JOURNAL OF FOOD PROCESS ENGINEERING, Issue 5 2010
PETER WAMBURA
ABSTRACT Peanut oil migrates to the outer surface during roasting, where it comes into contact with oxygen, leading to the oxidation reactions. Because of its cleaning effect, power ultrasound (sonication) was used for removing surface lipid of roasted peanuts. Georgia green runner-type peanuts were roasted at 178C for 15 min. Roasted peanuts were subjected to lipid extraction in n-hexane by sonication. Fluorescent and electron scanning micrographs revealed that the surface of sonicated peanuts was free of oil stains, as opposed to that of freshly roasted peanuts. These results showed that power ultrasound could remove the lipids from peanut surfaces very effectively. Details of microstructure of sonicated peanuts as was observed using scanning electron microscope reveal that 10 min sonication was sufficient to extract most of the lipids on the roasted peanut surfaces. Fluorescence and scanning electron microscopy are useful in peanut analysis because they can detect lipids in low concentration. PRACTICAL APPLICATIONS There is increasing interest of quick procedures to examine the surfaces of roasted peanut samples after undergoing treatments, such as removal of lipids. This research demonstrated the significant use of fluorescent and scanning electron microscopes to quickly study the extent of lipid removal from the surface of roasted peanuts after power ultrasound treatment (sonication). [source]

FLUORESCENCE-BASED MAXIMAL QUANTUM YIELD FOR PSII AS A DIAGNOSTIC OF NUTRIENT STRESS

JOURNAL OF PHYCOLOGY, Issue 4 2001
Jean-Paul Parkhill
In biological oceanography, it has been widely accepted that the maximum quantum yield of photosynthesis is influenced by nutrient stress. A closely related parameter, the maximum quantum yield for stable charge separation of PSII, (,PSII)m, can be estimated by measuring the increase in fluorescence yield from dark-adapted minimal fluorescence (Fo) to maximal fluorescence (Fm) associated with the closing of photosynthetic reaction centers with saturating light or with a photosynthetic inhibitor such as 3,-(3,4-dichlorophenyl)-1,,1,-dimethyl urea (DCMU). The ratio Fv/Fm (= (Fm, Fo)/Fm) is thus used as a diagnostic of nutrient stress. Published results indicate that Fv/Fm is depressed for nutrient-stressed phytoplankton, both during nutrient starvation (unbalanced growth) and acclimated nutrient limitation (steady-state or balanced growth). In contrast to published results, fluorescence measurements from our laboratory indicate that Fv/Fm is high and insensitive to nutrient limitation for cultures in steady state under a wide range of relative growth rates and irradiance levels. This discrepancy between results could be attributed to differences in measurement systems or to differences in growth conditions. To resolve the uncertainty about Fv/Fm as a diagnostic of nutrient stress, we grew the neritic diatom Thalassiosira pseudonana (Hustedt) Hasle et Heimdal under nutrient-replete and nutrient-stressed conditions, using replicate semicontinuous, batch, and continuous cultures. Fv/Fm was determined using a conventional fluorometer and DCMU and with a pulse amplitude modulated (PAM) fluorometer. Reduction of excitation irradiance in the conventional fluorometer eliminated overestimation of Fo in the DCMU methodology for cultures grown at lower light levels, and for a large range of growth conditions there was a strong correlation between the measurements of Fv/Fm with DCMU and PAM (r2 = 0.77, n = 460). Consistent with the literature, nutrient-replete cultures showed consistently high Fv/Fm (,0.65), independent of growth irradiance. Under nutrient-starved (batch culture and perturbed steady state) conditions, Fv/Fm was significantly correlated to time without the limiting nutrient and to nutrient-limited growth rate before starvation. In contrast to published results, our continuous culture experiments showed that Fv/Fm was not a good measure of nutrient limitation under balanced growth conditions and remained constant (,0.65) and independent of nutrient-limited growth rate under different irradiance levels. Because variable fluorescence can only be used as a diagnostic for nutrient-starved unbalanced growth conditions, a robust measure of nutrient stressed oceanic waters is still required. [source]

