Fluorescence Signal Strength Research Articles

Lemon Exosome-like Nanoparticles-Manipulated Probiotics Protect Mice from C.d iff Infection.

SummaryClostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.

Technical note: A muscle biopsy technique for stratifying cattle by skeletal muscle metabolic activity

Tissue biopsy metabolic activity, assessed using the oxidation-reduction indicator resazurin, may serve as a proxy to assess energy expenditure associated with maintenance in nongrowing animals or growth rate in growing animals. Herein, we evaluate the repeatability, practicality, and sensitivity of a resazurin-based assay for ranking bovine skeletal muscle biopsies based on metabolic activity. Six yearling Holstein heifers (body weight = 330 ± 11.3 kg) were fed 4 dietary treatments consisting of high or low rumen-degradable starch and fiber arranged factorially in a partially replicated Latin square design. Periods were 18 d, consisting of 3 d for diet transition, 14 d for diet adaptation, and 1 d for sample collection. Semitendinosus biopsies were collected into ice-cold Dulbecco's modified Eagle medium (Fisher Scientific, Hampton, NH) from each heifer during each period. Analysis was initiated within an hour of sample collection. To assess tissue metabolic rate, biopsies were transferred to Dulbecco's modified Eagle medium with resazurin and incubated at 37°C. Fluorescence of each sample was read at time 0 and at 15-min intervals for 2 h. Change in fluorescence was representative of skeletal muscle reducing equivalent production. Fluorescent signal strength increased with time and relative rank of treatments did not change with time; accordingly, future studies may compare fluorescence at a single time point. Change in fluorescence at 120 min was used for analysis of the fixed effects of fiber, starch, and animal when accounting for a random effect of period. Samples collected when animals were on a high-ruminally degradable starch diet were more metabolically active than samples collected from animals on low-starch diets. Significant differences in metabolic activity among individual animals were also identified. Average relative fluorescence was correlated with dry matter intake, average daily gain, and feed-to-gain ratio. The relative fluorescence tended to correlate with average daily gain (r = 0.749) and feed-to-gain ratio (r = -0.783); change in fluorescence did not correlate with dry matter intake. Although evaluated on a small sample size, this technique shows promise as a potential means of ranking animals by growth or feed efficiency. Further work on a larger experimental population is needed to confirm the usefulness of this assay as a consistent and reliable predictor of these important phenotypic parameters.

Enrichment and Quantification of Epitope-specific CD4+ T Lymphocytes using Ferromagnetic Iron-gold and Nickel Nanowires

Epitope-specific CD4+ T lymphocytes were magnetically enriched using ferromagnetic Ni and Fe-Au nanowires coated with a monomer containing a major histocompatibility complex class II-bound peptide epitope (pMHCII). The enriched lymphocytes were subsequently quantified using fluorescence-activated cell sorting (FACS). This was the first use of magnetic nanowires for cell sorting using FACS, and improvements in both specificity and fluorescent signal strength were predicted due to higher particle moments and lengths than conventional paramagnetic beads. Three different types of nanowires (Ni, Fe with Au tip and Fe-Au multilayers) were made by electrodeposition. Ni nanowires separated fewer T cells than Au tipped Fe nanowires, likely because Ni has a lower magnetic moment than Fe. Fe-Au multilayer nanowires separated more T cells than Au-tipped Fe nanowires because there was more monomer per nanowire. Also, increasing the amount of monomer increased the number of CD4+ cells separated. Compared to conventional paramagnetic beads, the nanowires had lower specificity for CD4+ T cells, but had stronger fluorescent signals due to more fluorophores per particle. This results in broader FACS baseline separation between the positive and negative cells, which is useful to detect T cells, even those with lower binding affinity for pMHCII ligands.

Integrating Metal-Enhanced Fluorescence and Surface Acoustic Waves for Sensitive and Rapid Quantification of Cancer Biomarkers from Real Matrices.

Metal-enhanced fluorescence (MEF) is utilized to lower the detection limit of carcinoembryonic antigen (CEA), a prognostic biomarker for colorectal cancer among others, in immunofluorescence assays. In addition, Rayleigh surface acoustic waves (SAWs) were utilized to remove nonspecifically bound proteins, improve mixing, and reduce incubation times. Fluorescence intensity was plasmonically enhanced by incubating silver nanocubes (AgNCs) of 50 nm edge-length on a SAW device. This increased sensor sensitivity by a factor of 6 and lowered the limit of detection to below 1 ng/mL in fluorescence detection of the antigen. Surface density of the AgNCs was optimized to produce the largest MEF, which increased the signal intensity by an order of magnitude. Acoustic streaming induced by Rayleigh SAWs was found to decrease antibody/antigen incubation times to 1/6th of the values without such micromixing, and to increase the fluorescence signal strength. Overall, the demonstrated results allow for construction of a sensor capable of detecting CEA rapidly in clinically relevant concentrations. Variables relevant for optimizing this sensor performance were identified, which will enable even better performance in immunofluorescence assays.

