Hjortsawyer1091

Optical contrast agents containing near-infrared (NIR) fluorophores are useful for visualizing biological landmarks, enzyme activities and biological processes in live animals and humans. Activatable (smart) quenched-fluorescent probes are sensors that become fluorescent after processing by an enzyme or in response to a physiological change (i.e., pH, ROS, etc.). Recently, there has been increased interest in developing activatable probes for research and clinical applications. This requires evaluation using in vivo animal models to gain insights into the pharmacodynamic and pharmacokinetic properties of a given probe. Important parameters to measure when evaluating quenched-fluorescent probes are signal brightness and signal-to-background ratios, which define the sensitivity and specificity of a probe. In this chapter, we discuss methods to evaluate activatable quenched-fluorescent probes in mouse models of cancer. Quantification of fluorescent signal intensity, calculation of tumor-to-background ratios, comparison of fluorescent activation in specific organ compartments, and fluorescence scanning of sectioned tissue will be discussed.Circadian rhythms are critical regulators of many physiological and behavioral functions. The use and abilities of small molecules to affect oscillations have recently received significant attention. find more These manipulations can be reversible and tunable, and have been used to study various biological mechanisms and molecular properties. Here, we outline procedures for assessment of cellular circadian changes following treatment with small molecules, using luminescent reporters. We describe reporter generation, luminometry experiments, and data analysis. Protocols for studies of accompanying effects on cells, including motility, viability, and anchorage-independent proliferation assays are also presented. As examples, we use indirubin-3'-oxime and two derivatives, 5-iodo-indirubin-3'-oxime and 5-sulfonic acid-indirubin-3'-oxime. In this case study, we analyze effects of these compounds on Bmal1 and Per2 (positive and negative core circadian elements) oscillations and provide step-by-step protocols for data analysis, including removal of trends from raw data, period estimations, and statistical analysis. The reader is provided with detailed protocols, and guidance regarding selection of and alternative approaches.Protein aggregation is a process that occurs through the self-assembly of misfolded proteins to form soluble oligomers and insoluble aggregates. While there has been significant interest in protein aggregation for neurodegenerative diseases, progress in this field of research has been limited by the lack of effective methods to detect and interrogate these species in live cells. To resolve this issue, we have developed a new imaging method named the AggTag to report on protein aggregation in live cells with fluorescence microscopy. The AggTag method utilizes a genetic fusion of a protein of interest (POI) to a protein tag to conjugate with the AggTag probe, which contains a fluorophore that turns on its fluorescence upon interaction with protein aggregates. Unlike the conventional methods, this method enables one to detect soluble misfolded oligomers that were previously invisible. Furthermore, the AggTag method has been applied for the simultaneous detection of co-aggregation between two different POIs by a dual-color and orthogonal tagging system. This chapter aims to provide step-by-step procedures of the AggTag method for researchers who intend to study aggregation of POIs in mammalian cell lines.Amino acid and acylcarnitine first-tier newborn screening typically employs derivatized or non-derivatized sample preparation methods followed by FIA coupled to triple quadrupole (TQ) MS/MS. The low resolving power of TQ instruments results in difficulties distinguishing nominal isobaric metabolites, especially those with identical quantifying product ions such as malonylcarnitine (C3DC) and 4-hydroxybutylcarnitine (C4OH). Twenty-eight amino acids and acylcarnitines extracted from dried blood spots (DBS) were analyzed by direct injection (DI)-HRMS on a Q-Exactive Plus across available mass resolving powers in SIM, in PRM at 17,000 full width at half maximum (FWHM), and a developed SIM/PRM hybrid MS method. Most notably, quantitation of C3DC and C4OH was successful by HRMS in non-derivatized samples, thus, potentially eliminating sample derivatization requirements. Quantitation differed between SIM and PRM acquired data for several metabolites, and it was determined these quantitative differences were due to collision energy differences or kinetic isotope effects between the unlabeled metabolites and the corresponding labeled isotopologue internal standards. Overall quantitative data acquired by HRMS were similar to data acquired on TQ MS/MS platform. A proof-of-concept hybrid DI-HRMS and SIM/PRM/FullScan method was developed demonstrating the ability to hybridize targeted newborn screening with metabolomic screening.Supercritical fluid chromatography is becoming more prevalent, particularly in industry. This is due to the inexpensive, and more importantly, environmentally benign carbon dioxide that is used as the major component of the mobile phase. Water is minimally miscible with carbon dioxide at temperatures and pressures commonly used in SFC. However, the introduction of a polar alcohol modifier component increases the solubility of water in carbon dioxide. Previously, the addition of small amounts of water in the mobile phase was shown to provide significant gains in efficiency in chiral supercritical fluid chromatography, especially with polar stationary phases. In this work, we report the effect of the addition of small amounts of water on efficiency and retention factor with four different SFC stationary phases used for achiral