Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

Three-dimensional fluorescence as a tool for investigating the dynamics of dissolved organic matter in the Lake Biwa watershed

Quantitative and qualitative characterizations of dissolved organic matter (DOM) were carried out at the watershed level in central Japan by measuring dissolved organic carbon (DOC) concentration and the three-dimensional excitation–emission matrix (3-D EEM). DOC concentration was low (mean 37 ± 19 µM C) in the upstream waters, whereas, in general, it increased toward the downstream areas (mean 92 ± 47 µM C). Significant variations in DOC concentration were detected among rivers and channels. DOC concentration in the epilimnion of Lake Biwa increased during the summer period and decreased during the winter period. The lake hypolimnion has lower DOC concentration (mean 87 ± 7 µM C) compared with the epilimnion (107 ± 15 µM C). Fulvic acid (FA)-like substances in the DOM were directly characterized by 3-D EEM. The fluorescence peak for upstream DOM was found in regions with longer wavelengths (excitation/emission 386 ± 6/476 ± 5 nm) compared with downstream and lake DOM (351 ± 12/446 ± 15 nm and 341 ± 6/434 ± 6 nm, respectively). The DOC concentration is correlated with fluorescence peak intensity of FA-like substances in DOM in river waters. Such a relationship was not found in lake DOM. A blueshift of the fluorescence peak from upstream to lake DOM was observed. A decrease in fluorescence intensities was also detected during the summer period. These results may suggest that the degradation of FA-like substances in DOM occurs from natural solar irradiation. Protein-like fluorescence was significantly detected in the lake epilimnion during the summer period. A linear relationship between DOC concentration and protein-like fluorescence indicated that an autochthonous input of DOM gave rise to the increase in DOC concentration in the lake epilimnion during the summer. These results may suggest that the 3-D EEM can be used as a tool for the investigation of DOM dynamics at the watershed level with concurrent measurement of DOC concentration and the fluorescence properties of fulvic acid-like and protein-like substances.     

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady‐state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high‐throughput drug screening campaigns.     

Two-dimensional fluorescence spectroscopy – a new tool for the determination of plant cell viability

 Two-dimensional fluorescence spectroscopy (2D-FS) has been used as a new method for determining the viability of tobacco cells (Nicotiana tabacum L.). Both horizontal beam geometry and a vertical set-up achieved with bifurcated fibres were tested. The latter arrangement enabled us to avoid the negative effect of cell sedimentation. Incubation of a tobacco BY-2 cell suspension with dimethylsulfoxide (DMSO) (0–10% v/v) resulted in cell samples differing in their viability – from fully viable (0–2% DMSO) to totally non-viable (8–10%DMSO). The validity of determining viability by means of measuring cell esterase activity by 2D-FS using fluorescein diacetate as a fluorogenic substrate was verified by comparison with microscopic evaluation of fluorescein fluorescence as well as with the routinely adopted trypan blue exclusion test. Received: 6 June 2000 / Revision received: 9 October 2000 / Accepted: 9 October 2000     

Delayed fluorescence as a universal tool for the measurement of circadian rhythms in higher plants

The plant circadian clock plays an important role in enhancing performance and increasing vegetative yield. Much of our current understanding of the mechanism and function of the plant clock has come from the development of Arabidopsis thaliana as a model circadian organism. Key to this rapid progress has been the development of robust circadian markers, specifically circadian-regulated luciferase reporter genes. Studies of the clock in crop species and non-model organisms are currently hindered by the absence of a simple high-throughput universal assay for clock function, accuracy and robustness. Delayed fluorescence (DF) is a fundamental process occurring in all photosynthetic organisms. It is luminescence-produced post-illumination due to charge recombination in photosystem II (PSII) leading to excitation of P680 and the subsequent emission of a photon. Here we report that the amount of DF oscillates with an approximately 24-h period and is under the control of the circadian clock in a diverse selection of plants. Thus, DF provides a simple clock output that may allow the clock to be assayed in vivo in any photosynthetic organism. Furthermore, our data provide direct evidence that the nucleus-encoded, three-loop circadian oscillator underlies rhythms of PSII activity in the chloroplast. This simple, high-throughput and non-transgenic assay could be integrated into crop breeding programmes, the assay allows the selection of plants that have robust and accurate clocks, and possibly enhanced performance and vegetative yield. This assay could also be used to characterize rapidly the role and function of any novel Arabidopsis circadian mutant.     