SOURCING OBSIDIAN FROM NEOLITHIC ÇATALHÖYÜK (TURKEY) USING ENERGY DISPERSIVE X-RAY FLUORESCENCE,

ARCHAEOMETRY, Issue 3 2007
T. CARTER
This paper details the chemical sourcing of 42 obsidian artefacts from a single Neolithic structure at Çatalhöyük (central Anatolia), using Energy Dispersive X-Ray Fluorescence (EDXRF). The chemical signatures of the samples match those of two geological sources in southern Cappadocia: East Göllü Da, and Nenezi Da,. The data provide a counterpoint for previous analyses at the site, and suggest possible intra-community distinctions with regard to shifts in raw material procurement and technical change. [source]

FLUORESCENCE ACTIVATED CELL SORTING OF TRANSIENTLY TRANSFECTED As4.1 CELLS SHOWS RENIN ENHANCER DIRECTS ON/OFF SWITCHING OF RENIN PROMOTER IN VITRO

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2008
Brian J MorrisArticle first published online: 27 FEB 200
SUMMARY 1The proximal promoter of the renin gene is weak and its activity is influenced by a strong, far-upstream enhancer. This and the ability of renin expression in renal afferent arteriolar cells to be ,recruited' under chronic stimulation is consistent with the on/off switching (variegation) model of gene expression. If true, this would provide an example in which variegation controls a physiologically regulable gene. 2The present study tested the hypothesis that renin promoter activity may accord with the variegation model, at least in individual juxtaglomerular (mouse As4.1) cells in vitro. 3As4.1 cells were transiently transfected with constructs containing the mouse renin (Ren-1c) enhancer adjacent to the Ren-1c promoter and a linked reporter gene encoding enhanced green fluorescent protein (EGFP). The EGFP signal from individual cells was monitored by fluorescence activated cell sorting. 4In the presence of the renin enhancer there was 10-fold higher EGFP expression in transfected cells compared with cells transfected with EGFP constructs containing the promoter alone. There was, moreover, an 8-fold increase in the number of EGFP expressing cells. However, EGFP expression in individual transfected cells was similar in the presence or absence of the enhancer. 5Results from the in vitro system used suggest that the Ren-1c enhancer does not regulate the rate of promoter activity, but rather increases the probability of achieving an active transcriptional state. Limitations of these findings are discussed. [source]

Rapid and easy semi-quantitative evaluation method for diacylglycerol and inositol-1,4,5-trisphosphate generation in orexin receptor signalling

ACTA PHYSIOLOGICA, Issue 3 2010
M. E. Ekholm
Abstract Aim:, Fluorescent protein-based indicators have enabled measurement of intracellular signals previously nearly inaccessible for studies. However, indicators showing intracellular translocation upon response suffer from serious limitations, especially the very time-consuming data collection. We therefore set out in this study to evaluate whether fixing and counting cells showing translocation could mend this issue. Methods:, Altogether three different genetically encoded indicators for diacylglycerol and inositol-1,4,5-trisphosphate were transiently expressed in Chinese hamster ovary cells stably expressing human OX1 orexin receptors. Upon stimulation with orexin-A, the cells were fixed with six different protocols. Results:, Different protocols showed clear differences in their ability to preserve the indicator's localization (i.e. translocation after stimulus) and its fluorescence, and the best results for each indicator were obtained with a different protocol. The concentration,response data obtained with cell counting are mostly comparable to the real-time translocation and biochemical data. Conclusion:, The counting method, as used here, works at single time point and looses the single-cell-quantitative aspect. However, it also has some useful properties. First, it easily allows processing of a 100- to 1000-fold higher cell numbers than real-time imaging producing statistically consistent population-quantitative data much faster. Secondly, it does not require expensive real-time imaging equipment. Fluorescence in fixed cells can also be quantitated, though this analysis would be more time-consuming than cell counting. Thirdly, in addition to the quantitative data collection, the method could be applied for identifying responsive cells. This might be very useful in identification of e.g. orexin-responding neurones in a large population of non-responsive cells in primary cultures. [source]