analysis namely FructoShell-N (native cyclofructan-6), SilicaShell (bare silica), PoroShell 120 EC C18 (octadecyl silica) and Xselect C18 SB. This is the first reported use of FructoShell-N, a cyclofructan derivatized phase for SFC applications. We devised a predictive test to determine which analytes show an increase in efficiency using their known chemical constants (logKow, pKa, PSA and Hsum). We also use discriminant analysis to elucidate the most important analyte parameters that contribute to "water enhanced" efficiency gains.This work compares the performance of transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), single particle inductively coupled plasma mass spectrometry (spICPMS) and flow injection (FI) coupled to spICPMS for the characterization of synthetic ferromagnetic Ni nanoparticles (NPs) prepared with and without polyvinylpyrrolidone (PVP) stabilizer. Whereas single NPs measurement by XRD yielded nominal diameters of 13.7 and 16.6 nm with and without PVP respectively, a diameter of 100-130 nm was obtained by TEM and SEM with or without PVP, indicating extensive agglomeration during preparation for microscopy. In contrast, without PVP stabilization, mean and mode sizes of respectively 35 ± 18 and 21 nm by spICPMS and 33 ± 15 and 20 nm by FI-spICPMS were obtained for suspensions of Ni NPs using external calibration with Ni standard solutions. With PVP stabilization, the mean and mode sizes respectively decreased to 27 ± 12 and 18 nm by spICPMS and 25 ± 10 and 16 nm by FI-spICPMS. Mass balance taking into account the amount of dissolved Ni was verified in all cases. No degradation in performance resulted from using FI-spICPMS instead of spICPMS, even though measurement of NPs mass by FI-spICPMS is done without knowledge of the transport efficiency and the sample uptake rate. This is the first time that spICPMS and FI-spICPMS are demonstrated to be suitable for the characterization of ferromagnetic NPs.Plants have excellent abilities to regenerate from detached tissues, in which various phytohormones play critical roles. It has been reported that jasmonate and auxin appeared sequentially during direct de novo root regeneration (DNRR) after leave detachment. However, the role of salicylic acid (SA) is still unknown in this procedure although it is another important plant hormone. We have demonstrated the potential of electrochemical sensors for real time screening of SA but the stability still needed to be improved. Herein a digital plotter was used to modify the carbon tape modified electrodes with pencil traces in order to improve the reproducibility. The modified electrodes in paper-based analytical devices were applied to monitor SA during direct DNRR. The drawing routines and the distances between two close traces were optimized. Our results showed that the carbon tape modified electrodes achieved more reproducible responses of SA. Combined with in vivo sampling, the results using our approach demonstrated that amounts of SA in the wild Arabidopsis thaliana leaves during direct DNRR reached highest at around 72 h after detachment and then decreased, implying that the wave of SA contents might follow that of auxin during direct DNRR. The application of the digital plotter offered a cost-effective and more reproducible method for preparation of disposable working electrodes, which might be extended for other biochemical assays.Functional DNAs-functionalized magnetic beads (MBs) offer great potential in bioanalysis field because of their target recognition and magnetic separation functions. However, the recognition capability and hybridization affinity of DNA probes often suffer from limited available space, poor probe conformation and non-selective adsorption. To overcome these limitations, we herein used aptamer-pendant DNA tetrahedron nanostructure-functionalized MBs (TETapt-tet MBs) to develop a target-response fluorescence method with tetracycline (TET) as a model. In the absence of TET, 6-carboxy-X-rhodamine-labeled complementary DNAs (ROX-cDNAs) were assembled on the surface of MBs. Upon the addition of target TET, the ROX-cDNAs were separated and released from the MBs to generate fluorescence signal. The limit of detection and limit of quantification for TET were found to be 6 pg mL-1 and 20 pg mL-1, respectively. Compared with ssDNA-functionalized MBs surface, the designed DNA tetrahedron nanostructure-based surface could decrease the hybridization time and reduce false positives, ensuring the accuracy of TET detection in complex samples. The presented method was successfully employed for TET detection in honey samples. Moreover, this functionalization strategy could be extended to detect multiple antibiotics by simply substituting different aptamer sequences. Therefore, the proposed method has great potential in the field of food safety and public health.Adenosine has received great attentions acting as a potential biomarker for monitoring lung cancer. Most of the reported studies for adenosine detection require large instruments and complicated procedures. Herein, a sensitive, rapid and in-situ colorimetric aptasensor was developed for adenosine detection. Moreover, a homemade biomimetic electronic-eye (E-eye) was established and utilized as a portable in-time detection equipment. The entire measurement can be completed within 20 min, including the combination of aptamer with adenosine or AuNPs and the detection of adenosine. Four different kinds of aptamer were compared and the results showed that the AuNPs-aptamer-biotin system was the most stable and with the widest detection range of 5.0 μM-60.0 μM and the lowest LOD of 0.17 μM. Moreover, the artificial urine samples were also tested with a linear range from 5.0 to 50.0 μM and a LOD of 0.48 μM. The results validated that the aptasensor together with the E-eye can be a promising platform for adenosine detection.