Papel das hemaglutininas 67-72p de Corynebacterium diphtheriae na ligação a proteínas plasmáticas, superfícies celulares, invasão e indução de apoptose

Corynebacterium diphtheriae pode ser isolado tanto de quadros de difteria clássica, quanto de infecções sistêmicas, como endocardite. O fibrinogênio (Fbn) e a fibronectina (Fn) são glicoproteínas presentes na matriz extracelular de tecidos conjuntivos. A influência destas proteínas na patogênese das infecções locais e invasivas causadas por C. diphtheriae é objeto de estudo devido ao fato do bacilo diftérico poder ser encontrado em lesões nas quais o Fbn e a Fn são predominantes, incluindo a pseudomembrana diftérica e vegetações cardíacas presentes na endocardite infecciosa. São crescentes as evidências de que o C. diphtheriae pode, além de aderir, ser internalizado por células em cultura. No presente estudo, investigou-se a participação de C. diphtheriae e das proteínas de superfície 67-72p na aderência à Fn e ao Fbn de plasma humano e a eritrócitos. A aderência às células HEp-2 e internalização também foram analisadas. A participação de 67-72p nos mecanismos de morte celular foi avaliada através das colorações por Azul de Tripan e 46-diamidino-2-fenil indol (DAPI), pelo ensaio de redução utilizando dimetil-tiazol-difenil tetrazólio (MTT) e por citometria de fluxo. As 67-72p foram extraídas da superfície da amostra toxigênica C. diphtheriae subsp. mitis CDC-E8392 através de processos mecânicos e precipitação com sulfato de amônio saturado. Análises por SDS-PAGE e immunoblotting detectaram a presença das bandas protéicas de 67 e 72kDa nas amostras toxinogênicas e atoxinogênicas analisadas, as quais pertenciam aos biotipos fermentador e não fermentador de sacarose. C. diphtheriae foi capazes não só de formar agregados na presença de plasma de coelho, mas também de converter Fbn em fibrina independentemente da presença do gene tox. No entanto, a amostra atoxinogênica ATCC 27010 (tox-) foi menos aderente ao Fbn do que a homóloga ATCC 27012 (tox+). A interação bacteriana com eritrócitos foi inibida somente pela Fn. Ligações entre Fn e/ou Fbn com 67-72p foram demonstradas por dot blotting, ELISA e/ou ensaios utilizando fluorescência. As 67-72p foram capazes de inibir as interações bacterianas com o Fbn, indicando que 67-72p podem participar do processo de aderência do patógeno aos tecidos do hospedeiro. Através da microscopia óptica, demonstrou-se a ligação de 67-72p adsorvidas em microesferas de látex com células HEp-2. Anticorpos de coelho do tipo IgG anti 67-72p interferiram somente com a expressão do padrão de aderência do tipo difuso, normalmente apresentado pela amostra CDC-E8392. A Microscopia Eletrônica de Transmissão (MET) e a inibição da internalização bacteriana pela IgG anti 67-72p ou por 67-72p indicaram o papel de 67-72p como invasina. Alterações do citoesqueleto de células HEp-2 com acumulação de actina polimerizada, induzida por microesferas sensibilizadas com 67-72p, foi observada pelo fluorescent actin staining (FAS) test. Foi visualizado um aumento no número de bactérias viáveis no compartimento intracelular após tratamento de células HEp-2 ou dos microrganismos com Fn. A presença de partículas de látex adsorvidas com 67-72p no interior de vacúolos frouxos em células HEp-2 sugeriu que estas proteínas podem causar efeito citotóxico. A avaliação através das colorações com Azul de Tripan, DAPI e os ensaios de redução utilizando MTT demonstraram um decréscimo na viabilidade de células tratadas com 67-72p. As mudanças morfológicas observadas 3 horas após o início do tratamento com 67-72p incluíram vacuolização, fragmentação nuclear e formação de corpúsculos apoptóticos. A citometria de fluxo revelou um decréscimo de 15,13% no volume/tamanho de células tratadas com 67-72p. Além disso, o ensaio utilizando Iodeto de Propídio (IP) e Anexina V (AV)-FITIC demonstrou que havia 66,1% de células vivas (IP-/AV-), 16,6% de células em apoptose inicial (IP-/AV+) e 13,8% de células em apoptose tardia ou necrose secundária. Em conclusão, as 67-72p estão diretamente envolvidas na interação com Fn e Fbn. As proteínas não fimbriais 67-72p são hemaglutininas implicadas na aderência a células respiratórias e na internalização. Além disso, estas proteínas podem atuar como fatores de virulência em potencial para induzir apoptose de células epiteliais nos estágios iniciais da difteria e nas infecções invasivas causadas pelo C. diphtheriae

Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of I-125-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with I-125-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the I-125 methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than I-125-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

Mechanisms involved in Escherichia coli and Serratia marcescens removal during activated sludge wastewater treatment

Wastewater treatment reduces environmental contamination by removing gross solids and mitigating the effects of pollution. Treatment also reduces the number of indicator organisms and pathogens. In this work, the fates of two coliform bacteria, Escherichia coli and Serratia marcescens, were analyzed in an activated sludge process to determine the main mechanisms involved in the reduction of pathogenic microorganisms during wastewater treatment. These bacteria, modified to express green fluorescent protein, were inoculated in an activated sludge unit and in batch systems containing wastewater. The results suggested that, among the different biological factors implied in bacterial removal, bacterivorous protozoa play a key role. Moreover, a representative number of bacteria persisted in the system as free-living or embedded cells, but their distribution into liquid or solid fractions varied depending on the bacterium tested, questioning the real value of bacterial indicators for the control of wastewater treatment process. Additionally, viable but nonculturable cells constituted an important part of the bacterial population adhered to solid fractions, what can be derived from the competition relationships with native bacteria, present in high densities in this environment. These facts, taken together, emphasize the need for reliable quantitative and qualitative analysis tools for the evaluation of pathogenic microbial composition in sludge, which could represent an undefined risk to public health and ecosystem functions when considering its recycling.

掺铒碲酸盐玻璃的热力学稳定性和光谱性质的研究

分析了掺Er^3+碲酸盐玻璃的热力学稳定性能，研究了掺Er^3+碲酸盐玻璃的吸收和荧光光谱性质；应用Judd-Ofelt理论计算了碲酸盐玻璃中Er^3+离子的强度参数Ω(Ω2=4．79×10^-20cm^2,Ω4=1．52×10^-20cm^2，Ω6=0．66×10^-20cm^2)，计算了离子的自发跃迁几率，荧光分支比；应用McCumber理论计算了Er^3+的受激发射截面(σe=10．40×10^-21cm^2)、Er^3+离子^4I13／2→^4I15／2发射谱的荧光半高宽(FWHM=65．5nm)

一种新型掺铒碲酸盐玻璃的光谱性质研究

研究了一种新型掺Er^3+碲酸盐玻璃的光谱性质；应用Judd-Ofelt理论计算了碲酸盐玻璃中Er^3+离子的强度参数Ω(Ω2=4．79×10^-20cm^2，Ω4=1．52×10^-20cm^2，Ω6=0．66×10^-20cm^2)，计算了离子的自发跃迁概率，荧光分支比；应用McCumber理论计算了Er^3+的受激发射截面(σe=10．40×10^-21cm^2)，Er^3+离子^4I13/2→^4I15/2发射谱的荧光半高宽(FWHM=65．5nm)及各能级的荧光寿命(^4I13/2能级为τrad

Influência da espessura do filme de cimento na resistência adesiva de pinos de fibra de vidro cimentados em condutos radiculares de dentes humanos

