金诺芬对表皮葡萄球菌及其生物膜的影响

目的 研究金诺芬对表皮葡萄球菌及其生物膜的作用。方法 利用微量稀释法药敏试验检测金诺芬对表皮葡萄球菌浮游菌生长的影响,并利用结晶紫染色和激光共聚焦显微镜观察金诺芬对表皮葡萄球菌生物膜形成的影响。结果 金诺芬对表皮葡萄球菌的MIC和MBC分别为0.125~0.250 μg/mL和2.000~4.000 μg/mL。同时,4 μg/mL金诺芬还能显著抑制表皮葡萄球菌标准菌株和临床菌株生物膜的形成（P<0.05）。通过激光共聚焦显微镜观察发现金诺芬能有效抑制生物膜的形成,降低生物膜的聚集,并增加死亡细菌的比例。结论 金诺芬能显著抑制表皮葡萄球菌浮游菌的增殖和生物膜的形成。     

Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration of spatial distribution of viability

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc.     

Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 348–354, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effect of cinnamaldehyde on biofilm formation and sarA expression by methicillin‐resistant Staphylococcus aureus

Aims: To investigate the antibiofilm effect of cinnamaldehyde on methicillin‐resistant Staphylococcus aureus (MRSA) and analyse the effect of subminimum inhibitory concentrations (MICs) of cinnamaldehyde on the expression of the biofilm‐related gene sarA. Methods and Results: The MICs and minimum bactericidal concentrations (MBCs) were determined using a microtitre broth dilution method. Biofilm susceptibility was determined using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) staining and colony forming unit (CFU) counting assays. Antibiofilm effects were studied with scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). SarA expression was assessed by real‐time PCR. MICs and MBCs were in the range 0·0625–0·5% (v/v). The killing effects were concentration dependent. At a concentration of 5× MIC, all strains in biofilm were decreased to lower than 20% of the control groups. SEM and CLSM images indicated that a 5× MIC concentration of cinnamaldehyde was able to detach and kill existing biofilms. Apart from strain JB‐06, real‐time PCR showed that the expression of sarA of all other strains was decreased upon exposure to sub‐MICs of cinnamaldehyde. Conclusions: These data showed the strong killing effect of cinnamaldehyde against MRSA within biofilms. Significance and Impact of the Study: This study indicated the potential of cinnamaldehyde as an inhibitory agent for use in MRSA biofilm‐related infections.     

Importance of biofilm formation for corrosion inhibition of SAE 1018 steel by axenic aerobic biofilms

To investigate if corrosion inhibition by aerobic biofilms is a general phenomenon, carbon steel (SAE 1018) coupons were exposed to a complex liquid medium (Luria–Bertani) and seawater-mimicking medium (VNSS) containing fifteen different pure-culture bacterial suspensions representing seven genera. Compared to sterile controls, the mass loss in the presence of these bacteria (which are capable of developing a biofilm to various degrees) decreased by 2- to 15-fold. The extent of corrosion inhibition in LB medium depended on the nature of the biofilm: an increased proportion of live cells, observed with confocal scanning laser microscopy (CSLM) and image analysis, decreased corrosion. Corrosion inhibition in LB medium was greatest with Pseudomonas putida (good biofilm formation), while metal coupons exposed to Streptomyces lividans in LB medium (poor biofilm formation) corroded in a manner similar to the sterile controls. Pseudomonas mendocina KR1 reduced corrosion the most in VNSS. It appears that only a small layer of active, respiring cells is required to inhibit corrosion, and the corrosion inhibition observed is due to the attached biofilm. Received 09 December 1996/ Accepted in revised form 19 March 1997     

Monohalogenated maleimides as potential agents for the inhibition of Pseudomonas aeruginosa biofilm

