Investigation of factors related to biofilm formation in Providencia stuartii.

Providencia stuartii is one of the Enterobacteriaceae species of medical importance commonly associated with urinary infections, which can also cause other ones, including uncommon ones, such as liver abscess and septic vasculitis. This bacterium stands out in the expression of intrinsic and acquired resistance to antimicrobials. Besides, it uses mechanisms such as biofilm for its persistence in biotic and abiotic environments. This study investigated the cellular hydrophobicity profile of clinical isolates of P. stuartii. It also analyzed genes related to the fimbrial adhesin in this species comparing with other reports described for other bacteria from Enterobacteriaceae family. The investigated isolates to form biofilm and had a practically hydrophilic cell surface profile. However, fimH and mrkD genes were not found in P. stuartii, unlike observed in other species of Enterobacteriaceae. These results show that P. stuartii has specificities regarding its potential for biofilm formation, which makes it difficult to destabilize the infectious process and increases the permanence of this pathogen in hospital units.

Observations of sesquiterpenes and their oxidation products in central Amazonia during the wet and dry seasons.

Biogenic volatile organic compounds (BVOCs) from the Amazon forest region represent the largest source of organic carbon emissions to the atmosphere globally. These BVOC emissions dominantly consist of volatile and intermediate-volatility terpenoid compounds that undergo chemical transformations in the atmosphere to form oxygenated condensable gases and secondary organic aerosol (SOA). We collected quartz filter samples with 12 h time resolution and performed hourly in situ measurements with a semi-volatile thermal desorption aerosol gas chromatograph (SV-TAG) at a rural site ("T3") located to the west of the urban center of Manaus, Brazil as part of the Green Ocean Amazon (GoAmazon2014/5) field campaign to measure intermediate-volatility and semi-volatile BVOCs and their oxidation products during the wet and dry seasons. We speciated and quantified 30 sesquiterpenes and 4 diterpenes with mean concentrations in the range 0.01-6.04 ngm-3 (1-670ppqv). We estimate that sesquiterpenes contribute approximately 14 and 12% to the total reactive loss of O3 via reaction with isoprene or terpenes during the wet and dry seasons, respectively. This is reduced from ~ 50-70 % for within-canopy reactive O3 loss attributed to the ozonolysis of highly reactive sesquiterpenes (e.g., ß-caryophyllene) that are reacted away before reaching our measurement site. We further identify a suite of their oxidation products in the gas and particle phases and explore their role in biogenic SOA formation in the central Amazon region. Synthesized authentic standards were also used to quantify gas- and particle-phase oxidation products derived from ß-caryophyllene. Using tracer-based scaling methods for these products, we roughly estimate that sesquiterpene oxidation contributes at least 0.4-5 % (median 1 %) of total submicron OA mass. However, this is likely a low-end estimate, as evidence for additional unaccounted sesquiterpenes and their oxidation products clearly exists. By comparing our field data to laboratory-based sesquiterpene oxidation experiments we confirm that more than 40 additional observed compounds produced through sesquiterpene oxidation are present in Amazonian SOA, warranting further efforts towards more complete quantification.

Impact of Preservation Conditions on Fatty Acids, Xanthan Gum Production and Other Characteristics of Xanthomonas campestris pv. mangiferaeindicae IBSBF 2103.

The conditions of storage, cultivation and maintenance of microbial cultures should preserve the microbiological homogeneity, phenotypic and genotypic characteristics to ensure better reproducibility of metabolic production. To evaluate the influence of the storage condition on the composition of cell fatty acids, genetic profile and biochemical characteristics of Xanthomonas campestris pv. mangiferaeindicae IBSBF 2103, as well as, to identify its relationship with the yielding and viscosity of the xanthan gum produced, this study monitored the strain preserved in two simple and widely used conditions, ultra-freezer (-80 °C) and refrigeration (3-8 °C) during 5 months. Were identified and quantified 13 fatty acids. The cells preserved at -80 °C showed more stable concentration of all fatty acids, producing more xanthan gum and with higher viscosity. The chromosomal analysis obtained with the enzyme XbaI revealed 17 distinct fragments with maximum size of 485 kilobases, without variations among the subcultures maintained in both storage conditions. The X. campestris pv. mangiferaeindicae subcultures preserved at -80 °C showed less pronounced phenotypic variations, which had positive influence in the qualitative and quantitative characteristics of the xanthan gum produced.

Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin ß2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

Sensitive bi-enzymatic biosensor based on polyphenoloxidases-gold nanoparticles-chitosan hybrid film-graphene doped carbon paste electrode for carbamates detection.

A bi-enzymatic biosensor (LACC-TYR-AuNPs-CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC-TYR-AuNPs-CS/GPE exhibited an improved Michaelis-Menten kinetic constant (26.9±0.5M) when compared with LACC-AuNPs-CS/GPE (37.8±0.2M) and TYR-AuNPs-CS/GPE (52.3±0.4M). Using 4-aminophenol as substrate at pH5.5, the device presented wide linear ranges, low detection limits (1.68×10(-9)±1.18×10(-10)-2.15×10(-7)±3.41×10(-9)M), high accuracy, sensitivity (1.13×10(6)±8.11×10(4)-2.19×10(8)±2.51×10(7)%inhibitionM(-1)), repeatability (1.2-5.8% RSD), reproducibility (3.2-6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8±0.3% (lemon) to 97.8±0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.

Laccase-Prussian blue film-graphene doped carbon paste modified electrode for carbamate pesticides quantification.

A novel enzymatic biosensor for carbamate pesticides detection was developed through the direct immobilization of Trametes versicolor laccase on graphene doped carbon paste electrode functionalized with Prussian blue films (LACC/PB/GPE). Graphene was prepared by graphite sonication-assisted exfoliation and characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. The Prussian blue film electrodeposited onto graphene doped carbon paste electrode allowed considerable reduction of the charge transfer resistance and of the capacitance of the device. The combined effects of pH, enzyme concentration and incubation time on biosensor response were optimized using a 2(3) full-factorial statistical design and response surface methodology. Based on the inhibition of laccase activity and using 4-aminophenol as redox mediator at pH 5.0, LACC/PB/GPE exhibited suitable characteristics in terms of sensitivity, intra- and inter-day repeatability (1.8-3.8% RSD), reproducibility (4.1 and 6.3% RSD), selectivity (13.2% bias at the higher interference:substrate ratios tested), accuracy and stability (ca. twenty days) for quantification of five carbamates widely applied on tomato and potato crops. The attained detection limits ranged between 5.2×10(-9)molL(-1) (0.002mgkg(-1) w/w for ziram) and 1.0×10(-7)molL(-1) (0.022mgkg(-1) w/w for carbofuran). Recovery values for the two tested spiking levels ranged from 90.2±0.1 (carbofuran) to 101.1±0.3% (ziram) for tomato and from 91.0±0.1% (formetanate) to 100.8±0.1% (ziram) for potato samples. The proposed methodology is appropriate to enable testing pesticide levels in food samples to fit with regulations and food inspections.

Biosensor based on multi-walled carbon nanotubes paste electrode modified with laccase for pirimicarb pesticide quantification.

This study focused on the development of a sensitive enzymatic biosensor for the determination of pirimicarb pesticide based on the immobilization of laccase on composite carbon paste electrodes. Multi-walled carbon nanotubes (MWCNTs) paste electrode modified by dispersion of laccase (3%, w/w) within the optimum composite matrix (60:40%, w/w, MWCNTs and paraffin binder) showed the best performance, with excellent electron transfer kinetic and catalytic effects related to the redox process of the substrate 4-aminophenol. No metal or anti-interference membrane was added. Based on the inhibition of laccase activity, pirimicarb can be determined in the range 9.90 × 10(-7) to 1.15 × 10(-5) mol L(-1) using 4-aminophenol as substrate at the optimum pH of 5.0, with acceptable repeatability and reproducibility (relative standard deviations lower than 5%). The limit of detection obtained was 1.8 × 10(-7) mol L(-1) (0.04 mg kg(-1) on a fresh weight vegetable basis). The high activity and catalytic properties of the laccase-based biosensor are retained during ca. one month. The optimized electroanalytical protocol coupled to the QuEChERS methodology were applied to tomato and lettuce samples spiked at three levels; recoveries ranging from 91.0 ± 0.1% to 101.0 ± 0.3% were attained. No significant effects in the pirimicarb electroanalysis were observed by the presence of pro-vitamin A, vitamins B1 and C, and glucose in the vegetable extracts. The proposed biosensor-based pesticide residue methodology fulfills all requisites to be used in implementation of food safety programs.

