Wide field scanning by optical fluorescence of oral squamous cell carcinoma (SCC): case report / Fotoevidenciação de campo amplo por fluorescência óptica de carcinoma de células escamosas (CEC) oral: estudo de casos

ABSTRACT Optical fluorescence detection aims to identify precursor lesions, little noticeable to the human eye, and oral cancer. Squamous cell carcinoma or spinocellular carcinoma is a malignant neoplasm that affects the mouth more. In this article, two clinical cases are analyzed, treated with the use of two types of equipment, namely: the photoevidenciation by optical fluorescence of light-emitting violet wavelength of 405nm, power of 100mW, which is luminous radiation, not Ionizing and in the adjuvant treatment, we used low-power laser therapy, power 100mW, with two wavelengths of 808nm infrared, for pain relief, and the red 660nm, for oral mucositis. In Photodynamic therapy, the Photosensitizing Chimiolux® (methylene blue) was used to control Candida albicans. From these cases, we discuss how a more assertive diagnostic hypothesis can save a life and save time, resources, and efforts for the correct diagnosis of the pathology compared to a biopsy and histopathology negative for neoplasia. We conclude that optical fluorescence has excellent social relevance due to its potential to help the professional not specialized in the establishment of early diagnosis of oral cancer. Early diagnosis improves the rates of death caused by this carcinoma, which would extend the post-diagnosis survival and decrease the financial and emotional costs for the patient and family.

RESUMO A detecção óptica por fluorescência visa identificar lesões precursoras, pouco perceptíveis ao olho humano, e do câncer oral. O carcinoma de células escamosas ou carcinoma espinocelular (CEC), é a denominação de uma neoplasia maligna que acomete mais a boca. Neste artigo, são analisados dois casos clínicos, tratados com a utilização de dois equipamentos, a saber: o de fotoevidenciação por fluorescência óptica de emissão de luz violeta de comprimento de onda de 405nm, potência de 100mW, que é radiação luminosa não ionizante; e no tratamento coadjuvante, utilizou-se a laserterapia de baixa potência, potência 100mW, com dois comprimentos de onda de infravermelho 808nm, para alívio de dor, e o vermelho 660nm, para as mucosites orais. Na terapia fotodinâmica, empregou-se o fotossensibilizador Chimiolux® (azul de metileno) para controle de Candida albicans. A partir desses casos, discutimos como uma hipótese diagnóstica mais assertiva pode salvar uma vida e poupar tempo, recursos e esforços para o correto diagnóstico da patologia se comparado a uma biópsia e histopatológico negativo para neoplasia. Por fim, concluímos que a fluorescência óptica tem grande relevância social devido a seu potencial de auxiliar o profissional não especialista no estabelecimento de um diagnóstico precoce do câncer oral, melhorando os índices de óbito causados por esse carcinoma, o que estenderia a sobrevida pós-diagnóstico e diminuiria os custos financeiros e emocionais do paciente e familiares.

Screening Aedes aegypti (Diptera: Culicidae) Populations From Pernambuco, Brazil for Resistance to Temephos, Diflubenzuron, and Cypermethrin and Characterization of Potential Resistance Mechanisms.

Resistance to chemical insecticides detected in Aedes aegypti (L.) mosquitoes has been a problem for the National Dengue Control Program (PNCD) over the last years. In order to provide deeper knowledge of resistance to xenobiotics, our study evaluated the susceptibility profile of temephos, diflubenzuron, and cypermethrin insecticides in natural mosquito populations from the Pernambuco State, associating these results with the local historical use of such compounds. Furthermore, mechanisms that may be associated with this particular type of resistance were characterized. Bioassays with multiple temephos and diflubenzuron concentrations were performed to detect and quantify resistance. For cypermethrin, diagnostic dose assays were performed. Biochemical tests were carried out to quantify the activity of detoxification enzymes. In addition, a screening of mutations present in the voltage-gated sodium channel gene (NaV) was performed in samples previously submitted to bioassays with cypermethrin. The populations under study were resistant to temephos and showed a positive correlation between insecticide consumption and the resistance ratio (RR) to the compound. For diflubenzuron, the biological activity ratio (BAR) ranged from 1.3 to 4.7 times, when compared to the susceptible strain. All populations showed resistance to cypermethrin. Altered enzymatic profiles of alpha, p-nitrophenyl acetate (PNPA) esterases and glutathione-S-transferases were recorded in most of these samples. Molecular analysis demonstrated that Arcoverde was the only population that presented the mutated form 1016Ile/Ile. These findings show that the situation is critical vis-à-vis the effectiveness of mosquito control using chemical insecticides, since resistance to temephos and cypermethrin is widespread in Ae. aegypti from Pernambuco.

Susceptibility profile of Aedes aegypti from Santiago Island, Cabo Verde, to insecticides.