New monohalogenated maleimide derivatives (with bromine, chlorine or iodine) were synthesized to test the effect of halogen atoms in inhibiting the formation of Pseudomonas aeruginosa biofilm. The evaluation of their biological activities clearly defines a structure–activity relationship. In this study, the bactericidal action of the three compounds was observed at the concentration range 0.3–5.0 mM on Luria-Bertani agar plates. The halogen atom of these molecules was critical in modulating the antibacterial activity, with a slightly higher effectiveness for chlorine. Confocal laser scanning microscopy was used to examine P. aeruginosa biofilms cultivated in flow cells. At concentration as low as 40 μM, the bromine and iodine compounds displayed a total inhibition towards the formation of bacterial biofilm. At this concentration, the bacterial attachment to glass surfaces was strongly affected by the presence of bromine and iodine whereas the chlorine derivative behaved as a bactericidal compound. A bioluminescent reporter strain was then used to detect the effect of the chemically synthesized maleimides on quorum sensing (QS) in P. aeruginosa. At the concentration range 10–100 μM, bioluminescence assays reveal that halogenated maleimides were able to interfere with the QS of the bacterium. Although the relationship between the weak inhibition of cell-to-cell communication (15–55% of the signal) and the high inhibition of biofilm formation has not been elucidated clearly, the results demonstrate that bromo- and iodo-N-substituted maleimides bromine and iodine may be used as new potent inhibitors that control bacterial biofilms.     

Membrane biofouling characterization: effects of sample preparation procedures on biofilm structure and the microbial community

Ensuring the quality and reproducibility of results from biofilm structure and microbial community analysis is essential to membrane biofouling studies. This study evaluated the impacts of three sample preparation factors (ie number of buffer rinses, storage time at 4°C, and DNA extraction method) on the downstream analysis of nitrifying biofilms grown on ultrafiltration membranes. Both rinse and storage affected biofilm structure, as suggested by their strong correlation with total biovolume, biofilm thickness, roughness and the spatial distribution of EPS. Significant variations in DNA yields and microbial community diversity were also observed among samples treated by different rinses, storage and DNA extraction methods. For the tested biofilms, two rinses, no storage and DNA extraction with both mechanical and chemical cell lysis from attached biofilm were the optimal sample preparation procedures for obtaining accurate information about biofilm structure, EPS distribution and the microbial community.     

Specific lectins map the distribution of fibronectin and beta 1-integrin on living MDCK cells

The expression and dynamics of bound fibronectin and the sialylated integral membrane protein, beta 1-integrin, were analyzed on the apical membrane of living MDCK cells. Fibronectin was identified by its specific binding of fluorescent peanut agglutinin and sialylated beta 1-integrin by its binding of Sambucus nigra agglutinin. Confocal epifluorescence microscopy and laser scanning cytometry determined the distribution and abundance of binding sites of the two fluorescently labeled lectins. Both fibronectin and beta 1-integrin were restricted to specific regions uniformly distributed over the entire apical surface. Apical-surface fibronectin binding varied much more between cells than did the expression of beta 1-integrin. Sialylated beta 1-integrin colocalized >92% with membrane microplicae while fibronectin was unrelated to these surface structures. This lack of colocalization of the proteins was confirmed by double-labeling experiments. From the maturation dependence of the fibronectin-binding capacity and the differences in protein turnover times, it was evident that fibronectin did not bind to sialylated beta 1-integrin. Furthermore, desialylation of beta 1-integrin uncovered additional fibronectin receptors on the apical membrane. We conclude that these lectins permit tracking of two membrane-associated glycoproteins in living cells and that fibronectin binds only to desialylated beta 1-integrin on MDCK cells.     

Bacillus cereus B-02对Botrytis cinerea拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拮抗机理。结果表明,处理菌丝表面形态受到严重破坏,发生强烈变形;菌丝细胞核、线粒体和细胞壁等亚细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较大液泡。此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响。     

Bacillus cereus B-02对Botrytis cinerea 拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拈抗机理.结果表明,处理菌丝表面形态受到严重破坏,发牛强烈变形;荫丝细胞核、线粒体和细胞壁等哑细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较人液泡.此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响.     