O presente estudo visa avaliar a influência da espessura do filme de cimento sobre a resistência de união de pinos de fibra de vidro em diferentes regiões do conduto radicular (cervical, médio e apical), cimentados com cimento resinoso autoadesivo, com e sem a adição de Rodamina B, por meio do teste de push-out. Quarenta raízes foram incluídas em resina epóxi, submetidas a tratamento endodôntico e obturadas com guta percha e cimento endodôntico sem eugenol. Após sete dias, os condutos foram desobstruídos e aleatoriamente divididos em 4 grupos (n=10), de acordo com as brocas do sistema de pinos de fibra WhitePost DC (FGM) usadas: (G1) broca #2; (G2) broca #3; (G3) broca #4; (G4) broca #2. O preparo foi realizado a uma profundidade de 10 mm. A cimentação foi realizada com o cimento resinoso autoadesivo RelyX U100 (3M ESPE), e apenas nos grupos G1, G2 e G3 uma pequena quantidade de Rodamina B em pó foi usada como corante no cimento. Após uma semana, cada raiz foi seccionada em máquina de corte, obtendo-se 6 fatias de 1 0,1 mm de espessura. Antes do ensaio de push-out, imagens digitais foram obtidas, por meio de um estereomicroscópio, de ambas as faces de cada fatia, para determinação do raio dos pinos e da espessura do filme de cimento. Após o ensaio mecânico, novas imagens foram obtidas para determinação do modo de falha. Para determinar a espessura de cimento, foi desenvolvida uma rotina (macro) no software KS 400. Os dados foram estatisticamente analisados com análise de variância (ANOVA) 2 fatores (influência do diâmetro da broca e influência dos terços) e Kruskal-Wallis (influência da espessura do filme de cimento). Comparações múltiplas foram realizadas com o teste Duncan. Todos os testes foram aplicados com α=0,05. Houve diferenças significantes entre os grupos em relação ao diâmetro da broca (p<0,0001), sendo G2 (14,62 5,15 MPa) > G1 (10,04 5,13 MPa) > G3 (7,68 6,14 MPa). O terço do conduto exerceu influência significativa sobre a resistência adesiva (p<0,0001), sendo os maiores valores obtidos no terço apical. As espessuras do filme de cimento foram estatisticamente diferentes nos grupos. Os maiores valores de espessura de cimento foram obtidos no G3 (248,78 μm), seguido de G2 (185,91 μm) e G1 (110,16 μm), sendo o último estatisticamente semelhante ao G4 (119,99 μm). Os resultados de G1(10,04 5,13 MPa) e G4 (8,89 + 5,18 MPa) foram estatisticamente semelhantes, indicando que a presença da Rodamina B não influencia significativamente na resistência de união. O tipo de falha predominante no G1 foi mista, no G2, adesiva entre pino e cimento, e no G3 e G4, adesiva entre cimento e dentina. O diâmetro da broca influenciou significativamente nos resultados de resistência de união ao teste de push-out. O ligeiro aumento na espessura do filme de cimento promoveu um aumento nos valores de resistência, quando comparado com filmes de cimento muito finos ou muito espessos.

Intense infrared luminescence in transparent glass-ceramics containing beta-Ga2O3 : Ni2+ nanocrystals

Transparent glass-ceramics containing beta-Ga2O3:Ni2+ nanocrystals were synthesized and characterized by X-ray diffraction, transmission electron microscopy, and electron energy loss spectroscopy. Intense broad-band luminescence centering at 1200 nm was observed when the sample was excited by a diode laser at 980 nm. The room-temperature fluorescent lifetime was 665 mu s, which is longer than the Ni2+-doped ZnAl2O4 and LiGa5O8 glass-ceramics and is also comparable to the Ni2+-doped LiGa5O8 single crystal. The intense infrared luminescence with long fluorescent lifetime may be ascribed to the high crystal field hold by Ni2+ and the moderate lattice phonon energy of beta-Ga2O3. The excellent optical properties of this novel material indicate that it might be a promising candidate for broad-band amplifiers and room-temperature tunable lasers.

Transparent Ni2+-doped lithium-alumino-silicate glass-ceramics for broadband near-infrared light source

Broadband infrared luminescence centred at around 1300 nm with full-width at half maximum of about 342 nm was observed from transparent Ni2+-doped lithium-alumino-silicate glass-ceramics embedded with beta-eucryptite crystallines. The room temperature fluorescent lifetime was 98 mu s. The transparent glass-ceramics may have potential applications in a widely tunable laser and a super-broadband optical amplifier for optical communications.

Bismuth-doped zinc aluminosilicate glasses and glass-ceramics with ultra-broadband infrared luminescence

Broadband infrared luminescence covering the optical telecommunication wavelength region of 0, E and S bands was observed from bismuth-doped zinc aluminosilicate glasses and glass-ceramics. The spectroscopic properties of the glasses and glass-ceramics depend on the thermal-treatment history. With the appearance of gahnite (ZnAl2O4) crystalline phase, the fluorescent peak moves to longer wavelength, but the fluorescent intensity decreases. The similar to 1300 nm fluorescence with a FWHM larger than 250 nm and a lifetime longer than 600 mu s possesses these optical materials with potential applications in laser devices and broadband amplifiers. The broad infrared luminescence from the bismuth-doped zinc aluminosilicate glasses and glass-ceramics might be from BiO or bismuth clusters rather than from Bi5+ and Bi3+. (c) 2005 Elsevier B.V. All rights reserved.

Blue upconversion Luminescence in Tm3+/Yb3+-Codoped New Oxyfluoride Tellurite Glass

The thermal stability, Raman spectrum and upconversion properties of Tm^(3+)/Yb^(3+) co-doped new oxyfluoride tellurite glass are investigated. The results show that Tm^(3+)/Yb^(3+) co-doped oxyfluoride tellurite glass possesses good thermal stability, lower phonon energy, and intense upconversion blue luminescence. Under 980-nm laser diode (LD) excitation, the intense blue (475 nm) emission and weak red (649 nm) emission corresponding to the 1G4 -> 3H6 and 1G4 -> 3F4 transitions of Tm^(3+) ions respectively, were simultaneously observed at room temperature. The possible upconversion mechanisms are evaluated. The intense blue upconversion luminescence of Tm^(3+)/Yb^(3+) co-doped oxyfluoride tellurite glass can be used as potential host material for the development of blue upconversion optical devices.