Identification of microbial and proteomic biomarkers in early childhood caries.

The purpose of this study was to provide a univariate and multivariate analysis of genomic microbial data and salivary mass-spectrometry proteomic profiles for dental caries outcomes. In order to determine potential useful biomarkers for dental caries, a multivariate classification analysis was employed to build predictive models capable of classifying microbial and salivary sample profiles with generalization performance. We used high-throughput methodologies including multiplexed microbial arrays and SELDI-TOF-MS profiling to characterize the oral flora and salivary proteome in 204 children aged 1-8 years (n = 118 caries-free, n = 86 caries-active). The population received little dental care and was deemed at high risk for childhood caries. Findings of the study indicate that models incorporating both microbial and proteomic data are superior to models of only microbial or salivary data alone. Comparison of results for the combined and independent data suggests that the combination of proteomic and microbial sources is beneficial for the classification accuracy and that combined data lead to improved predictive models for caries-active and caries-free patients. The best predictive model had a 6% test error, >92% sensitivity, and >95% specificity. These findings suggest that further characterization of the oral microflora and the salivary proteome associated with health and caries may provide clinically useful biomarkers to better predict future caries experience.

Avaliação da superfície dentinária após diferentes técnicas de acabamento / Evaluation of dentin surface after different finishing techniques

O objetivo deste trabalho foi comparar, in vitro, diferentes técnicas de acabamento de preparos por meio da análise da rugosidade superficial e microscopia eletrônica de varredora (MEV). Cinquenta e seis pré-molares humanos foram divididos em quatro grupos, (n= 10): Grupo I - sem acabamento (grupo controle); Grupo II - ponta diamantada 2135 F e 2135 FF; Grupo III - ponta 2135 acopladas em um contra ângulo multiplicador (Kavo Koncept 1:5) e Grupo IV - ponta CVD cilíndrica 8.2137 (CVDentus- Clorovale) em aparelho de ultra-som (Profi II - Dabi Atlante). Após o acabamento, dez espécimes de cada grupo foram submetidos à leitura rugosimétrica e quatro foram analisados no MEV (Microscopia eletrônica de varredura). Sessenta e quatro micrografias foram analisadas por doze examinadores. Os valores de Ra (média±dp) foram: GI- 1,46±0,2; GII- 0,87±0,3; GIII- 1,6±0,4 e GIV- 1,34±0,2. Após teste de t-Student, observou-se que não houve diferença significativa entre os grupos I (controle), III (multiplicador) e IV (CVD). Entretanto, houve diferença entre o grupo I e II; Grupo II e III; GII e I. Após teste de Mann-Whitney para análise em MEV, observou-se que não houve diferença significativa entre os grupos I (controle), II (ponta diamantada fina e extra-fina) e III (multiplicador). Conclui-se que o grupo que apresentou menor valor de rugosidade (Ra) foi o grupo II, realizado com pontas diamantadas 2135 F e 2135FF montadas em alta rotação. Para análise em MEV, concluiu-se que a superfície produzida pelo grupo IV (CVD) foi a mais rugosa.