Changes in yield ofin-vivo fluorescence of chlorophyll a as a tool for selective herbicide monitoring

Triazines and derivatives of phenylurea, which are often found in outdoor water samples, induce specific changes in the yield of thein-vivo chlorophyll -fluorescence of PSII. These changes are correlated quantitatively with the concentration of the herbicides and can therefore be used to set-up a low-price monitor system. In order to detect selectively the herbicide-sensitive part of the fluorescence emission a pulse amplitude modulated fluorimeter was used. The bioassay system was optimised with respect to test organism, growing and measuring conditions. The relationship between fluorescence yield and herbicide concentrations were experimentally determined for the triazines atrazine and simazine and the phenylurea herbicide DCMU and mathematically fitted (r=0.99). The I₅₀-values were 0.9 µM for DCMU, 2.2 µM for simazine and 3.3 µM for atrazine. The detection limit of about 0.5 µM clearly shows that the sensitivity of this bioassay system is too low to reach the requirements of the drinking water regulation. However, due to its insensitivity against complex water matrices, there is good hope to combine this fluorometric bioassay with a potent herbicide preconcentration method like a solid-phase extraction procedure.Author for correspondence     

The diagnostic and prognostic values of microRNA-196a in cancer

MicroRNA-196a (miR-196a) was previously reported to be up-regulated in cancers, and it has the diagnostic and prognostic values in cancers. Whereas, the conclusion was still unclear according to the published data. To assess such roles of miR-196a in cancers, the present study was conducted based on published data and online cancer-related databases. To identify the relevant published data, we searched articles in databases and then the relevant data were extracted to evaluate the correlation between miR-196a expression and diagnosis, prognosis for cancer patients. The pooled results showed that miR-196a was a valuable diagnostic biomarker in cancer (area under curve (AUC) = 0.87, 95% CI: 0.84–0.90; sensitivity (SEN) = 0.73, 95% CI: 0.64–0.81; specificity (SPE) = 0.90, 95% CI: 0.81–0.95), which was consistent with the data from databases (breast cancer: miR-196a-3p: AUC = 0.77, 95% CI: 0.74–0.79; miR-196a-5p: AUC = 0.71, 95% CI: 0.66–0.75; pancreatic cancer: miR-196a-3p: AUC = 0.80, 95% CI: 0.73–0.87; miR-196a-5p: AUC = 0.61, 95% CI: 0.51–0.71). In addition, the pooled result revealed that elevated miR-196a expression in tumor tissues (HR = 2.54, 95% CI: 1.79–3.61, P_{Heterogeneity}=0.000, I² = 75.8%) or serum/plasma (HR = 4.06, 95% CI: 2.67–6.18, P_{Heterogeneity}=0.668, I² = 0%) of patients was an unfavorable survival biomarker, which was consistent with the data from databases (adrenocortical carcinoma: HR = 5.70; esophageal carcinoma: HR = 1.93; brain lower grade glioma: HR = 2.91; {"type":"entrez-geo","attrs":{"text":"GSE40267","term_id":"40267"}}GSE40267: HR = 2.47, 95% CI: 1.2–5.07; TCGA: HR = 1.82, 95% CI: 1.21–2.74; {"type":"entrez-geo","attrs":{"text":"GSE19783","term_id":"19783"}}GSE19783: HR = 4.24, 95% CI: 1–18.06). In short, our results demonstrated that miR-196a in tumor tissue or serum/plasma could be used as a prognostic and diagnostic values for cancers.     

A fluorescence quenching test for the detection of flavonoid transformation

A novel fluorescence quenching test for the detection of flavonoid degradation by microorganisms was developed. The test is based on the ability of the flavonoids to quench the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Several members of the anthocyanidins, flavones, isoflavones, flavonols, flavanones, dihydroflavanones, chalcones, dihydrochalcones and catechins were tested with regard to their quenching properties. The anthocyanidins were the most potent quenchers of DPH fluorescence, while the flavanones, dihydroflavanones and dihydrochalcones, quenched the fluorescence only weakly. The catechins had no visible impact on DPH fluorescence. The developed test allows a quick and easy differentiation between flavonoid-degrading and flavonoid-non-degrading bacteria. The investigation of individual reactions of flavonoid transformation with the developed test system is also possible.     

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.     

Evaluation of chlorophyll fluorescence as a tool for salt stress detection in roses

The induction kinetic of the chlorophyll (Chl) fluorescence and the F_v/F_m ratio have been tested in order to find out the suitability of this technique to evaluate damage caused by salinity in plants of Rosa hybrida cv. Ilseta grafted on R. manetti growing in a greenhouse under non-saturating irradiance. Under these conditions salinity induced changes in plants morphology, nutrient and Chl contents and in the gas exchange parameters, but not in the F_v/F_m ratio. The Rfd index did not reveal more information. The F_v/F_m ratio as well as the fluorescence induction curves were more affected by salinity when an irradiation stress was added, therefore as an indicator of salt stress in roses, Chl fluorescence is of limited use when the plants are grown without additional stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.     