Effect of various alkaline-earth metal oxides on the broadband infrared luminescence from bismuth-doped silicate glasses

We report on the effect of various alkaline-earth metal oxides on the broadband infrared luminescence covering 1000-1600 nm wavelength region from bismuth-doped silicate glasses. The full width at half maximum (FWHM) of the infrared luminescence and the fluorescent lifetime is more than 200 nm and 400 mu s, respectively. The fluorescent intensity decreases with increasing basicity of host glasses. Besides the broadband infrared luminescence, luminescence centered at 640 nm was also observed, which should be ascribed to Bi2+ rather than to the familiar Bi3+. We suggest that the infrared luminescence should be assigned to the X-2 (2)Pi (3/2) -> X-1 (2)Pi(1/2) transition of BiO molecules dispersed in the host glasses. (c) 2006 Elsevier Ltd. All rights reserved.

Effects of Yb ion concentration on the spectral properties of lead silica glasses

The spectral properties in different concentration of Yb ions (0.5-5 mol%)-doped silica glasses are explored in this paper. The glasses are prepared by traditional melting method. The absorption spectra and the fluorescent lifetime (tau(f)) are measured at room temperature and low temperature (18 K). The stimulated cross-section (sigma(emi)) and potential laser properties (beta(min), I-sat, I-min) are calculated based on the absorption spectra. The absorption cross-section (sigma(abs)) are in the range 1.08 x 10(-20) - 1.18 x 10(-20) cm(2) in different glasses, the fluorescence lifetime (tau(f)) change from 1.9 to 1.2 ms with the increase of Yb3+ concentration. The potential laser properties indicate that lead silica glass is a good host for highly Yb ion doping glass. (c) 2005 Elsevier B.V. All rights reserved.

Spectroscopic and fluorescence decay behaviors of Yb3+ -doped SiO2-PbO-Na2O-K2O glass

The spectroscopic and fluorescent decay behaviors of Yb3+-doped SiO2-PbO-Na2O-K2O glass is reported in this work. Yb2O3 contents of 1, 1.5 and 2mol% are added into the glass. Through the measurement of absorption and fluorescence spectra, and fluorescent decay rate at room temperature and at low temperature (18 K), it is found that the nonradiative decay rate of Yb3+ ions is mainly determined by the interaction between residual hydroxyl groups and Yb3+ ions. Concentration quenching effect can be omitted in this glass up to the Yb3+ ion concentration of 8.98 x 10(20)/cm(3). Multiphonon decay rate is also very small because of the large energy gap between F-2(5/2) and F-2(7/2) levels of Yb3+ ions. (c) 2004 Elsevier B.V. All rights reserved.

Enhanced broadband near-infrared luminescence in transparent silicate glass ceramics containing Yb3+ ions and Ni2+-doped Li Ga5 O8 nanocrystals

Spectral properties of Yb3+/Ni2+ codoped transparent silicate glass ceramics containing LiGa5O8 nanocrystals were investigated. The near-infrared emission intensity of Ni2+ was largely increased with Yb3+ codoping due to Yb3+-> Ni2+ energy transfer. The qualitative calculation of the energy transfer constant Cs-a and rate Ps-a showed that the Yb3+-> Ni2+ energy transfer was much greater than in the opposite direction. Yb3+/Ni2+ codoped glass ceramics with 0.75 mol % Yb2O3 exhibited a near-infrared emission with full width at half maximum of 290 nm and fluorescent lifetime of 920 mu s. The glass ceramics are promising for broadband optical amplification.

Preparation and spectroscopic properties of Yb-doped and Yb-Al-codoped high silica glasses

Yb-doped and Yb-Al-codoped high silica glasses have been prepared by sintering nanoporous glasses. The absorption, fluorescent spectra and fluorescent lifetimes have been measured and the emission cross-section and minimum pump intensities were calculated. Codoping aluminum ions enhanced the fluorescence intensity of Yb-doped high silica glass obviously. The emission cross-sections of Yb-doped and Yb-Al-codoped high silica glasses were 0.65 and 0.82 pm(2), respectively. The results show that Yb-Al-codoped high silica glass has better spectroscopic properties for a laser material. The study of high silica glass doped with ytterbium is helpful for its application in Yb laser systems, especially for high-power and high-repetition lasers. (C) 2007 Elsevier B.V. All rights reserved.