In 2009, Cabo Verde diagnosed the first dengue cases, with 21,137 cases reported and Aedes aegypti was identified as the vector. Since the outbreak, chemical insecticides and source reduction were used to control the mosquito population. This study aimed to assess the susceptibility of A. aegypti populations from Santiago, Cabo Verde to insecticides and identify the mechanisms of resistance. Samples of A. aegypti eggs were obtained at two different time periods (2012 and 2014), using ovitraps in different locations in Santiago Island to establish the parental population. F1 larvae were exposed to different concentrations of insecticides (Bacillus thuringiensis var israelensis (Bti), diflubenzuron and temephos) to estimate the lethal concentrations (LC90) and calculate the respective rate of resistance (RR90). Semi-field tests using temephos-ABATE(®) were performed to evaluate the persistence of the product. Bottle tests using female mosquitoes were carried out to determine the susceptibility to the adulticides malathion, cypermethrin and deltamethrin. Biochemical and molecular tests were performed to investigate the presence of metabolic resistance mechanisms, associated with the enzymes glutathione S-transferases (GSTs), esterases and mixed-function oxidases (MFO) and to detect mutations or alterations in the sodium channel and acetylcholinesterase genes. A. aegypti mosquitoes from Santiago exhibited resistance to deltamethrin, cypermethrin (mortality<80%) and temephos (RR90=4.4) but susceptibility to malathion (mortality≥98%), Bti and diflubenzuron. The low level of resistance to temephos did not affect the effectiveness of Abate(®). The enzymatic analysis conducted in 2012 revealed slight changes in the activities of GST (25%), MFO (18%), α-esterase (19%) and ß-esterase (17%), but no significant changes in 2014. Target site resistance mutations were not detected. Our results suggest that the A. aegypti population from Santiago is resistant to two major insecticides used for vector control, deltamethrin and temephos. To our knowledge, this is the first report of temephos resistance in an African A. aegypti population. The low level of temephos resistance was maintained from 2012-2014, which suggested the imposition of selective pressure, although it was not possible to identify the resistance mechanisms involved. These data show that the potential failures in the local mosquito control program are not associated with insecticide resistance.

TGF-ß1 promotes cerebral cortex radial glia-astrocyte differentiation in vivo.

The major neural stem cell population in the developing cerebral cortex is composed of the radial glial cells, which generate glial cells and neurons. The mechanisms that modulate the maintenance of the radial glia (RG) stem cell phenotype, or its differentiation, are not yet completely understood. We previously demonstrated that the transforming growth factor-ß1 (TGF-ß1) promotes RG differentiation into astrocytes in vitro (Glia 2007; 55:1023-33) through activation of multiple canonical and non-canonical signaling pathways (Dev Neurosci 2012; 34:68-81). However, it remains unknown if TGF-ß1 acts in RG-astrocyte differentiation in vivo. Here, we addressed the astrogliogenesis induced by TGF-ß1 by using the intraventricular in utero injection in vivo approach. We show that injection of TGF-ß1 in the lateral ventricles of E14,5 mice embryos resulted in RG fibers disorganization and premature gliogenesis, evidenced by appearance of GFAP positive cells in the cortical wall. These events were followed by decreased numbers of neurons in the cortical plate (CP). Together, we also described that TGF-ß1 actions are region-dependent, once RG cells from dorsal region of the cerebral cortex demonstrated to be more responsive to this cytokine compared with RG from lateral cortex either in vitro as well as in vivo. Our work demonstrated that TGF-ß1 is a critical cytokine that regulates RG fate decision and differentiation into astrocytes in vitro and in vivo. We also suggest that RG cells are heterogeneous population that acts as distinct targets of TGF-ß1 during cerebral cortex development.

Septin C-terminal domain interactions: implications for filament stability and assembly.

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K(D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K(D) of 4 µM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Insecticide resistance in Aedes aegypti populations from Ceará, Brazil.

BACKGROUND: Organophosphates and pyrethroids are used widely in Brazil to control Aedes aegypti, the main vector of dengue viruses, under the auspices of the National Programme for Dengue Control. Resistance to these insecticides is widespread throughout Brazil. In Ceará the vector is present in 98% of districts and resistance to temephos has been reported previously. Here we measure resistance to temephos and the pyrethroid cypermethrin in three populations from Ceará and use biochemical and molecular assays to characterise resistance mechanisms. RESULTS: Resistance to temephos varied widely across the three studied populations, with resistance ratios (RR(95)) of 7.2, 30 and 192.7 in Juazeiro do Norte, Barbalha and Crato respectively. The high levels of resistance detected in Barbalha and Crato (RR(95) ≥ 30) imply a reduction of temephos efficacy, and indeed in simulated field tests reduced effectiveness was observed for the Barbalha population. Two populations (Crato and Barbalha) were also resistant to cypermethrin, whilst Juazeiro do Norte showed only an altered susceptibility. The Ile1011Met kdr mutation was detected in all three populations and Val1016Ile in Crato and Juazeiro do Norte. 1011Met was significantly associated with resistance to cypermethrin in the Crato population. Biochemical tests showed that only the activity of esterases and GSTs, among the tested detoxification enzymes, was altered in these populations when compared with the Rockefeller strain. CONCLUSIONS: Our results demonstrate that two A. aegypti populations from Ceará are under strong selection pressure by temephos, compromising the field effectiveness of this organophosphate. Our results also provide evidence that the process of reducing resistance to this larvicide in the field is difficult and slow and may require more than seven years for reversal. In addition, we show resistance to cypermethrin in two of the three populations studied, and for the first time the presence of the allele 1016Ile in mosquito populations from northeastern Brazil. A significant association between 1011Met and resistance was observed in one of the populations. Target-site mechanisms seem not to be implicated in temephos resistance, reinforcing the idea that for the studied populations, detoxification enzymes most likely play a major role in the resistance to this insecticide.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 µM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization.

