RESUMO
Triatominae are hematophagous insects involved in the transmission of Chagas disease. Among the 19 genera of the subfamily, those with the highest epidemiological importance regarding the dissemination of Trypanosoma cruzi are Panstrongylus, Rhodnius, and Triatoma. Of these three genera, Rhodnius presents the greatest difficulties for specific identification. Thus, there is a need to overcome the difficulties in identifying phenotypes of similar species of this genus. In the present study, the MALDI-TOF MS methodology was used to identify 12 Rhodnius species, among the 21 admitted. The MALDI-TOF MS methodology allowed specific characterization through the identification of peptides and proteins, starting from four different methods of extraction: (A) acetonitrile/formic acid (ACN/AF), (B) acetonitrile/trifluoroacetic acid (ACN/TFA), (C) isopropyl/formic acid (IPA/AF), and (D) methanol/formic acid (MeOH/AF), and four types of MALDI-TOF matrices: α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 6-aza-2-thiothymine (ATT), and 2,6-dihydroxyacetophenone (DHAP). The experiments were performed by combining the four solvents and four matrices to select the best MALDI extraction/matrix. The application of the MALDI-TOF MS technique, through the digital mass spectrometry approach combined with chemometric tools, such as partial least squares-discriminant analysis (PLS-DA), was able to discriminate 12 species of Rhodnius genus, which are difficult to identify using morphological characteristics. Thus, in view of the results obtained, the methodology described in the present article can be applied with speed and efficiency for the discrimination of Triatominae species. Graphical Abstract.
Assuntos
Proteínas de Insetos/química , Peptídeos/química , Rhodnius/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetofenonas/química , Animais , Ácidos Cumáricos/química , Formiatos/químicaRESUMO
Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.
Assuntos
Cacau/microbiologia , Lipídeos/química , Proteínas de Plantas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cacau/metabolismoRESUMO
Sand flies are vectors of great public health importance, since they constitute a group of hematophagous insects responsible for etiological agents transmission of zoonotic diseases such a visceral leishmaniasis. In face of the expansion of these diseases, efficient control strategies are needed which depend on comprehending the sand fly eco-epidemiology. In this regard, MALDI-TOF mass spectrometry has been used for bacteria, fungi and yeast detection studies through peptide/protein profiles. However, little is known about interference of biological factors associated with vector ecology, such as blood meal preferences and even sand fly age on the peptide/protein profiles. Thus, the present study aimed to evaluate the differences in peptide/protein profiles of the sand fly Lutzomyia longipalpis, by means of MALDI-TOF, due to the sand fly's age, sex, blood meal source and Leishmania infantum infection. Sample preparation was made removing both head and last abdomen segments keeping the thorax, its appendices and the rest of the abdomen. Five specimens per pool were used to obtain peptide/protein extract of which 1 µL solution was deposited over 1 µL MALDI matrix dried. Characteristic spectra were analyzed using principal coordinate analysis as well as indicator species analysis to discriminate differences in sand flies's peptide/protein profile by sex, age, blood meal source and L. infantum infection. The results show that the evaluated variables produced distinct peptide/protein profiles, demonstrated by the identification of specific diagnostic ions. It was found that the interference of biological factors should be taken into account when using the MALDI-TOF analysis of sand fly species identification and eco-epidemiological applications in field studies. Based on our results, we believe that it is possible to identify infected specimens and the source of blood meal in a collection of wild sand flies, serving to measure infectivity and understand the dynamics of the vector's transmission chain. Our results may be useful for epidemiological studies that look at the ecology of sand flies and leishmaniasis, as well as for raising awareness of biological characteristics' impact on peptide/protein profiles in sand fly species identification.