The use of outer filter effects for Cu(2+) quantitation: a unique example for monitoring nonfluorescent molecule with fluorescence

This article concerns a new and precise strategy for the determination of Cu(2+) based on a color reaction and outer filter effects (OFEs). Cu(2+) can react with sodium diethyldithiocarbamate trihydrate (DDTC) to form a DDTC-Cu(2+) complex with a significant absorption at 447 nm. Being positively correlated with Cu(2+), the absorption could be treated as the basis for the determination of Cu(2+). When cuvettes containing the complex were fixed in the light path of a fluorescence spectrophotometer, the excitation/emitted light were absorbed by the OFEs, similar to absorption mechanisms of inner filter effects. Under suitable conditions, OFEs from the complex could quantitatively reduce the fluorescence intensities of quinine sulfate and acridine yellow by absorbing the excitation or emission light. Compared with traditional absorption spectroscopy (with a detection limit at 0.9 μmol/L), indirect OEF techniques showed increased sensitivities by about 1 order of magnitude. The strategy could be extended to many different systems where components absorb the excitation wavelength and/or emission wavelength of fluorescers.     

Development of a quantum dot molecularly imprinted polymer sensor for fluorescence detection of atrazine

Atrazine is a common agricultural pesticide which has been reported to occur widely in surface drinking water, making it an environmental pollutant of concern. In the quest for developing sensitive detection methods for pesticides, the use of quantum dots (QDs) as sensitive fluorescence probes has gained momentum in recent years. QDs have attractive and unique optical properties whilst coupling of QDs to molecularly imprinted polymers (MIPs) has been shown to offer excellent selectivity. Thus, the development of QD@MIPs based fluorescence sensors could provide an alternative for monitoring herbicides like atrazine in water. In this work, highly fluorescent CdSeTe/ZnS QDs were fabricated using the conventional organometallic synthesis approach and were then encapsulated with MIPs. The CdSeTe/ZnS@MIP sensor was characterized and applied for selective detection of atrazine. The sensor showed a fast response time (5 min) upon interaction with atrazine and the fluorescence intensity was linearly quenched within the 2–20 mol L^?1 atrazine range. The detection limit of 0.80 × 10^?7 mol L^?1 is comparable to reported environmental levels. Lastly, the sensor was applied in real water samples and showed satisfactory recoveries (92–118%) in spiked samples, hence it is a promising candidate for use in water monitoring.     

Suppressive effect of spinach extract on the formation of melanin in B16 mouse melanoma cells

A non-dialyzable extract of fresh spinach leaves exhibited a strong antioxidant activity towards oxidation of linoleic acid and suppressed the melanin formation of a mouse melanoma cell line, B16 melanoma 4A5, without any significant effect on the proliferation of cells.     

The microbial detection array combined with random Phi29-amplification used as a diagnostic tool for virus detection in clinical samples

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples.     

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.     

Chlorophyll fluorescence transients of Photosystem II membrane particles as a tool for studying photosynthetic oxygen evolution

The rise of the chlorophyll fluorescence yield of Photosystem II (PS II) membranes as induced by high-intensity actinic light comprises only two distinct phases: (1) the initial O-J increase and (2) the subsequent J-P increase. Partial inhibition of the PS II donor side by heating or washing procedures which remove peripheral PS II proteins or cofactors of the oxygen-evolving complex results in decrease of magnitude and rate of the J-P phase. The rate constant of the J-P increase is directly proportional to the steady-state rate of oxygen evolution; complete suppression of the J-P phase corresponds to full inhibition. A characteristic dip after J-level is observed only in Tris-washed or severely heated PS II membranes; manganese release correlates with appearance of the dip after J-level as verified by EPR spectroscopy. Presence of stabilizing cosolutes (glycine betaine, sucrose) or addition of donor-side cofactors (bicarbonate, chloride, calcium) to PS II membranes before heating (47 °C, 5 min) diminishes J-P phase suppression and prevents dip appearance, whereas the addition after heating is without effect. In conclusion, analysis of chlorophyll fluorescence transients of PS II membranes is a potentially useful tool for investigations on photosynthetic oxygen evolution. A decreased rate of the J-P phase can be employed as a convenient indicator for partial inhibition of oxygen-evolution activity; the appearance of a dip after J-level is suggestive of manganese release. This revised version was published online in June 2006 with corrections to the Cover Date.     

A sensitive fluorescence quenching method for the detection of tartrazine with acriflavine in soft drinks

In this work, a simple, rapid, sensitive, selective spectrofluorimetric method was applied to detect tartrazine. The fluorescence of acriflavine could be efficiently quenched by tartrazine. The method manifested real time response as well as presented satisfied linear relationship to tartrazine. The linear response range of tartrazine (R² = 0.9995) was from 0.056 to 5 μmol L^?1. The detection limit (3σ/k) was 0.017 μmol L^?1, indicating that this method could be applied to detect traces of tartrazine. The accuracy and precision of the method was further assured by recovery studies via a standard addition method, with percentage recoveries in the range of 96.0% to 103.0%. Moreover, a quenching mechanism was investigated systematically by the linear plots at varying temperatures based on the Stern–Volmer equation, fluorescence lifetime and UV–visible absorption spectra, all of which proved to be static quenching. This sensitive, selective assay possessed a great application prospect for the food industry owing to its simplicity and rapidity for the detection of tartrazine.