Papers by Emanuel Carrilho
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Chromatographia, 2001
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Quim Nova, 2006
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Microb Pathog, 2009
Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in severa... more Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant–pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X. fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with α-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed “cyanogenesis”, which catalyzes the dissociation of α-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X. fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X. fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs.
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Electrophoresis, 2010
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Molecular Plant Pathology, 2009
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Quim Nova, 2008
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ELECTROPHORESIS, 2015
One of the problems with 2DE is that proteins present in low amounts in a sample are usually not ... more One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a sample pre-purified using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie brilliant blue dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign twelve new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue. This article is protected by copyright. All rights reserved.
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Química Nova
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Analytica Chimica Acta, 2015
Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-μL scale... more Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-μL scale polymerase chain reaction (PCR) on microchips, has been integrated with DNA extraction and separation on a glass microchip in a fully integrated micro Total Analysis System by Easley et al., in 2006. IR-PCR has been demonstrated on both glass and PMMA microdevices where the fabrication (bonding) is not trivial. Polyester-toner (PeT) microfluidic devices have significant potential as cost-effective, disposable microdevices as a result of the ease of fabrication (∼$0.25 USD and <10 min per device) and availability of commercial substrates. For the first time, we demonstrate here the thermal cycling in PeT microchips on the IR-PCR system. Undesirable IR absorption by the black-toner bonding layer was eliminated with a spatial filter in the form of an aluminum foil mask. The solution heating rate for a black PeT microchip using a tungsten lamp was 10.1 ± 0.7 °C s(-1) with a cooling rate of roughly -12 ± 0.9 °C s(-1) assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the λ-phage genome (in 11 min) and a 1500 bp region of Azospirillum brasilense. Using a centrosymmetric chamber configuration in a multichamber PeT microchip, homogenous temperature distribution over all chambers was achieved with inter-chamber temperature differences at annealing, extension and denaturing steps of less than ±2 °C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human β-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human β-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of ∼10%. Development of PeT microchips for IR-PCR has the potential to provide rapid, low-volume amplification while also integrating PCR with extraction upstream and separation/detection downstream.
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Pesquisa Agropecuaria Brasileira
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Research conducted focuses on applications of Multi Walled Carbon Nanotubes (MWCNTs) serving as w... more Research conducted focuses on applications of Multi Walled Carbon Nanotubes (MWCNTs) serving as working electrodes for cyclic voltammetry (CV) and impedance spectroscopy. Expectations of increased electrochemical properties of the working electrodes were confirmed and the analysis of CNTs electrical properties was obtained. Conditions for the MWCNTs are as follows: after acidic functionalization, MWCNTs bonded with N-hydroxysuccinimide, and MWCNTs bonded with the AChE protein. Iron cyanide was chosen as the optimal analyte solution. Concentration of MWCNTs on the working electrode was also investigated. With CV an increase in capacitance, sensitivity, and sensibility was noticed. When compared to the unmodified graphite carbon electrode the modified electrode yielded lower resistivity, and higher capacitance. When compared to NHS and the AChE protein, the functionalized CNTs yielded a higher capacitance, increased sensitivity and sensibility with decrease in surface roughness. Throu...
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Scientific reports, 2015
A new protocol for fabrication of glass microchips is addressed in this research paper. Initially... more A new protocol for fabrication of glass microchips is addressed in this research paper. Initially, the method involves the use of an uncured SU-8 intermediate to seal two glass slides irreversibly as in conventional adhesive bonding-based approaches. Subsequently, an additional step removes the adhesive layer from the channels. This step relies on a selective development to remove the SU-8 only inside the microchannel, generating glass-like surface properties as demonstrated by specific tests. Named sacrificial adhesive layer (SAB), the protocol meets the requirements of an ideal microfabrication technique such as throughput, relatively low cost, feasibility for ultra large-scale integration (ULSI), and high adhesion strength, supporting pressures on the order of 5 MPa. Furthermore, SAB eliminates the use of high temperature, pressure, or potential, enabling the deposition of thin films for electrical or electrochemical experiments. Finally, the SAB protocol is an improvement on SU-...
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Nature Protocols, 2015
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Genetics and molecular research : GMR, 2014
Cytokines are small cell-signaling proteins that play an important role in the immune system, par... more Cytokines are small cell-signaling proteins that play an important role in the immune system, participating in intracellular communication. Four candidate genes of the cytokine family (IL2, IL4, IL13, and IFNG) were selected to identify Single Nucleotide Polymorphisms (SNPs) that might be associated with resistance to gastrointestinal endoparasites in goats. A population of 229 goats, F2 offspring from an F1 intercross was produced by crossing pure Saanen goats, considered as susceptible to gastrointestinal endoparasites, with pure Anglo-Nubian goats, considered resistant. Blood was collected for DNA extraction and fecal samples were also collected for parasite egg count. Polymorphisms were prospected by sequencing animals with extreme phenotype for fecal egg count (FEC) distribution. The association between SNPs and phenotype was determined by using the Fisher exact test with correction for multiple tests. Three of the 10 SNPs were identified as significant (P ≤ 0.03). They were fo...
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Phytopathology, 2008
Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges gra... more Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were...
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ELECTROPHORESIS, 2015
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Journal of High Resolution Chromatography, 1989
Abstract The present paper introduces an instrumental procedure based on column liquid chromatogr... more Abstract The present paper introduces an instrumental procedure based on column liquid chromatography for the total analysis of petroleum heavy ends and similar samples. The procedure avoids the precipitation of asphaltenes and asphaltols. One sequence of ...
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Papers by Emanuel Carrilho