Force propagation and force generation in cells,

CYTOSKELETON, Issue 9 2010
Oliver Jonas
Abstract Determining how forces are produced by and propagated through the cytoskeleton (CSK) of the cell is of great interest as dynamic processes of the CSK are intimately correlated with many molecular signaling pathways. We are presenting a novel approach for integrating measurements on cell elasticity, transcellular force propagation, and cellular force generation to obtain a comprehensive description of dynamic and mechanical properties of the CSK under force loading. This approach uses a combination of scanning force microscopy (SFM) and Total Internal Reflection Fluorescence (TIRF) microscopy. We apply well-defined loading schemes onto the apical cell membrane of fibroblasts using the SFM and simultaneously use TIRF microscopy to image the topography of the basal cell membrane. The locally distinct changes of shape and depth of the cytoskeletal imprints onto the basal membrane are interpreted as results of force propagation through the cytoplasm. This observation provides evidence for the tensegrity model and demonstrates the usefulness of our approach that does not depend on potentially disturbing marker compounds. We confirm that the actin network greatly determines cell stiffness and represents the substrate that mediates force transduction through the cytoplasm of the cell. The latter is an essential feature of tensegrity. Most importantly, our new finding that, both intact actin and microtubule networks are required for enabling the cell to produce work, can only be understood within the framework of the tensegrity model. We also provide, for the first time, a direct measurement of the cell's mechanical power output under compression at two femtowatts. © 2010 Wiley-Liss, Inc. [source]

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: Cellular localization, spatial expression and overexpression

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2008
Takahiro Katayama
It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A,D) showing sequence similarity to human homologues of Sir2 (SIRT1,3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription,polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum. [source]

Determination of vigabatrin in human plasma by means of CE with LIF detection

ELECTROPHORESIS, Issue 19 2007
Alessandro Musenga
Abstract A method has been developed for the quantitation of the antiepileptic drug vigabatrin (VGB) in human plasma. It is based on CE with LIF detection. The effect of the pH of the buffer and of N -methylglucamine (GLC) as BGE constituent was investigated. The final BGE consisted of 50,mM borate buffer, pH,9.0, with 100,mM GLC and enabled separation within 12,min at 20,kV voltage. An SPE procedure was used for the pretreatment of biological samples, based on mixed-mode lipophilic-cation exchange cartridges, followed by a derivatization step with 6-carboxyfluorescein- N -succinimidyl ester (CFSE). Fluorescence was excited by an Ar-ion laser (,exc,=,488,nm). Linearity was observed in the 10,120,,g/mL plasma concentration range. Extraction yield was >96%, precision (expressed as RSD) <6.7% and accuracy (recovery) was between 97.0 and 101.6%. The method has been successfully applied to the analysis of VGB in plasma of epileptic patients undergoing therapy with the drug. [source]

Light-emitting diode-induced fluorescence detection of native proteins in capillary electrophoresis

ELECTROPHORESIS, Issue 21 2005
Chanan Sluszny
Abstract A continuous-wave 280 nm light-emitting diode (LED) was used as the excitation source for native fluorescence detection of proteins in CE. The operating current and temperature of the LED were optimized in order to achieve high luminescence power. It was found that a forward current of 30,mA and a temperature of approximately 5°C gave the best S/N. By using a set of two ball lenses to focus light from the LED, we achieved a spot of approximately 200,,m with a power of 0.1,0.2,mW on the detection window. Fluorescence was collected with a ball lens at 90° angle through a bandpass filter onto a photomultiplier tube. In CZE an LOD of 20,nM for conalbumin was reached. In capillary gel electrophoresis all eight proteins from a commercial standard kit were detected with high S/N. For a 10,,g/mL total protein mixture, S/N was better than 3 for all proteins in solution. Further improvement in LOD should be possible on utilization of an LED with higher luminescence power. [source]

Visualization of Differential Gene Expression , Using Fluorescence-Based cDNA-AFLP

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2004
S. Gigliotti
Abstract cDNA-AFLP is one of the techniques developed to study differentially expressed genes. This recent technique is advantageous because it does not need prior sequence knowledge and is reliable due to highly stringent PCR conditions. The traditional cDNA-AFLP method uses radioactively labelled products and is characterised by high sensitivity and resolution. Here, the use of Cy5-labelled primers to detect products on polyacrylamide gels is reported. This non-radioactive method, based on fluorescence, is shown to be faster and the recovery of interesting bands is easier. The study of the differential gene expression of the interaction between potato and Phytophthora infestans was used for the valuation of this method. Different gene expression profiles , such as up-regulation, down-regulation or point expression , were obtained. Moreover, this technique was shown to be highly reproducible. [source]

Widespread occurrence of an intranuclear bacterial parasite in vent and seep bathymodiolin mussels