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

非培养细胞的贴壁方法及细胞内Ca^2＋观测

本文介绍非培养细胞贴壁的简易方法和细胞内Ｃａ＾２＋观察。以ｆｌｕｏ－３／ＡＭ进行染色,在３７℃孵育箱内孵育６０分钟,用激光共聚焦显微镜监测细胞内Ｃａ＾２＋的荧光信号。结果表明本方法可成功地应用于共聚焦显微镜测定细胞的研究。     

Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces

Staphylococcus epidermidis is a significant nosocomial pathogen in predisposed hosts because of its capability of forming a biofilm on indwelling medical devices. The initial stage of biofilm formation has a key role in S. epidermidis abiotic surface colonization. Recently, many strategies have been developed to create new anti-biofilm surfaces able to control bacterial adhesion mechanisms. In this work, the self-assembled amphiphilic layers formed by two fungal hydrophobins (Vmh2 and Pac3) have proven to be able to reduce the biofilm formed by different strains of S. epidermidis on polystyrene surfaces. The reduction in the biofilm thickness on the coated surfaces and the preservation of cell vitality have been demonstrated through confocal laser scanning microscope analysis. Moreover, the anti-biofilm efficiency of the self-assembled layers on different medically relevant materials has also been demonstrated using a CDC biofilm reactor.     

变应性鼻炎鼻分泌物中细胞内DNA和RNA代谢的观测

本文介绍应变性鼻炎鼻分泌物中细胞内DNA和RNA代谢变化的观测方法。以鼻分泌物中的多种炎性细胞为实验标本,经细胞贴壁、0.1%吖橙染色标记,37℃孵育15min;采用激光扫描共聚焦显微镜对细胞内DNA,RNA形态和含量进行观测。结果表明：本方法应用于变应性鼻炎鼻分泌物细胞内DNA、RNA代谢的研究是切实可行的。     

Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells

Alveolar type II (ATII) cells in the peripheral human lung spontaneously differentiate toward ATI cells, thus enabling air‐blood barrier formation. Here, linear Raman and coherent anti‐Stokes Raman scattering (CARS) microscopy are applied to study cell differentiation of freshly isolated ATII cells. The Raman spectra can successfully be correlated with gradual morphological and molecular changes during cell differentiation. Alveolar surfactant rich vesicles in ATII cells are identified based on phospholipid vibrations, while ATI‐like cells are characterized by the absence of vesicular structures. Complementary, CARS microscopy allows for three‐dimensional visualization of lipid vesicles within ATII cells and their secretion, while hyperspectral CARS enables the distinction between cellular proteins and lipids according to their vibrational signatures. This study paves the path for further label‐free investigations of lung cells and the role of the pulmonary surfactant, thus also providing a basis for rational development of future lung therapeutics.      

Acridine orange distribution and fluorescence spectra in myoblasts and single muscle fibers

Acridine orange (AO) fluorescence spectra in nuclei and cytoplasm of living myoblasts L6J1 and frog single muscle fibers have been studied using spectral scanning system of Leica TCS SL confocal microscope. AO fluorescence spectra in salt solutions dependent on free AO concentrations or in complex with DNA have also been obtained. Myoblast nuclei fluoresced in the green spectral region with maximum at about 530 nm; nucleoli had the brightest fluorescence. The fluorescence of nuclear chromatin was not uniform. Similar fluorescence of nuclei and nucleoli was observed in frog single muscle fibers. Uniform, weak, green fluorescence was observed in the myoblast cytoplasm. In the sarcoplasm of muscle fibers, AO green fluorescence was seen in A discs. In the cytoplasm of myoblasts and muscle fibers stained with AO, different red, yellow, and green fluorescent granules, which were acidic organelles, were visualized. The comparison of AO fluorescence spectra in living cells with AO fluorescence spectra in buffer solutions with different AO concentrations and AO in complex with DNA enables the estimation of the AO concentration in acidic granules. It is important for the evaluation of these cellular organelles functions in intracellular transport, adaptation, and apoptosis, as well as in a number of pathological processes.     

