—with improved cost-effectiveness when compared to existing technologies.

Evaluating the effects of the adulterants in milk using direct-infusion high-resolution mass spectrometry

Milk is an extremely complex food, capable of providing essential nutrients as well as being an important source of energy, and high-quality proteins and fats. Due to advances in technology, and to meet the increasing demand, production costs have increased, turning milk into a target of adulterations. Routine methods usually applied to certify the quality of the milk are restricted to microbiological tests, and assays that attest the nutritional composition within the expected values. However, potentially harmful byproducts generated by adulterating substances in general are not detected through these methodologies. In this contribution, we simulated the adulteration of freshly produced milk samples with four adulterants whose use already had reported for extended shelf life: formaldehyde, hydrogen peroxide, sodium hydroxide, and sodium hypochlorite. These samples were submitted to direct-infusion high-resolution mass spectrometry analysis and multivariate statistical analysis. This approach allows the characterization of a series of molecules modified by the adulterants, what demonstrates how these species affect the nutritious characteristics of this product.