ENVIRONMENTAL MICROBIOLOGY, Issue 5 2009
Frank U. Zielinski
Summary Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite "Candidatus Endonucleobacter bathymodioli" that invades the nuclei of deep-sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur- and methane-oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep-sea ecosystems. We first discovered the intranuclear parasite "Ca. E. bathymodioli" in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid-Atlantic Ridge. Using primers and probes specific to "Ca. E. bathymodioli" we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of "Ca. E. bathymodioli" showed that the infection of a nucleus begins with a single rod-shaped bacterium which grows to an unseptated filament of up to 20 ,m length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by "Ca. E. bathymodioli" were those of the gill bacteriocytes. These cells contain the symbiotic sulfur- and methane-oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the "Ca. E. bathymodioli" belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates. [source]

Fluorescence in situ hybridization analysis of hindgut bacteria associated with the development of equine laminitis

ENVIRONMENTAL MICROBIOLOGY, Issue 10 2008
Gabriel J. Milinovich
No abstract is available for this article. [source]

Abundance and activity of Chloroflexi -type SAR202 bacterioplankton in the meso- and bathypelagic waters of the (sub)tropical Atlantic

ENVIRONMENTAL MICROBIOLOGY, Issue 7 2008
Marta M. Varela
Summary The contribution of Chloroflexi -type SAR202 cells to total picoplankton and bacterial abundance and uptake of d - and l -aspartic acids (Asp) was determined in the different meso- and bathypelagic water masses of the (sub)tropical Atlantic (from 35°N to 5°S). Fluorescence in situ hybridization (FISH) revealed that the overall abundance of SAR202 was , 1 × 103 cells ml,1 in subsurface waters (100 m layer), increasing in the mesopelagic zone to 3 × 103 cells ml,1 and remaining fairly constant down to 4000 m depth. Overall, the percentage of total picoplankton identified as SAR202 increased from < 1% in subsurface waters to 10,20% in the bathypelagic waters. On average, members of the SAR202 cluster accounted for about 30% of the Bacteria in the bathypelagic waters, whereas in the mesopelagic and subsurface waters, SAR202 cells contributed < 5% to total bacterial abundance. The ratio of d -Asp : l -Asp uptake by the bulk picoplankton community increased from the subsurface layer (d -Asp : l -Asp uptake ratio , 0.03) to the deeper layers reaching a ratio of ,1 at 4000 m depth. Combining FISH with microautoradiography to determine the proportion of SAR202 cells taking up d -Asp versus l -Asp, we found that ,,30% of the SAR202 cells were taking up l -Asp throughout the water column while d -Asp was essentially not taken up by SAR202. This d -Asp : l -Asp uptake pattern of SAR202 cells is in contrast to that of the bulk bacterial and crenarchaeal community in the bathypelagic ocean, both sustaining a higher fraction of d -Asp-positive cells than l -Asp-positive cells. Thus, although the Chloroflexi -type SAR202 constitutes a major bathypelagic bacterial cluster, it does not contribute to the large fraction of d -Asp utilizing prokaryotic community in the meso- and bathypelagic waters of the North Atlantic, but rather utilizes preferentially l -amino acids. [source]

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera)

ENVIRONMENTAL MICROBIOLOGY, Issue 6 2008
Dominique Yue
Summary American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae -specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death. [source]

Heterotrophic symbionts of phototrophic consortia: members of a novel diverse cluster of Betaproteobacteria characterized by a tandem rrn operon structure