The objective of this study was to compare, in vitro, different techniques for finishing preparations of facets indirect through the analysis of surface roughness and electron microscopy sweeper (SEM). Forty human premolars were divided into 4 groups according to the finishing of the cavity preparation for indirect facets: Group I (n = 10) - unfinished (control group), Group II (n = 10) - diamond bur 2135 F and 2135FF, Group III (n = 10)- tip in 2135 engaged in a multiplier (Contra angle multiplier Koncept Kavo 1:5) and Group IV (n = 10) tip cylindrical CVDcat. 8.2137 (CVDentus - Clorovale) in equipment ultrasound (Profi II- Dabi Atlante). The reading was held rugosimetric relief meter Mitutoyo SJ-201. For SEM analysis were prepared 16 teeth were divided into four groups according to the finishing technique described above, with four teeth in each group, totaling 64 images, analyzed by twelve examiners. The values of Ra (mean ± SD) were: GI-1.46 ± 0.2, GII - 0.87 ± 0.3, GIII, 1.6 ± 0.4 and GIV-1.34 ± 0.2 . After test t-test (Figure 1), it was observed that there was no significant difference between groups I (control),III (multiplier) and IV (CVD). However, there was difference between group I and II, Group II and III, GII and I. After the Mann-Whitney test for analysis by SEM, it was observed that there was no significant difference between groups I (control), II (thin diamond point) and III (multiplier). However, there was difference between group I, II, III with group IV (CVD), with p <0.001. It follows that the group with lowest roughness (Ra) (smoother) the group II was performed with diamond burs and 2135 F 2135FF mounted on heavy rotation. For SEM analysis, itwas concluded that surface produced by the group IV (CVD) was the most wrinkled. The values assigned by examiners through the analysis of SEM were low averaging less than 5.0 for all groups.

O impacto do aumento dos incisivos superiores na atratividade e rejuvenescimento do sorriso / Impact of increasing upper incisors length on smiles attractiveness and youthful appearance

O propósito desta pesquisa foi avaliar a atratividade e percepção da idade do sorriso por leigos e cirurgiões-dentistas frente ao aumento do comprimento de incisivos superiores em fotografias aproximadas e frontais da face. Foram obtidas fotografas iniciais e finais do sorriso aproximado e da face. Os resultados apresentaram uma diferença estatisticamente signifcativa (P menor ou igual a 0,001) com maior atratividade na fotografia final para leigos e cirurgiões-dentistas. O aumento do comprimento dos incisivos superiores proporcionou sete anos de rejuvenescimento considerando a percepção dos cirurgiões-dentistas, quatro anos para os leigos e um impacto positivo na atratividade entre as fotos iniciais e finais, sendo uma alternativa de tratamento estético para alcançar um sorriso agradável.

The purpose of this study was to evaluate atractiveness and perception of aging on smile by laypersons and dental professionals on close-up and facial photographs. The results show that increasing superior incisors length provides seven years of youthful appearance by dental professionals, four years by laypersons and positive impact on atractiveness between initial and final photographs, providing an alternative treatment that enhances an agreeable smile.

Effects of natural flavones on membrane properties and citotoxicity of HeLa cells / Efeitos de flavonas naturais em propriedades de membranas e em citotoxicidade de células HeLa

The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM) decreased approximately 30 percent the rate and total amplitude of valinomycin induced swelling and 60-100 percent the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM) reduced 35 percent the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM) promoted a reduction of 40 percent in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.

O objetivo deste estudo foi avaliar se eupafolina e hispidulina, flavonas extraídas do Eupatorium littorale Cabrera, Asteraceae, possuíam a capacidade de alterar propriedades das membranas biológicas e promover efeitos citotóxicos. Eupafolina (50-200 µM) reduziu em aproximadamente 30 por cento a velocidade e amplitude do inchamento mitocondrial induzido por valinomicina e 60-100 por cento o inchamento mitocondrial dependente de substrato. Além disso, eupafolina na dose de 200 µM reduziu a transição de permeabilidade mitocondrial em 35 por cento entretanto, a hispidulina não alterou este parâmetro em todas as doses testadas. A avaliação da transição de fase dos lipossomas de DMPC com a sonda DPH demonstrou que ambas as flavonas afetam a fase gel e fluida. Quando lipossomas de membranas mitocondriais e a sonda DPH-PA foram utilizados, houve aumento da polarização de fluorescência promovido pela hispidulina. Eupafolina e hispidulina, na dose de 100 µM, promoveram 40 por cento de redução da viabilidade de células HeLa em 24 h. Nossos resultados sugerem que eupafolina e hispidulina têm efeitos citotóxicos que podem ser explicados em parte pelas alterações promovidas por estas flavonas sobre propriedades de membranas biológicas e sobre a bioenergética mitocondrial.

Eupafolin: Effect on mitochondrial energetic metabolism.