Assuntos
Psychodidae , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Psychodidae/parasitologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Feminino , Masculino , Peptídeos/análise , Proteínas de Insetos/análise , Insetos Vetores/parasitologia , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/transmissãoRESUMO
The purpose of this study was to use traditional physical assessments combined with a metabolomic approach to compare the anthropometric, physical fitness level, and serum fasting metabolic profile among U22 soccer players at different competitive levels. In the experimental design, two teams of male U22 soccer were evaluated (non-elite = 20 athletes, competing in a regional division; elite = 16 athletes, competing in the first division of the national U22 youth league). Earlobe blood samples were collected, and metabolites were extracted after overnight fasting (12 h). Untargeted metabolomics through Liquid Chromatograph Mass Spectrometry (LC-MS) analysis and anthropometric evaluation were performed. Critical velocity was applied to determine aerobic (CV) and anaerobic (ARC) capacity. Height (non-elite = 174.4 ± 7.0 cm; elite = 176.5 ± 7.0 cm), body mass index (non-elite = 22.1 ± 2.4 kg/m2; elite = 21.9 ± 2.3 kg/m2), body mass (non-elite = 67.1 ± 8.8 kg; elite = 68.5 ± 10.1 kg), lean body mass (non-elite = 59.3 ± 7.1 kg; elite = 61.1 ± 7.9 kg), body fat (non-elite = 7.8 ± 2.4 kg; elite = 7.3 ± 2.4 kg), body fat percentage (non-elite = 11.4 ± 2.4%; elite = 10.5 ± 1.7%), hematocrit (non-elite = 50.2 ± 4.0%; elite = 51.0 ± 4.0%), CV (non-elite = 3.1 ± 0.4 m/s; elite = 3.0 ± 0.2 m/s), and ARC (non-elite = 129.6 ± 55.7 m; elite = 161.5 ± 61.0 m) showed no significant differences between the elite and non-elite teams, while the multivariate Partial Least Squares Discriminant Analysis (PLS-DA) model revealed a separation between the elite and non-elite athletes. Nineteen metabolites with importance for projection (VIP) >1.0 were annotated as belonging to the glycerolipid, sterol lipid, fatty acyl, flavonoid, and glycerophospholipid classes. Metabolites with a high relative abundance in the elite group were related in the literature to a better level of aerobic power, greater efficiency in the recovery process, and improvement of mood, immunity, decision making, and accuracy, in addition to acting in mitochondrial preservation and electron transport chain maintenance. In conclusion, although classical physical assessments were not able to distinguish the teams at different competitive levels, the metabolomics approach successfully indicated differences between the fasting metabolic profiles of elite and non-elite teams.
RESUMO
The Eugenia calycina Cambess, also known as pitanga-do-cerrado, is an unexplored Brazilian fruit used by native people for the treatment of diabetes. The aim of this study was to identify the phenolic compounds from the leaves, pulp and seed of Eugenia calycina by using LC-MS-based targeted and untargeted analysis. The LC-MS-based targeted quantitative analysis showed a high phenolic content in all plant parts in which the ellagic acid was the main phenolic compound with values of 8244.53 µg/g dw (leaves), 5054.43 µg/g dw (pulp) and 715.42 µg/g dw (seed). The leaves, pulp and seed showed a high total phenolic content of 20371.96, 7139.70 and 2204.75 µg/g dw, respectively. In addition, the LC-MS-based targeted analysis showed ellagic acid, myricitrin and epicatechin gallate as the main phenolic compounds in the Eugenia calycina. The LC-MS-based untargeted analysis showed the phytochemical profile of the leaves, pulp and seed in which 153 phytochemicals from different chemical classes were annotated, including organic acids, phenolic acids, flavonoids, and other compounds. The Eugenia calycina has high potential as a plant-based food due to its phytochemical profile and high phenolic content.
Assuntos
Eugenia , Brasil , Cromatografia Líquida , Humanos , Extratos Vegetais , Folhas de Planta , Sementes , Espectrometria de Massas em TandemRESUMO
Obesity is a chronic disease with rising worldwide prevalence and largely associated with several other comorbidities, such as cancer, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. Hepatic steatosis, a hallmark of NAFLD, is strongly correlated with obesity and has been correlated with changes in the gut microbiota, which can promote its development through the production of short-chain fatty acids (SCFAs) that regulate insulin resistance, bile acid, choline metabolism, and inflammation. Recent studies have suggested a controversial role for the inflammasome/caspase-1 in the development of obesity and non-alcoholic steatohepatitis (NASH). Here, we evaluated the role of inflammasome NLRP3 and caspases 1/11 in the establishment of obesity and hepatic steatosis in diet-induced obese mice, correlating them with the global lipid profile of the liver and gut microbiota diversity. After feeding wild-type, caspases 1/11, and NLRP3 knockout mice with a standard fat diet (SFD) or a high-fat diet (HFD), we found that the caspases 1/11 knockout mice, but not NLRP3 knockout mice, were more susceptible to HFD-induced obesity, and developed enhanced hepatic steatosis even under SFD conditions. Lipidomics analysis of the liver, assessed by MALDI-MS analysis, revealed that the HFD triggered a significant change in global lipid profile in the liver of WT mice compared to those fed an SFD, and this profile was modified by the lack of caspases 1/11 and NLRP3. The absence of caspases 1/11 was also correlated with an increased presence of triacylglycerol in the liver. Gut microbial diversity analysis, using 16S rRNA gene sequencing, showed that there was also an increase of Proteobacteria and a higher Firmicutes/Bacteroidetes ratio in the gut of caspases 1/11 knockout mice fed an HFD. Overall, mice without caspases 1/11 harbored gut bacterial phyla involved with weight gain, obesity, and hepatic steatosis. Taken together, our data suggest an important role for caspases 1/11 in the lipid composition of the liver and in the modulation of the gut microbial community composition. Our results further suggest that HFD-induced obesity and the absence of caspases 1/11 may regulate both lipid metabolism and gut microbial diversity, and therefore may be associated with NAFLD and obesity.
Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Microbioma Gastrointestinal , Metabolismo dos Lipídeos , Fígado/metabolismo , Obesidade/enzimologia , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Caspase 1/genética , Caspases Iniciadoras/genética , Dieta Hiperlipídica , Ácidos Graxos Voláteis/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/microbiologiaRESUMO
Leishmaniasis is a group of diseases caused by protozoan parasites from the genus Leishmania. There are estimated 1.3 million new cases annually with a mortality of 20,000-30,000 per year, when patients are left untreated. Current chemotherapeutic drugs available present high toxicity and low efficacy, the latter mainly due to the emergence of drug-resistant parasites, which makes discovery of novel, safe, and efficacious antileishmanial drugs mandatory. The present work reports the synthesis, characterization by ESI-MS, 1H and 13C NMR, and FTIR techniques as well as in vitro and in vivo evaluation of leishmanicidal activity of guanidines derivatives presenting lower toxicity. Among ten investigated compounds, all being guanidines containing a benzoyl, a benzyl, and a substituted phenyl moiety, LQOF-G2 (IC50-ama 5.6⯵M; SIâ¯=â¯131.8) and LQOF-G7 (IC50-ama 7.1⯵M; SIâ¯=â¯87.1) were the most active against L. amazonensis intracellular amastigote, showing low cytotoxicity to the host cells according to their selectivity index. The most promising compound, LQOF-G2, was further evaluated in an in vivo model and was able to decrease 60% of the parasite load in foot lesions at a dose of 0.25â¯mg/kg/day. Moreover, this guanidine derivative demonstrated reduced hepatotoxicity compared to other leishmanicidal compounds and did not show nephrotoxicity, as determined by the analyses of biomarkers of hepatic damage and renal function, which make this compound a potential new hit for therapy against leishmaniasis.
Assuntos
Antiprotozoários/farmacologia , Guanidinas/farmacologia , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Feminino , Guanidinas/síntese química , Guanidinas/química , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-AtividadeRESUMO
A method was developed for the simultaneous analysis of 14 pesticide residues (clofentezine, carbofuran, diazinon, methyl parathion, malathion, fenthion, thiabendazole, imazalil, bifenthrin, permethrin, prochloraz, pyraclostrobin, difenoconazole and azoxystrobin) in mango fruit, based on solid-phase micro extraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS). Different parameters of the method were evaluated, such as fiber type, extraction mode (direct immersion and headspace), temperature, extraction and desorption times, stirring velocities and ionic strength. The best results were obtained using polyacrylate fiber and direct immersion mode at 50 degrees C for 30 min, along with stirring at 250 rpm and desorption for 5 min at 280 degrees C. The method was validated using mango samples spiked with pesticides at concentration levels ranging from 33.3 to 333.3 microg kg(-1). The average recoveries (n=3) for the lowest concentration level ranged from 71.6 to 117.5%, with relative standard deviations between 3.1 and 12.3%, respectively. Detection and quantification limits ranged from 1.0 to 3.3 microg kg(-1) and from 3.33 to 33.33 microg kg(-1), respectively. The optimized method was then applied to 16 locally purchased mango samples, all of them containing the pesticides bifenthrin and azoxystrobin in concentrations of 18.3-57.4 and 12.7-55.8 microg kg(-1), respectively, although these values were below the MRL established by Brazilian legislation. The method proved to be selective, sensitive, and with good precision and recovery rates, presenting LOQ below the MRL admitted by Brazilian legislation.