ENVIRONMENTAL MICROBIOLOGY, Issue 11 2007
Kristina R. Pfannes
Summary Phototrophic consortia represent the most highly developed type of interspecific association of bacteria and consist of green sulfur bacterial epibionts attached around a central colourless rod-shaped bacterium. Based on 16S rRNA gene sequencing, the central bacterium of the consortium ,Chlorochromatium aggregatum' was recently shown to represent a novel and phylogenetically isolated lineage of the Comamonadaceae within the ,-subgroup of the Proteobacteria. To date, 19 types of phototrophic consortia are distinguished based on the different 16S rRNA gene sequences of their epibionts, but the diversity and phylogenetic relationships of the heterotrophic partner bacteria are still unknown. We developed an approach based on the specific rrn (ribosomal RNA) operon structure of the central bacterium of ,C. aggregatum' to recover 16S rRNA gene sequences of other central bacteria and their close relatives from natural consortia populations. Genomic DNA of the central bacterium of ,C. aggregatum' was first enriched several hundred-fold by employing a selective method for growth of consortia in a monolayer biofilm followed by a purification of the genome of the central bacterium by cesium chloride-bisbenzimidazole equilibrium density gradient centrifugation. A combination of inverse PCR, cloning and sequencing revealed that two rrn operons of the central bacterium are arranged in a tandem fashion and are separated by an unusually short intergenic region of 195 base pairs. This rare gene order was exploited to screen various natural microbial communities by PCR. We discovered a diverse and previously unknown subgroup of Betaproteobacteria in the chemoclines of freshwater lakes. This group was absent in other freshwater and soil samples. All the 16S rRNA gene sequences recovered are related to that of the central bacterium of ,C. aggregatum'. Fluorescence in situ hybridization indicated that two of these sequences originated from central bacteria of different phototrophic consortia, which, however, were only distantly related to the central bacterium of ,C. aggregatum'. Based on a detailed phylogenetic analysis, these central bacterial symbionts of phototrophic consortia have a polyphyletic origin. [source]

Changes in equine hindgut bacterial populations during oligofructose-induced laminitis

ENVIRONMENTAL MICROBIOLOGY, Issue 5 2006
G. J. Milinovich
Summary In the horse, carbohydrate overload is thought to play an integral role in the onset of laminitis by drastically altering the profile of bacterial populations in the hindgut. The objectives of this study were to develop and validate microbial ecology methods to monitor changes in bacterial populations throughout the course of experimentally induced laminitis and to identify the predominant oligofructose-utilizing organisms. Laminitis was induced in five horses by administration of oligofructose. Faecal specimens were collected at 8 h intervals from 72 h before to 72 h after the administration of oligofructose. Hindgut microbiota able to utilize oligofructose were enumerated throughout the course of the experiment using habitat-simulating medium. Isolates were collected and representatives identified by 16S rRNA gene sequencing. The majority of these isolates collected belonged to the genus Streptococcus, 91% of which were identified as being most closely related to Streptococcus infantarius ssp. coli. Furthermore, S. infantarius ssp. coli was the predominant oligofructose-utilizing organism isolated before the onset of lameness. Fluorescence in situ hybridization probes developed to specifically target the isolated Streptococcus spp. demonstrated marked population increases between 8 and 16 h post oligofructose administration. This was followed by a rapid population decline which corresponded with a sharp decline in faecal pH and subsequently lameness at 24,32 h post oligofructose administration. This research suggests that streptococci within the Streptococcus bovis/equinus complex may be involved in the series of events which precede the onset of laminitis in the horse. [source]

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants

ENVIRONMENTAL MICROBIOLOGY, Issue 3 2006
Yunhong Kong
Summary The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2,6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus -related PAOs and Actinobacteria -related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus -related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed. [source]

Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries

ENVIRONMENTAL MICROBIOLOGY, Issue 11 2002
Andreas Schramm
Summary A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated Td values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. [source]

Novel Metallosupramolecular Networks Constructed from CuII, NiII, and CdII with Mixed Ligands: Crystal Structures, Fluorescence, and Magnetism

EUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 6 2006
Miao Du
Abstract Reactions of mixed ligands succinic acid (H2suc) and bent dipyridines, such as 2,5-bis(3-pyridyl)-1,3,4-oxadiazole (3-bpo) and its 4- N -donor analog (4-bpo), with inorganic CuII, NiII, and CdII salts yield three new metal-organic coordination frameworks {[Cu(suc)(3-bpo)(H2O)2]·(H2O)1.75}n (1), {[Ni(suc)(4-bpo)(H2O)2]·(H2O)5}n (3), and {[Cd2(suc)2(3-bpo)2(H2O)2]·(H2O)6.75}n (4), in which the metal centers are linked by bridging ligands 3-bpo/4-bpo and suc2, along two directions to form 2D infinite networks. The corrugated 2D nets of 1 and 4, obtained under hydrothermal conditions, align in an interdigitated manner with the presence of significant aromatic-stacking interactions to result in similar 3D architectures. The 2D sheets in 3 are extended by interlayer hydrogen bonds to afford a 3D structure. However, when succinic acid is replaced by fumaric acid (H2fum) in the reaction with 3-bpo and CuII salt, a metallacyclophane [Cu(Hfum)2(3-bpo)(H2O)]2·(3-bpo)2·(H2O)6 (2) is generated. The binuclear coordinated motifs are hydrogen-bonded to the lattice water chains to furnish a unique 3D channel-like framework, in which the guest 3-bpo molecules are accommodated. The thermal stabilities of these new materials were investigated by thermogravimetric analysis (TGA) of mass loss. The magnetic coupling in complexes 1,3 is antiferromagnetic and very small, which is as expected considering the long organic bridges between the paramagnetic centers. The solid-state luminescence properties of 4 reveal an intense fluorescence emission at 378 nm. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source]

