Citrus sudden death (CSD) is a disease that has affected millions of orange trees in Brazil, leading to economic losses in the order of billions of US dollars. This article examines the effects of CSD on the fatty acid composition of triacylglycerides (TAG) extracted from rootstock and scion bark. The fatty acid profile determined by gas chromatography showed a reduction in oleic and linolenic acids, and an increase in the saturated fatty acids and linoleic acid content, which was in line with the severity of CSD. The reduction in linolenic acid content was related to its role in the biosynthesis of jasmonic acid, which is involved in responses to abiotic and biotic stresses, as well as senescence. These alterations in the fatty acid profile were also used to classify plants both with and without CSD symptoms by using chemometric means. This method represents an alternative to support the diagnosis of CSD disease.

ABSTRACT Metabolomics uses several analytical tools to identify the chemical diversity of metabolites present in organisms. These metabolites are low molecular weight molecules (<1500 Da) classified as a final or intermediary product of metabolic processes. The application of this omics technology has become prominent in inferring physiological conditions through reporting on the phenotypic state; therefore, the introduction of metabolomics into clinical studies has been growing in recent years due to its efficiency in discriminating pathophysiological states. Regarding endocrine diseases, there is a great interest in verifying comprehensive and individualized physiological scenarios, in particular for growth hormone deficiency (GHD). The current GHD diagnostic tests are laborious and invasive and there is no exam with ideal reproducibility and sensitivity for diagnosis neither standard GH cut-off point. Therefore, this review was focussed on articles that applied metabolomics in the search for new biomarkers for GHD. The present work shows that the applications of metabolomics in GHD are still limited, since the little complementarily of analytical techniques, a low number of samples, GHD combined to other deficiencies, and idiopathic diagnosis shows a lack of progress. The results of the research are relevant and similar; however, their results do not provide an application for clinical practice due to the lack of multidisciplinary actions that would be needed to mediate the translation of the knowledge produced in the laboratory, if transferred to the medical setting.

Dengue is one of the most prevalent infectious tropical diseases in the world, with high incidence in over 100 countries. Rapid and reliable diagnosis of dengue is of great importance for public health. To simplify molecular diagnostics, isothermal amplification techniques have recently emerged as an alternative to conventional methods of deoxyribonucleic acid (DNA) amplification. Here, we developed a one-step method for dengue virus detection from real sample based on RT-LAMP (reverse transcription-loop mediated isothermal amplification) in a disposable microdevice. The reaction was thermally controlled with a thermoblock for 15 min at 72 ºC. At the end of the incubation time, we either removed the solution for detection of fragments by gel electrophoresis or added DNA intercalator for visual detection on-chip. Our results demonstrated that it is possible to detect dengue virus through RT-LAMP directly from a serum sample, without previous ribonucleic acid (RNA) extraction. The success of RT-LAMP was confirmed in reactions initiated with 0.8 fg µL-1 of RNA, which represents 200 copies of RNA per µL. RT-LAMP in a polyester-toner (PeT) microdevice is a simple and inexpensive method that allows for rapid detection of dengue virus with high reliability and great potential for point-of-care applications.

Short-chain fatty acids (SCFA) are compounds produced during fermentation of gut microbiota. Acetic, propionic and butyric acids are the most important SCFA produced from non-digestible foods. We developed and validated a rapid and low-cost capillary electrophoretic (CE) method for determination of acetic, propionic and butyric acids in mice feces (100 mg of sample). Electrophoretic separation was performed for SCFA determination in feces samples during 10 min. The method showed good linearity for all analytes (determination coefficient, r2 > 0.98); recovery from 74.1 to 109.8%, while the intra- and interday precision essays were suitable (relative standard deviation (RSD) < 10%); and limits of detection and quantification of 0.13-0.43 mM for acetic acid, 0.09-0.29 mM for propionic acid and 0.03-0.09 mM for butyric acid. Real samples of mice dextran sulfate sodium-induced colitis feces treated with jabuticaba (Plinia cauliflora) aqueous extract were performed successfully.