Effect of bevacizumab combined with chemotherapy at different sequences in the gastric-cancer-bearing nude mice.

To observe changes in the growth of fluorescence-labelled tumour cells in nude mice using small animal in vivo imaging technology and to compare the anti-tumour effects of the administration of bevacizumab monoclonal antibodies combined with chemotherapy at different time sequences. Different time sequences of administration of bevacizumab monoclonal antibodies combined with the 5-fluorouracil and cisplatin (FP) chemotherapy regimen were used for intervention treatment of tumour growth in a subcutaneous xenograft model of human gastric cancer in nude mice. Tumour growth, that is, tumour volume, was evaluated with the changes in fluorescence signal strength and the inhibition rate. Compared with the control group (normal saline), experimental groups had a certain inhibition rate, while the tumour inhibition rate in the group with a bevacizumab treatment for 24 h followed by the FP chemotherapy regimen was the highest (68.42%). Moreover, the fluorescence signal strength changed significantly in all of the experimental groups. At the 3rd week of bevacizumab administration, the fluorescence signal value in the group with a bevacizumab treatment for 24 h followed by the FP chemotherapy regimen was the lowest, indicating this is the best treatment out of five groups. Bevacizumab monoclonal antibodies combined with chemotherapy had synergistic effects. The small animal in vivo imaging system could dynamically obtain long and short diameters of tumours and their fluorescence signal values; compared with traditional methods that calculate tumour inhibition rates by weighing tumours, this method was more sensitive and more objective for drug evaluation.

The effects of humidity on combustion characteristics of a nonpremixed syngas flame

With the rapid development of economy, environmental protection and energy shortage are becoming more and more acute in China. The Integrated Gasification humid air (IGHAT) cycle is highlighted in providing efficient and clean options for meeting future coal-utilizing power generation needs. Syngas combustion in humid air is a bottleneck problem in IGHAT cycle for power generation. This paper carried out experiments on a non-premixed model combustor, equipped with a double-swirled syngas burner. Planar laser induced fluorescence (PLIF) of OH radical measurement is adopted to identify main reaction zones and burnt gas regions as well. With the temperature and emission measurements in the exhaust section, some important characteristics of the syngas flame are investigated overall. And the effects of the CO/H2 molar ratio consisting of syngas fuel are investigated under different humidity. With the increase of CO/H2 ratios, the concentration field of OH radicals is gradually away from the nozzle exit, and the nozzle exit almost no existence of OH radicals, forming a typical lifted flame. In addition, fluorescent signal strength of OH radicals pronounced weakening, the flame gradually appeared W type distribution and more and more obvious with the increased of humidification amount. At the same time there are almost no changes of the average exhaust temperature of combustor or CO and NOx emissions. The study can provide a reliable database for high moisture gas turbine combustor design and combustion numerical simulation.

BODIPY-Based Fluorescent Probes for Sensing Protein Surface-Hydrophobicity.

Mapping surface hydrophobic interactions in proteins is key to understanding molecular recognition, biological functions, and is central to many protein misfolding diseases. Herein, we report synthesis and application of new BODIPY-based hydrophobic sensors (HPsensors) that are stable and highly fluorescent for pH values ranging from 7.0 to 9.0. Surface hydrophobic measurements of proteins (BSA, apomyoglobin, and myoglobin) by these HPsensors display much stronger signal compared to 8-anilino-1-naphthalene sulfonic acid (ANS), a commonly used hydrophobic probe; HPsensors show a 10- to 60-fold increase in signal strength for the BSA protein with affinity in the nanomolar range. This suggests that these HPsensors can be used as a sensitive indicator of protein surface hydrophobicity. A first principle approach is used to identify the molecular level mechanism for the substantial increase in the fluorescence signal strength. Our results show that conformational change and increased molecular rigidity of the dye due to its hydrophobic interaction with protein lead to fluorescence enhancement.