Squaraine-Doped Functional Nanoprobes: Lipophilically Protected Near-Infrared Fluorescence for Bioimaging

ADVANCED FUNCTIONAL MATERIALS, Issue 17 2010
Yong-Deok Lee
Abstract Hydrophobically stabilized near-IR fluorescence from self-assembled nanoprobes composed of amphiphilic poly(maleic anhydride- alt -octadec-1-ene) (PMAO) and lipophilized squaraine dopants is reported. From comparative studies with varying lipophilicity of squaraine dyes as well as of nanoparticulate polymer matrices, it is found that dual protection by simultaneous lipophilization of the dye-polymer pair greatly improves the chemical stability of labile squaraine dyes, to produce efficient NIR fluorescence in physiological aqueous milieux. The surface properties of negatively charged PMAO nanoparticles are readily modified by coating with an amine-rich cationic glycol chitosan with biofunctionality. Efficient cellular imaging and in vivo sentinel lymph node mapping with size and surface-controlled nanoprobes demonstrate that lipophilic dual protection of NIR fluorescence and the underlying functional nanoprobe approach hold great potential for bioimaging applications. [source]

Nanoarrays: Cooperative Near-Field Surface Plasmon Enhanced Quantum Dot Nanoarrays (Adv. Funct.

ADVANCED FUNCTIONAL MATERIALS, Issue 16 2010
Mater.
Abstract Fluorescence from quantum dots (QDs) sandwiched between colloidal gold nanoparticles and lithographically created metal nanoarrays is studied using engineered peptides as binding agents. For optimized structures, a 15-fold increase is observed in the brightness of the QDs due to plasmon-enhanced fluorescence. This enhanced brightness is achieved by systematically tuning the vertical distance of the QD from the gold nanoparticles using solid-specific peptide linkers and by optimizing the localized surface plasmon resonance by varying the geometric arrangement of the patterned gold nanoarray. The size and pitch of the patterned array affect the observed enhancement, and sandwiching the QDs between the patterned features and colloidal gold nanoparticles yields even larger enhancements due to the increase in local electromagnetic hot spots induced by the increased surface roughness. The use of bifunctional biomolecular linkers to control the formation of hot spots in sandwich structures provides new ways to fabricate hybrid nanomaterials of architecturally induced functionality for biotechnology and photonics. [source]

Cooperative Near-Field Surface Plasmon Enhanced Quantum Dot Nanoarrays

ADVANCED FUNCTIONAL MATERIALS, Issue 16 2010
Kirsty Leong
Abstract Fluorescence from quantum dots (QDs) sandwiched between colloidal gold nanoparticles and lithographically created metal nanoarrays is studied using engineered peptides as binding agents. For optimized structures, a 15-fold increase is observed in the brightness of the QDs due to plasmon-enhanced fluorescence. This enhanced brightness is achieved by systematically tuning the vertical distance of the QD from the gold nanoparticles using solid-specific peptide linkers and by optimizing the localized surface plasmon resonance by varying the geometric arrangement of the patterned gold nanoarray. The size and pitch of the patterned array affect the observed enhancement, and sandwiching the QDs between the patterned features and colloidal gold nanoparticles yields even larger enhancements due to the increase in local electromagnetic hot spots induced by the increased surface roughness. The use of bifunctional biomolecular linkers to control the formation of hot spots in sandwich structures provides new ways to fabricate hybrid nanomaterials of architecturally induced functionality for biotechnology and photonics. [source]