Due to the expansion of sugarcane crops and the high costs of agricultural inputs, an alternative for fertilization of the soil is the use of humic substances (HS) because they improve its physical-chemical properties. The objective of this study was to assess the biological degradation of fipronil in soil supplemented with HS. For determination of fipronil and its metabolites, we validated a quick, easy, cheap, effective, rugged, and safe gas chromatography-mass spectrometry (QuEChERS/GC-MS) method, which yielded detection and quantification limits of 15.0 and 62.5 ng g-1, respectively. The method presented linearity of 0.99, precision between 1.5 and 10.9%, while the recovery ranged from 78 to 98%, with precision (relative standard deviation (RSD)) < 5%. The strain B. thailandensis of Burkholderia sp. grew under typical conditions for degradation of fipronil. Eight treatments assessed the degradation by B. thailandensis in the presence of HS. The metabolites produced in the experiment, although detected, could not be quantified because they were below the limit of quantification of the method. Our results showed that B. thailandensis has the potential to degrade fipronil, and that HS is not toxic to the microbiota, and does not inactivate fipronil.

Acute lymphoblastic leukemia (ALL) is a fatal disorder in most cases and is the most common cancer in young patients. ALL features an uncontrolled proliferation and maturation arrest of lymphoid progenitor cells (lymphoblast) in the bone marrow. An increase of reactive species production occurs in ALL with an involvement of oxidative stress (OS) and cellular damage. However, there is a lack of information about variations in young patients. The biomarker widely used to measure the OS is the main product of lipid peroxidation, the malondialdehyde (MDA). This biomarker, when evaluated by a chromatographic method and the data analyzed by multivariate analysis with other biochemical parameters of ALL patient charts, shows the distinction of healthy and ALL patients, and a great correlation with the risk of disease.

Abstract Congenital anomalies are already the second cause of infant mortality in Brazil, as in many other middle-income countries in Latin America. Birth defects are a result of both genetic and environmental factors, but a multifactorial etiology has been more frequently observed. Here, we address the environmental causes of birth defects – or teratogens – as a public health issue and present their mechanisms of action, categories and their respective maternal-fetal deleterious effects. We also present a survey from 2008 to 2013 of Brazilian cases involving congenital anomalies (annual average of 20,205), fetal deaths (annual average of 1,530), infant hospitalizations (annual average of 82,452), number of deaths of hospitalized infants (annual average of 2,175), and the average cost of hospitalizations (annual cost of $7,758). Moreover, we report on Brazilian cases of teratogenesis due to the recent Zika virus infection, and to the use of misoprostol, thalidomide, alcohol and illicit drugs. Special attention has been given to the Zika virus infection, now proven to be responsible for the microcephaly outbreak in Brazil, with 8,039 cases under investigation (from October 2015 to June 2016). From those cases, 1,616 were confirmed and 324 deaths occurred due to microcephaly complications or alterations on the central nervous system. Congenital anomalies impact life quality and raise costs in specialized care, justifying the classification of teratogens as a public health issue.

ABSTRACT Paper-based devices present low-cost and are versatile, making them very attractive for clinical analysis. To manufacture those devices wax patterns are printed on paper surface and upon heating the wax permeates through the entire thickness of the paper, creating hydrophobic barriers that delimit test areas. Antibodies produced in rabbits against canine distemper virus (CDV) were physically adsorbed on the surface of gold nanoparticles (AuNPs) and incubated with CDV viral antigens, forming the immunocomplex. Anti-CDV antibodies were immobilized into the microchannels by physical adsorption, forming the test region. The test solution containing conjugated AuNPs was applied at the bottom of the microchannel and it was eluted with a phosphate buffer solution 0.01 M pH 7.4. When the solution containing the AuNPs reached the test zone the recognition of antigens contained on the immunocomplex occurred with the consequent development of a red line, which represents a positive outcome for the test. This method demonstrated the success of physical immobilization of antibodies on AuNPs and the physical immobilization of antibodies on cellulose’s surface. This colorimetric assay brings simplicity and versatility to clinical analyses, presenting potential for CDV diagnosis.

Foi utilizada a reação em cadeia da polimerase via transcriptase reversa (RT-PCR) seguida por eletroforese capilar em microchip para avaliar a viabilidade de esporos de Alicyclobacillus acidoterrestris após a inativação com saponina e temperatura. O método demonstrou ser adequado para tal propósito, além de ser mais rápido e sensível que a tradicional técnica de plaqueamento usado como padrão na indústria de alimentos. Os limites de quantificação e detecção foram 0,0107 e 0,0039 ng µL-1 de DNA amplificado, respectivamente. O coeficiente de correlação (r) entre plaqueamento e o método desenvolvido foi 0,9977, o qual indica uma excelente correspondência entre eles. Assim, é possível avaliar com confiança a viabilidade da bactéria usando a reação de RT-PCR para detecção. Para avaliação do potencial deste método molecular sobre microbiologia tradicional na inativação de A. acidoterrestris foi utilizado saponina como agente de inativação, potencializado com temperatura.