Effects of α ⁃asarone on expressions of proteins in hippocampus of rats with seizures induced by kainic acid

Objective To explore the effects of α ⁃ asarone on expressions of proteins in hippocampus of rats with seizures induced by kainic acid (KA), and find the drug action targets of α ⁃ asarone. Methods Twenty healthy male Sprague ⁃ Dawley (SD) rats were given KA by intraperitoneal injection to induce epilepsy, and were randomly divided into 2 groups: the KA group (n = 10) and α⁃asarone (24 mg/kg) group (n = 10). In α⁃asarone group α⁃asarone were administered once daily by intraperitoneal injection in 7 consecutive days. Bicinchoninic acid (BCA) method was used to measure the protein expression in hippocampus before and after α⁃asarone therapy. The endogenous international normalized ratio (INR) was calculated according to the standardization of hybridization fluorescence signal strength, and was used to measure the difference of protein expression between the two groups. INR ≥ 1.30 indicated the protein expression up ⁃ regulated while INR ≤ 0.77 indicated the protein expression down ⁃ regulated. Results Thirty⁃five differentially expressed proteins were found. In comparison with KA group 5 proteins presented higher expression, while 30 lower expression in α⁃asarone group, and most of them involved in signal transduction, cell cycle regulation, gene transcription and regulation, apoptosis, and neurobiological functions. Conclusion α ⁃ Asarone may regulate a variety of protein expression in hippocampus of rats with seizures induced by kainic acid, and it may indicate that the drug has multiple therapeutic targets. DOI：10.3969/j.issn.1672⁃6731.2012.05.016

SYTO dyes and EvaGreen outperform SYBR Green in real-time PCR

BackgroundReal-time PCR can be carried out using either probes or DNA dyes. SYBR Green has been used the most, but it suffers from several drawbacks. Numerous other DNA dyes are commercially available, but with limited structural information. Dye behavior in real time PCR is difficult to predict, so empirical data are needed. In the work described here, a panel of 23 different DNA dyes--including green, orange, and red SYTO dyes, EvaGreen, and SYBR Green--were evaluated with respect to their performance in real time PCR.FindingsData were analyzed for reaction inhibition, effects on amplicon melting temperature, fluorescent signal strength, and reaction efficiency. This is the first report of reaction efficiency using alternatives to SYBR Green. Results indicated substantial variation in performance even within the SYTO dye family. EvaGreen and the SYTO dyes 13, 16, 80, and 82 performed better than SYBR Green in general, and high reaction efficiencies can be achieved using these dyes.ConclusionsEmpirical data were generated for 23 DNA dyes. This paper confirms and extends previous findings that among commercially available DNA dyes, EvaGreen and certain SYTO dyes are the most desirable alternatives to the commonly used SYBR Green in real-time PCR.

Electronic Microarrays in DNA Computing

DNA Computing is a rapidly-developing interdisciplinary area which could benefit from more experimental results to solve practical problems with the current biological tools. In this study, we have integrated microelectronics and molecular biology techniques for the storage of information and basic arithmetic operations via DNA. Using 16 different complementary sequences of DNA, we stored 4 bits of information on an electronic microarray and read the data via the fluorescent signal strength coming from the microarray pads. We also showed the possibility of addition and subtraction of quantities of fluorescently tagged DNA determined via their fluorescent signal strength. We conclude that the hybrid technology we employed, based on a matured Si-CMOS platform, has the potential to strengthen the pursuit of DNA Computation as well as finding its own niche applications.

Abstract 2621: Development of Fluoro-UK-101, an immunoproteasome-targeting activity-based fluorescent probe