Helically ,-Stacked Conjugated Polymers Bearing Photoresponsive and Chiral Moieties in Side Chains: Reversible Photoisomerization-Enforced Switching Between Emission and Quenching of Circularly Polarized Fluorescence

ADVANCED FUNCTIONAL MATERIALS, Issue 8 2010
Hiroyuki Hayasaka
Abstract Novel multifunctional conjugated polymers, [poly(p -phenylene)s and poly(bithienylene-phenylene)s with (R)- and (S)-configurations], which have fluorescence, chirality, and photoresponsive properties, have been designed and synthesized. The polymers are composed of ,-conjugated main chains, where poly(p -phenylene) and poly(bithienylene-phenylene) are fluorescence moieties, and the side chains of the photochromic dithienylethene moiety are linked with chiral alkyl groups. The polymer films exhibit right- or left-handed circularly polarized fluorescence (CPF) and also show reversible quenching and emitting behaviors as a result of photochemical isomerization of the dithienylethene moiety upon irradiation with ultraviolet and visible light. This is the first report realizing the reversible switching of CPF using chirality and photoresponsive properties. [source]

High Efficiency Blue Organic LEDs Achieved By an Integrated Fluorescence,Interlayer,Phosphorescence Emission Architecture

ADVANCED FUNCTIONAL MATERIALS, Issue 4 2010
Tianhang Zheng
Abstract This paper presents a new strategy to develop efficient organic light-emitting devices (OLEDs) by doping fluorescent- and phosphorescent-type emitters individually into two different hosts separated by an interlayer to form a fluorescence,interlayer,phosphorescence (FIP) emission architecture. One blue OLED with FIP emission structure comprising p -bis(p - N,N -diphenylaminostyryl)benzene (DSA-Ph) and bis[(4,6-di-fluorophenyl)-pyridinate- N,C2']picolinate (FIrpic) exhibiting a peak luminance efficiency of 15.8,cd A,1 at 1.54,mA cm,2 and a power efficiency of 10.2,lm W,1 at 0.1,mA cm,2 is successfully demonstrated. The results are higher than those of typical phosphorescent OLEDs with a single emission layer by 34% and 28%, respectively. From experimental and theoretical investigations on device performance, and the functions of the used emitters and interlayer, such enhancement should ascribe to the appropriate utilization of the two types of emitters. The fluorescent emitter of DSA-Ph is used to facilitate the carrier transport, and thus accelerate the generation of excitons, while the phosphorescent emitter of FIrpic could convert the generated excitons into light efficiently. The method proposed here can be applied for developing other types of red, green, and white OLEDs. [source]

Light-Triggered Self-Assembly of a Spiropyran-Functionalized Dendron into Nano-/Micrometer-Sized Particles and Photoresponsive Organogel with Switchable Fluorescence

ADVANCED FUNCTIONAL MATERIALS, Issue 1 2010
Qun Chen
Abstract The synthesis, self-assembly, and spectroscopic investigations of spiropyran (SP)-functionalized dendron 1 are reported. Under UV light irradiation, assembly of 1 into nano-/microparticles occurs due to the transformation of the closed form of SP into the open merocyanine (MC) form. The formation of these nano-/microparticles is confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments in addition to the confocal laser scanning microscopy (CLSM) measurements. These nano-/microparticles exhibit relatively strong red emission. It is interesting to note that the direct cooling of the toluene/benzene solution of 1 to 0,°C leads to gel formation. Multivalent ,,, interactions due to the dendron in 1 may be the driving-force for the gelation. The UV light irradiation cannot destroy the gel phase, and in fact, the gel,gel transition is successfully realized. The purple-blue gel exhibits relatively strong red fluorescence; moreover, the fluorescence can be reversibly switched by alternating UV and visible light irradiation. The results clearly indicate that the MC form after aggregation becomes more stable and fluorescent. [source]

Further characterization of the first seminoma cell line TCam-2

GENES, CHROMOSOMES AND CANCER, Issue 3 2008
Jeroen de Jong
Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs. © 2007 Wiley-Liss, Inc. [source]

Frequency and characterization of HMGA2 and HMGA1 rearrangements in mesenchymal tumors of the lower genital tract