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 alpha-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 alpha-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.

The stability and aggregation properties of the GTPase domain from human SEPT4.

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTP. At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease.

An intermediate structure in the thermal unfolding of the GTPase domain of human septin 4 (SEPT4/Bradeion-beta) forms amyloid-like filaments in vitro.

SEPT4 is a member of the mammalian septin family of GTPases. Mammalian septins are conserved proteins which form heteropolymers in vivo and which are implicated in a variety of cellular functions such as cytokinesis, exocytosis, and vesicle trafficking. However, their structural properties and modes of action are largely unknown. There is a limited, but as yet inconclusive, amount of experimental data suggesting that SEPT4 may accumulate in tau-based filamentous deposits and cytoplasmic inclusions in Alzheimer's and Parkinson's disease, respectively. Here we report an intermediate structure of the GTPase domain of human SEPT4 (SEPT4-G) during unfolding transitions induced by temperature. This partially unfolded intermediate, which is rich in beta-sheet and free of bound nucleotide, was plagued by irreversible aggregation. The aggregates have the ability to bind specific dyes such as Congo red and thioflavin-T, suggesting they are amyloid in nature. Under electron microscopy, fibers of variable diameter extending for several micrometers in length can be visualized. This is the first report of amyloid formation by a septin or domain thereof, and the capacity of SEPT4-G to form such fibrillar aggregates may shed some light on the current discussion concerning the formation of homo- and heteropolymers of septins in vitro.

Dissection of a human septin: definition and characterization of distinct domains within human SEPT4.

The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.

In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis.

A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.

Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides.

A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 A resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 A. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (VM = 2.5 A3 Da(-1)). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 A resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.

Structure of the cytosolic Cu,Zn superoxide dismutase from Schistosoma mansoni.

Cu,Zn superoxide dismutase (Cu,Zn SOD) is an essential enzyme for protecting cells from the toxic effects of reactive oxygen species. In humans, two distinct Cu,Zn SOD genes are located on chromosomes 4 and 21 and mutations in the latter have been associated with familial amyotrophic lateral sclerosis. Similarly, schistosomes (trematode parasites responsible for the chronically debilitating disease schistosomiasis) also produce two distinct Cu,Zn SODs, in this case one cytosolic and one bearing a signal peptide. The crystal structure of the cytosolic form of the enzyme from the human trematode Schistosoma mansoni (SmCtSOD) was solved and refined to a resolution of 2.2 A (space group P2(1)2(1)2(1), R = 17.6% and R(free) = 24.1%) and 1.55 A (space group P2(1), R = 15.7% and R(free) = 17.1%). This is the first report of a crystal structure of a Cu,Zn superoxide dismutase derived from a human parasite. Alternate positions for the catalytic copper and its water ligand were refined for the 1.55 A SmCtSOD model, but the most interesting structural differences between SmCtSOD and the human homologue reside in the loops used for electrostatic guidance of the substrate to the enzyme active site.

Abrus pulchellus type-2 RIP, pulchellin: heterologous expression and refolding of the sugar-binding B chain.

Abrus pulchellus type-2 RIP, or pulchellin, is a heterodimeric glycoprotein found in A. pulchellus seeds. These chimerolectins, like all type-2 RIPs, are characterized as highly toxic proteins with enzymatic and lectin properties performed by two separate polypeptide subunits. Intending to obtain pure and homogeneous protein for structural and biological studies, the A. pulchellus type-2 RIP lectin subunit or pulchellin binding chain encoding gene fragment (PBC) was cloned. Oligonucleotides based on the sequence homologies between other RIPs like abrin and ricin were synthesized and used to amplify the complete PBC from A. pulchellus genomic DNA. The amplification product was inserted into plasmid pET28a to express the recombinant PBC (rPBC) in Escherichia coli BL21(DE3). The rPBC was expressed as inclusion bodies that were recovered and denatured in a buffer containing urea. Repeated dialysis rounds against the oxidation buffer, which presented the redox pair cysteine-cystine, D-galactose, and decreasing urea concentrations, conducted the protein refolding. The refolding process of rPBC was successfully confirmed by biological assays and circular dichroism.