Abstract Introduction: This study was conducted to develop the first activity-based fluorescent probe targeting the immunoproteasome subunit LMP2. The immunoproteasome is an alternate form of the constitutive proteasome which is composed of unique catalytic subunits. While immunoproteasomes were believed to primarily function in generating peptides for antigen presentation on MHC class I molecules, antibody-based studies have revealed the expression of immunoproteasome catalytic subunits in a variety of cancers. However, antibodies are incapable of distinguishing between active and inactive forms of these proteins, a limitation that can be overcome using activity-based probes. Experimental Procedures: Fluorescent compounds were synthesized by derivatization of the LMP2-specific inhibitor UK-101 at the P2 position, where lysine was substituted for alanine. The free amino group on the side chain of lysine was coupled to linkers of various lengths. Fluorescein was then coupled to each linker to yield the fluorescent probes. These compounds were characterized in prostate cancer cell lines via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescence (IF) microscopy. One analog was selected for further characterization. Prostate cancer cells were treated with an LMP2-selective dose of this analog, Fluoro-UK-101, and the co-localization of this fluorescent probe with antibodies specific for proteasome subunits and intracellular organelles was examined via IF. Data Summary: The structures of the synthesized fluorescent derivatives of UK-101 were confirmed via mass spectrometry and nuclear magnetic resonance spectroscopy. Treatment of prostate cancer cells with these compounds gave punctate staining in a dose-dependent manner, indicating the cell permeability and activity of these probes. The impact of the linker length on LMP2 binding specificity and fluorescent signal strength was determined via SDS-PAGE and IF microscopy. Doses at which these fluorescent analogs specifically target LMP2, but not other proteasome subunits, were identified via SDS-PAGE. One analog, Fluoro-UK-101, was selected based on binding specificity and IF intensity. Additionally, IF microscopy techniques using the selected fluorescent analog, Fluoro-UK-101, revealed differences in LMP2 localization patterns among prostate cancer cell lines. Conclusions: We have successfully developed Fluoro-UK-101, an activity-based fluorescent probe that selectively binds the immunoproteasome catalytic subunit LMP2. Fluoro-UK-101 allows the visualization of catalytically active immunoproteasomes in intact cells. Modification of the fluorophore will facilitate its application in a rapid readout assay to detect tumors in animal models using LMP2 as a biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2011-2621

Electronic Microarrays in DNA Computing

DNA Computing is a rapidly-developing interdisciplinary area which could benefit from more experimental results to solve practical problems with the current biological tools. In this study, we have integrated microelectronics and molecular biology techniques for the storage of information and basic arithmetic operations via DNA. Using 16 different complementary sequences of DNA, we stored 4 bits of information on an electronic microarray and read the data via the fluorescent signal strength coming from the microarray pads. We also showed the possibility of addition and subtraction of quantities of fluorescently tagged DNA determined via their fluorescent signal strength. We conclude that the hybrid technology we employed, based on a matured Si-CMOS platform, has the potential to strengthen the pursuit of DNA computation as well as finding its own niche applications.

Long-Range Surface Plasmon-Enhanced Fluorescence Spectroscopy Biosensor for Ultrasensitive Detection of E. coli O157:H7

A new biosensor platform for the detection of bacterial pathogens based on long-range surface plasmon-enhanced fluorescence spectroscopy (LRSP-FS) is presented. The resonant excitation of LRSP modes provides an enhanced intensity of the electromagnetic field, which is directly translated to an increased strength of fluorescence signal measured upon the capture of target analyte at the sensor surface. LRSPs originate from a coupling of surface plasmons across a thin metallic film embedded in dielectrics with similar refractive indices. With respect to regular surface plasmon-enhanced fluorescence spectroscopy, the excitation of LRSPs offers the advantage of a larger enhancement of the evanescent field intensity and a micrometer probing depth that is comparable to the size of target bacterial pathogens. The potential of the developed sensor platform is demonstrated in an experiment in which the detection of E. coli O157:H7 was carried out using sandwich immunoassays. The limit of detection below 10 cfu mL(-1) and detection time of 40 min were achieved.

Enhancement effect of nano Fe 3O 4 to the drug accumulation of doxorubicin in cancer cells

Tetraheptylammonium capped magnetic nano Fe 3O 4 was synthesized and used in the study of in vitro drug accumulation inside leukemia K562 cell lines. Confocal fluorescence microscopy methods were used to detect the fluorescent signal strength of doxorubicin in the absence and presence of nano Fe 3O 4. Atomic force microscopy (AFM) was used to characterize the surface structure of the leukemia cells. In addition, MTT assay was used to evaluate the safety and effectiveness of the presence of nano Fe 3O 4. Our results demonstrate the synergistic enhancement effect of the nano Fe 3O 4 on respective drug uptake of doxorubicin for drug sensitive and drug resistant leukemia K562 cells. These observations indicate that the tetraheptylammonium-capped nano Fe 3O 4 could efficiently enhance the relevant drug permeation into cancer cells through internalization/endocytosis processes and result in significantly enhanced doxorubicin uptake in respective leukemia K562 cells.

Stability of isooctane mixtures with 3-pentanone or biacetyl as fluorescence tracers in combustion experiments

Evidence is presented in the literature that common fluorescence tracer/fuel mixtures used in engine experiments, 3-pentanone/isooctane and biacetyl/isooctane, may decompose during extended use and storage. Investigations presented here show that preferential evaporation of the tracer and not chemical decomposition is responsible for observed decreases in fluorescence signal strength in these experiments.