GENES, CHROMOSOMES AND CANCER, Issue 11 2007
Fabiola Medeiros
Mesenchymal tumors of the lower genital tract predominantly occur in women of reproductive age and are mainly represented by aggressive angiomyxoma (AAM) and angiomyofibroblastoma (AMF). Whether these tumors are different phenotypic expressions of the same biological entity is still debatable. Genetic rearrangements of HMGA2 have been reported in a few cases of AAM but its frequency and clinicobiological implications have not been studied systematically. We evaluated 90 cases of mesenchymal tumors of the lower genital tract that comprised 42 AAMs, 18 AMFs, 6 cellular angiofibromas, 5 fibroepithelial stromal polyps, 15 genital leiomyomas, 3 superficial angiomyxomas, and 1 spindle cell lipoma. Fluorescence in situ hybridization was used to identify rearrangements of HMGA2 and its homologue HMGA1. HMGA2 rearrangements were identified in 14 AAMs (33%) and in 1 vaginal leiomyoma. All other tumors were negative for HMGA2 rearrangements. HMGA1 rearrangement was not found in any of the cases. RT-PCR confirmed transcriptional upregulation of HMGA2 only in tumors with HMGA2 rearrangements. Standard cytogenetic analyses were performed in two AAMs and one AMF. One AAM had a t(1;12)(p32;q15); the other tumors had normal karyotypes. Mapping and sequence analysis of the breakpoint showed fusion to the 3, untranslated region of HMGA2 to genomic sequences derived from the contig NT 032977.8 on chromosome 1p32. Our findings support the hypothesis that AAM and AMF are distinct biological entities. The diagnostic usefulness of HMGA2 rearrangements to differentiate between AAM and other tumors of the lower genital tract may be limited due to the their low frequency. © 2007 Wiley-Liss, Inc. [source]

Chromosome 8 BAC array comparative genomic hybridization and expression analysis identify amplification and overexpression of TRMT12 in breast cancer,

GENES, CHROMOSOMES AND CANCER, Issue 7 2007
Virginia Rodriguez
Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors. To fine map such genomic changes by comparative genomic hybridization (CGH), a high resolution (100 kb) chromosome 8 array that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC (bacterial artificial chromosome) DNA. The BAC array CGH resolved the two known amplified regions (8q21 and 8q24) of a breast cancer cell line (SKBR3) into nine separate regions including six amplicons and three deleted regions, all of which were verified by Fluorescence in situ hybridization. The extent of the gain/loss for each region was validated by qPCR. CGH was performed with a total of 8 breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis. A 1.2-Mb region (125.3,126.5 Mb) and a 1.0-Mb region (128.1,129.1 Mb) in 8q24 were amplified in 7/8 cell lines. A global expression analysis was performed to evaluate expression changes associated with genomic amplification/deletion: a novel gene, TRMT12 (at 125.5 Mb), amplified in 7/8 cell lines, showed highest expression in these cell lines. Further analysis by RT-qPCR using RNA from 30 breast tumors showed that TRMT12 was overexpressed >2 fold in 87% (26/30) of the tumors. TRMT12 is a homologue of a yeast gene encoding a tRNA methyltransferase involved in the posttranscriptional modification of tRNAPhe, and exploring the biological consequence of its altered expression, may reveal novel pathways in tumorigenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. Published 2007 Wiley-Liss, Inc. [source]

FISH studies identify the i(20q,) anomaly as a der(20)del(20)(q11q13)idic(20)(p11)

GENES, CHROMOSOMES AND CANCER, Issue 6 2006
Tianyu Li
Fluorescence in situ hybridization (FISH) analyses were performed on six of seven patients who had been reported in 2004 to have an i(20q,) anomaly expressed as ider(20)(q10)del(20)(q11q13). The i(20q,) was investigated with a series of probes: a centromere-specific probe for chromosome 20, two paint probes for 20p and 20q, and a panel of locus-specific probes prepared from BAC/PAC clones mapped to 20p. The results showed that: (1) i(20q,) was a dicentric chromosome; (2) both of its arms comprised a deleted 20q and a small part of 20p near the centromere of chromosome 20; and (3) the breakpoints and reunion sites of i(20q,) differed, residing in the region 20p11.21,20p11.22 delineated by BAC/PAC clones RP11-96L6 and RP13-401N8. Thus, i(20q,) could be more precisely described as a der(20)del(20)(q11q13)idic(20)(p11). © 2006 Wiley-Liss, Inc. [source]