This study evaluated the effects of flavone eupafolin (6-methoxy 5,7,3',4'-tetrahydroxyflavone), extracted from dry leaves of Eupatorium litoralle. Eupafolin (25-200microM) promoted inhibition of the respiratory rate in state 3, in the presence of glutamate or succinate. During succinate oxidation, it was found that only state 4 respiratory rate was stimulated approximately 30% by eupafolin (100microM) and ADP/O ratio and RCC were reduced with all doses. When glutamate was used as substrate, RCC was similarly reduced. Eupafolin caused a reduction of enzymatic activities between complexes I and III of the respiratory chain. Cytochrome c oxidase and ATPase activities were not affected. Using voltammetry cyclic analysis, eupafolin give rise to irreversible oxidation with an anodic peak potential at +0.08V (SHE). We also observed that eupafolin can undergo oxidation catalyzed by EDTA-Fe, promoting cytochrome c reduction in the presence of NADH, resulting in the production of the superoxide radical and hydrogen peroxide. All together, the results could explain the cytotoxic effects observed previously with the eupafolin.

Sensitivities of the alternative respiratory components of potato tuber mitochondria to thiol reagents and Ca2+.

Plant mitochondria differ from those of mammals, since they incorporate an alternative electron transport pathway, which branches at ubiquinol to an alternative oxidase (AOX), characteristically inhibited by salicylhydroxamic acid (SHAM). Another feature of plant mitochondria is that besides complex I (EC 1.6.5.3) they possess alternative NAD(P)H-dehydrogenases insensitive to rotenone. Many stress conditions are known to alter the expression of the alternative electron transport pathway in plant mitochondria. In the present study we investigated the effects of some thiol reagents and Ca(2+) on potato mitochondrial respiratory chain presenting different activities of the alternative respiratory components AOX and external NADH dehydrogenase, a condition induced by previous treatment of potato tubers (Solanum tuberosum L., cv. Bintje) to cold stress. The results showed that Ca(2+) presented an inhibitory effect on AOX pathway in potato mitochondria energized with NADH or succinate, which was only now observed when the cytochrome pathway was inhibited by cyanide. When the cytochrome pathway was functional, Ca(2+) stimulated the external NADH dehydrogenase. Diamide was a potent AOX inhibitor and this effect was only now observed when the cytochrome pathway was inactive, as was the case for the calcium ion. Mersalyl inhibited the externally located NADH dehydrogenase and had no effect on AOX activity. The results may represent an important function of Ca(2+) on the alternative mitochondrial enzymes NADH-DH(ext) and AOX.

Antimelanoma activity of 1,3,4-thiadiazolium mesoionics: a structure-activity relationship study.

The effect of a series of 4-phenyl-5-(2'-Y, 4'-X or 4'-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides was evaluated against B16-F10 murine melanoma cells in vitro and against tumors resulting from implanted B16-F10 cells in C57BL/6 mice. These compounds differ from each other only at the cinnamoyl ring substituent (MI-J, X=OH; MI-2,4diF, X=Y=F; MI-4F, X=F and MI-D, X=NO2). The results were compared with those obtained for MI-D, which has already been shown to be a potent and promising drug against melanoma. On exposure of B16-F10 cells to MI-D, MI-2,4diF and MI-4F, all of them at the same micromolar concentration (50 microM) decreased the cell viability to 8, 50 and 22%, respectively, while MI-J did not show any significant effect under the same conditions. However, low doses such as 10 microM MI-D were sufficient to impair cell growth over 72 h, but for MI-2,4diF and MI-4F the effect on B16-F10 proliferation was only observed at a concentration of 25 microM. Furthermore, MI-4F had a slightly better effect than MI-2,4diF in vitro; its effect on tumor growth in vivo was not significant. MI-D inhibited tumor growth by 77%. The greater effectiveness of MI-D compared with MI-2,4diF, MI-4F and MI-J against B16-F10 melanoma cells is probably due to its stronger electron-withdrawing group (NO2), which increases the positive charge on the mesoionic ring and allows extensive conjugation of the side-chain with the exocyclic moiety. This seems to be important for degree of anti-tumor activity of these compounds.