Optical and spectroscopic methods for biofilm examination and monitoring

Optical and spectroscopic methods for biofilmexamination and monitoring are reviewed.Biofilm examination techniques includemicroscopic methods, coupled with imageanalysis and with oligonucleotide ribosomal RNAprobing methods (fluorescence in situhybridization). Microscopic examinationtechniques are especially advantageous inextracting biofilm structural and architecturalparameters, as well as structure-functionrelationships of the biofilm microbialpopulation. Spectroscopic techniques are ableto elicit biofilm chemical and metabolicpatterns, as well as biofilm activity. They areof outstanding importance for on-line,non-invasive biofilm monitoring, especiallywhen coupled with chemometric algorithms forspectra calibration and pattern recognition.The paper emphasises the importance of thecombination of novel and established analyticaltechniques, as well as their integration withpowerful computational methods for theautomation of biofilm monitoring.     

Effects of sodium fluoride (NaF) on the cilia and microtubular system of Tetrahymena

The effect of the nucleophilic reagent NaF on the microtubular system of Tetrahymena was studied by using scanning electron microscopy (SEM), confocal microscopy, and flow cytometry. Treatments with 40 mM NaF significantly reduced the amount of alpha-tubulin while 80 mM treatment did not alter its quantity. One possible explanation for this alpha-tubulin overexpression is that the higher amount of alpha-tubulin enables this organism to carry out the appropriate function of the cytoskeleton under this undesirable influence of higher amounts of 80 nM NaF. However, the amount of acetylated tubulin increased in a dose-dependent manner. The cilia became fragile under the effect of 80 mM NaF. Confocal microscopy revealed that after 40 mM NaF treatment transversal microtubule bands (TMs) and longitudinal microtubule bands (LMs) as well as basal bodies (BBs) were extremely strong decorated with anti-acetylated tubulin antibody and TM-localization abnormalities were visible. In the 80 mM NaF-treated cells, the deep fiber of oral apparatus was very strongly labeled, while the TMs and LMs were less decorated with anti-acetylated tubulin antibody, and LM deformities were visible. It is supposed that post-translational tubulin modifications (e.g., acetylation) defend the microtubules against the NaF-induced injury. NaF is able to influence the activity of several enzymes and G-proteins, therefore is capable to alter the structure, metabolism, and the dynamics of microtubular system. The possible connection of signaling and cytoskeletal system in Tetrahymena is discussed.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

基于高通量共聚焦激光扫描显微镜方法定量分析副溶血性弧菌生物被膜结构

【目的】副溶血性弧菌是水产品中常见的食源性致病菌,生物被膜的形成对副溶血性弧菌的环境生存和传播至关重要。这项工作的目的是评估临床和环境中分离出的44株副溶血性弧菌菌株形成的生物被膜的结构多样性。【方法】该研究基于共聚焦激光扫描显微镜的高通量方法,使用与高分辨率成像兼容的96孔微量滴定板,结合结构分析软件ISA-2来研究生物被膜形成和结构,分析22株食品与22株临床来源的副溶血性弧菌菌株形成的生物被膜结构参数(生物体积、平均厚度、粗糙系数)。【结果】CLSM图像显示,44株副溶血性弧菌菌株在培养48h后能够形成3D结构,进一步比较分析了临床来源菌株与环境来源菌株形成的生物被膜结构异同,发现临床菌株生物被膜的变异系数比环境菌株生物被膜的变异系数小,且同时携带tdh和trh两种毒力因子的菌株生物被膜变异性最小。凝聚层次聚类分析结果显示,副溶血性弧菌生物被膜可以分为致密且表面光滑(39%)、斑驳且表面粗糙(27%)、疏松且表面坑洼(34%),临床菌株易形成致密且表面光滑和斑驳且表面粗糙的生物被膜,而环境菌株易形成致密且表面光滑和疏松且表面坑洼的生物被膜。【结论】该研究深入了解了副溶血性弧菌生物...