It was used reverse-transcriptase polymerase chain reaction (RT-PCR) followed by capillary electrophoresis on a microchip to probe the viability of Alicyclobacillus acidoterrestris spores after inactivation with saponin and heat. The method was shown to be suitable for the purpose, and was faster and more sensitive than the traditional plating technique, the standard of the food industry. The limits of quantification and detection were 0.0107 and 0.0039 ng µL-1 of amplified DNA, respectively. The correlation coefficient (r) between traditional plating and our method was 0.9977, which indicates an excellent correspondence between them. Consequently, it was possible to assess, with confidence, the viability of bacteria using the RT-PCR reaction for detection. It was evaluated the potential of this molecular method over traditional microbiology in the inactivation of A. acidoterrestris by saponin as an effective agent, potentiated by heat.

Duas técnicas analíticas baseadas no equilíbrio de partição, extração sortiva em barra de agitação (SBSE) e microextração por sorvente empacotado (MEPS) foram avaliadas para a determinação de antidepressivos inibidores seletivos da recaptação de serotonina (SSRI): fluoxetina, sertralina, citalopram e paroxetina, em amostra de plasma por eletroforese capilar em meio não aquoso (NACE) com detecção espectrofotométrica. Os métodos SBSE/NACE e MEPS/NACE apresentaram linearidade na faixa de concentração do limite de quantificação (LOQ) até 500 ng mL-1. Os valores de LOQ variaram de 10 a 30 ng mL-1, em ambas as técnicas, dependendo do fármaco. A precisão interensaio dos métodos apresentou coeficiente de variação inferior a 12%. Os resultados analíticos de validação demonstraram que os métodos são adequados para análises de antidepressivos ISRS em níveis terapêuticos e, consequentemente, para fins de monitorização terapêutica. Estes métodos reduziram os volumes de amostras biológicas, a utilização de solventes orgânicos e o número de etapas do preparo das amostras.

Two approaches based on partition equilibrium, stir bar sorptive extraction (SBSE) and microextraction by packed sorbent (MEPS) were evaluated for determination of selective serotonin reuptake inhibitor antidepressants (SSRI): fluoxetine, sertraline, citalopram and paroxetine in plasma sample by non-aqueous capillary electrophoresis (NACE) with spectrophotometric detection. The SBSE/NACE and MEPS/NACE methods showed linear range from the LOQ up to 500 ng mL-1. LOQ values ranged from 10 to 30 ng mL(-1)1 in both techniques, depending on the compound. The inter-assay precision of the methods presented coefficient of variation lower than 12%. According to the analytical validation results, the methods are adequate for SSRI antidepressant analyses at therapeutic levels, consequently, amenable to therapeutic drug monitoring. These methods reduced the volumes of biological samples, the use of organic solvents and the number of steps involved in sample preparation.

The aim of this work was to evaluate two paternity cases by microchip electrophoresis and the validation of the methodology by comparison of the results with those obtained in a commercial genetic analyzer. It was observed that when working with tetranucleotide regions, in which the minimal difference between the alleles was only four base pairs, the commercial microchip system did not present the resolution and repeatability needed. Nevertheless, the relative standard deviation was between 0 and 1.2% and the fragments detected were within the expected size ranges as described in the literature.

Em espectrometria de massas (MS) no modo de ionização eletrospray (ESI), as amostras podem ser analisadas com um método prévio de separação (cromatografia líquida ou eletroforese capilar) ou por infusão direta da amostra. A injeção direta apresenta um grande benefício que é a redução do volume de amostra consumido e a possibilidade de amostragem contínua por um período estendido. Este maior tempo de amostragem possibilita a análise completa através de fragmentações sucessivas por espectrometria de massas, crítico quando se busca elucidação estrutural de novos compostos, ou quando se dispõe de um analisador de massas do tipo captura de íons. Neste trabalho, descrevemos uma metodologia de deposição de filme estável na extremidade cônica dos dispositivos de boro-silicato, visando o desenvolvimento de nanoinjetores estáticos. A formação de uma camada dupla de titânio e ouro proporcionou uma excelente fixação do filme, resultando em um nanoinjetor para amostragem por ionização/formação do aerossol no sistema de espectrometria de massas. O objetivo deste filme é manter o contato elétrico no sistema. Os nanoinjetores apresentaram repetibilidade e estabilidade elevadas por mais de 100 min de amostragem contínua com apenas 10 µL de amostra.