Fluorometric assay for detection and quantitation of polyamidoamine dendrimers

Polyamidoamine dendrimers are protein-like nanomaterials that have many potential biological applications. Research studies using dendrimers often require their detection and quantitation. This article describes a simple, inexpensive, and rapid fluorometric assay based on the ability of dendrimers to bind the fluorescent probe, 8-anilino-1-naphthalene-sulfonic acid. This interaction leads to an enhanced fluorescence intensity of the dye that shows a linear relationship with dendrimer concentration. The procedure is applicable to dendrimers that have various surface functional groups and internal cores. Amine- and hydroxyl-terminated dendrimers gave the best calibration curves under alkaline conditions (pH 7.4). Carboxyl-terminated dendrimers required acidic conditions for their measurement. The assay for dendrimers with an amine surface and an ethylene diamine core was accurate between 0 and 100 μM and produced a within-run coefficient of less than 5%. Under the assay conditions used, the fluorescence signal strength was dependent on type of surface group and core length of the dendrimer.

Binary Solvent Effects in Capillary Zone Electrophoresis with Ultrasensitive Near-IR Fluorescence Detection of Related Tricarbocyanine Dyes and Dye-Labeled Amino Acids

We have investigated the effects of mixed organic/aqueous carrier buffers on the chromatographic efficiency and detectabilipy of native near-infrared (near-IR) dyes and near-IR dye-labeled amino acids separated using capillary electrophoresis (CE). The near-IR fluorescence detector for this application incorporated a solid-state Ti/sapphire laser for excitation and a single-photon avalanche diode photodetector. The on-column mass detection limits for a series of cationic tricarbocyanine near-IR dyes varied from 350 to 5500 ymol in running buffers comprised of 95/5 methanol/water (pH=9.1). In predominately aqueous running buffers, the detection limits degraded with significant losses in the separation efficiencies. Two near-IR dyes with similar structures but different charges (cationic and anionic) were used to investigate efficiency losses with binary methanol/water running buffers. The results indicated that solute/wall interactions were a major contribution to zone broadening for the cationic dye in high water compositions, resulting in significant losses in the numbers of theoretical plates, while the anionic dye did not exhibit these types of interactions. In order to demonstrate the usefulness of near-IR fluorescence in CE applications, a series of amino acids were labeled with a near-IR fluorescent dye containing an isothiocyanate fuctional group and separated using CE with mixed methanol/water buffers. The fluorescence signal strength and the chromatographic resolution were found to be improved in high methanol compositions in the running buffer

Computerized quantification of immunofluorescence-labeled axon terminals and analysis of co-localization of neurochemicals in axon terminals with a confocal scanning laser microscope.

The confocal scanning laser microscope (CSLM) offers improved optical resolution and contrast, high photometric precision, and the ability to make optical sections. These benefits were explored for use in quantitative analysis of immunofluorescence-labeled axon terminals. Guidelines were obtained for adjustments of the CSLM parameters. In the present applications, bleaching of the fluorescence did not represent a serious obstacle to analysis with the CSLM. A method was developed to distinguish the background fluorescence from the specific fluorescence labeling. This procedure made way for the development of automated quantification of immunolabeled axon terminals. The automated procedures substantially reduced the man-hour expenditure for analysis and provided highly reproducible quantifications compared with manual methods. The increased resolution and contrast of the CSLM allowed measurements of the fluorescence signal strength of individual axon terminals. The CSLM also allowed detection of co-localized neurochemicals in axon terminals.

Selection of fluorescence lidar operating parameters for SNR maximization

Fluorescence lidar when applicable offers one of the most sensitive methods for measuring the concentration of trace constituents of the atmosphere. In the conduct of a fluorescence lidar experiment, one can control a number of parameters which can be used to optimize the SNR. In this paper the optimum division of laser pulses centered on and off the fluorescence excitation wavelength is calculated as a function of the ratio of the fluorescence signal strength to the strength of fluorescence from interfering species. For strong interference signals the time should be divided equally on and off the line. For strong fluorescence signals the time on line is proportional to the square root of the on-line off-line signal ratio. The optimization of the integration time for varying values of signal-to-background and signal-to-interference ratios, atmospheric attenuation, laser energy variations, background measurement time, and on-line off-line time division is also considered.