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample.

Os sideróforos provenientes de Xylella fastidiosa de videiras foram investigados. Tais metabólitos seqüestram ferro, um elemento essencial, do hospedeiro, o que os torna um potencial fator de patogenicidade. Em um meio de cultura em placa com limitação de ferro, tais sideróforos foram detectados pela reação com o complexo cromoazurol S (CAS). Diferentes métodos de análise instrumentais foram utilizados para caracterização dos sideróforos, como: cromatografia de afinidade por metal imobilizado (IMAC), cromatografia micelar eletrocinética capilar (MEKC) e espectrometria de massas com ionização por electrospray (ESI-MS). Os resultados obtidos confirmaram a produção de sideróforos. A extração do(s) composto(s) por IMAC com Fe3+ imobilizado foi uma etapa importante. O(s) sideróforo(s) não foi separado por eletroforese capilar de zona, indicando sua neutralidade sob os pHs investigados. As análises por MEKC apresentaram um pico diferente (quando comparadas à análise do controle), com caráter levemente hidrofóbico. A espectrometria de massas mostrou que os compostos alvos podem ter uma massa molecular relativa dentro da esperada para sideróforos, como: 875, 1004 e 1092 Da.

Siderophore molecules from grapevine Xylella fastidiosa were investigated. Such metabolites sequester iron, an essential element, from the host, making them a potential factor of pathogenicty. In an iron-limited medium, siderophores were detected in culture plates of X. fastidiosa containing the complex Chromeazurol S (CAS). A combination of different instrumental analyses was used for siderophore(s) characterization, such as: immobilized metal affinity chromatography (IMAC), micelar electrokinetic capillary chromatography (MEKC) and electrospray-mass spectrometry (ESI-MS). The results show that grapevine X. fastidiosa produced siderophore(s), as confirmed by the CAS plate assay. The extraction of the compound(s) using IMAC with immobilized Fe3+ was important for analyte purification. The chelator was not separated by capillary zone electrophoresis, indicating the possibility of a neutral compound under the investigated pHs. MEKC runs presented a different peak (when compared to the control analysis) which represented a slightly hydrophobic compound. Mass spectrometry showed that the compound(s) may have a relative molecular mass within the expected range for siderophore molecules, such as 875, 1004 and 1092 Da.

DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo.

DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.

No presente trabalho, três colunas capilares, uma sem recobrimento interno e duas com recobrimentos internos diferentes foram utilizadas na separação de fragmentos de DNA: poli(vinil álcool) (PVA) e poli(dimetilacrilamida) (PDMA) - ambos de recobrimento covalente - foram comparados para a separação de DNA utilizando soluções poliméricas. As separações foram realizadas usando hidroxietilcelulose (HEC) (90-105 kDa) nas concentrações entre 0,00 e 2,00% m/v. Os resultados indicaram que a eficiência de separação foi maior no capilar de PVA do que no de PDMA, em todas as concentrações de HEC testadas. Ainda, uma resolução superior também foi observada com o capilar de PVA, já que com o capilar de PDMA o formato dos picos não se mostrou reprodutível quando corridas subseqüentes foram realizadas. Contrariamente ao relatado na literatura, nenhuma separação foi conseguida com o capilar sem revestimento interno.

In this work three capillary columns, one with uncoated inner wall and two with covalently-bound internal coatings - poly(vinyl alcohol) (PVA) and poly(dimethylacrylamide) (PDMA) - both covalently covered - were used to separate DNA fragments and compared to DNA separation using replaceable polymer solutions. The separations were performed using hydroxyethylcellulose (HEC) (90-105 kDa) in concentrations ranging from 0.00 to 2.00% m/v. The results indicated that the separation efficiency was higher in the PVA capillary than in the PDMA in all evaluated concentrations of HEC. In addition, higher resolution was also observed in PVA-coated capillary since in PDMA the shape of the peaks was not reproducible when subsequent runs were performed. Contrary to what has previously been reported in the literature, no reasonable separation was possible in bare fused silica.