Measuring the potency labelling of onabotulinumtoxinA (Botox(®)) and incobotulinumtoxinA (Xeomin (®)) in an LD50 assay.

PubMed

Dressler, Dirk; Mander, Gerd; Fink, Klaus

2012-01-01

The biological potency of botulinum toxin (BT) drugs is determined by a standardised LD50 assay. However, the potency labelling varies vary amongst different BT drugs. One reason for this may be differences in the LD50 assays applied. When five unexpired batches of onabotulinumtoxinA (Botox(®)) and incobotulinumtoxinA (Xeomin(®)) are compared in the Xeomin(®) batch release assay, the potency variability of both BT drugs fell within the range allowed by the European Pharmacopoiea. Statistical analyses failed to detect differences in the potency labelling of both products. Although the existence of a conversion ratio has been questioned recently, our experimental data are in line with previous clinical experience showing that Botox(®) and Xeomin(®) can be compared using a 1:1 conversion ratio. Identical potency labelling allows easy exchange of both BT drugs in a therapeutic setting, and direct comparison of efficacy, adverse effects and costs.

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

PubMed

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-05-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

A novel label-free cell-based assay technology using biolayer interferometry.

PubMed

Verzijl, D; Riedl, T; Parren, P W H I; Gerritsen, A F

2017-01-15

Biolayer interferometry (BLI) is a well-established optical label-free technique to study biomolecular interactions. Here we describe for the first time a cell-based BLI (cBLI) application that allows label-free real-time monitoring of signal transduction in living cells. Human A431 epidermoid carcinoma cells were captured onto collagen-coated biosensors and serum-starved, followed by exposure to agonistic compounds targeting various receptors, while recording the cBLI signal. Stimulation of the epidermal growth factor receptor (EGFR) with EGF, the β 2 -adrenoceptor with dopamine, or the hepatocyte growth factor receptor (HGFR/c-MET) with an agonistic antibody resulted in distinct cBLI signal patterns. We show that the mechanism underlying the observed changes in cBLI signal is mediated by rearrangement of the actin cytoskeleton, a process referred to as dynamic mass redistribution (DMR). A panel of ligand-binding blocking and non-blocking anti-EGFR antibodies was used to demonstrate that this novel BLI application can be efficiently used as a label-free cellular assay for compound screening and characterization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats

PubMed Central

OLIVEIRA, Bruna Vasconcelos; BARROS SILVA, Paulo Goberlânio; NOJOSA, Jacqueline de Santiago; BRIZENO, Luiz André Cavalcante; FERREIRA, Jamile Magalhães; SOUSA, Fabrício Bitú; MOTA, Mário Rogério Lima; ALVES, Ana Paula Negreiros Nunes

2016-01-01

ABSTRACT Diabetes mellitus (DM) is a disease associated with delayed wound healing of oral ulcers by increased expression of proinflammatory cytokines and cellular apoptosis. Objective to evaluate the influence of Tumor Necrosis Factor alpha (TNF-α) and apoptosis in rats with DM treated with chamomile extract or triamcinolone. Material and Methods Wistar male rats (210.0±4.2 g) were divided into five groups: negative control group (NCG) without diabetes; positive control group (PCG) with DM (alloxan, 45 mg/kg); and groups treated with chamomile extract (normoglycemic= NCG group and diabetic= DCG group) and with triamcinolone (TG). Traumatic ulcers were performed on all animals that received topical triamcinolone, chamomile extract or saline 12/12 hours for ten days. Results On days five and ten the animals were euthanized and the ulcers were analyzed by light microscopy, TUNEL assay, and immunohistochemically (TNF-α). The NCG (p=0.0062), PCG (p=0.0285), NCG (p=0.0041), and DCG (p<0.0001) groups were completely healed on the 10th day, however, there was no healing on the TG (p=0.5127) group. The TNF-α expression showed a significant reduction from the 5th to the 10th day in NCG (p=0.0266) and DCG (p=0.0062). In connective tissue, the TUNEL assay showed a significant reduction in the number of positive cells in NCG (p=0.0273) and CNG (p=0.0469) and in the epithelium only in CDG (p=0.0320). Conclusions Chamomile extract can optimize the healing of traumatic oral ulcers in diabetic rats through the reduction of apoptosis in the epithelium and TNF-α expression. PMID:27383710

Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining

PubMed Central

Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

2011-01-01

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke. PMID:21245871

Evaluation of TaqMan qPCR System Integrating Two Identically Labelled Hydrolysis Probes in Single Assay

PubMed Central

Nagy, Alexander; Vitásková, Eliška; Černíková, Lenka; Křivda, Vlastimil; Jiřincová, Helena; Sedlák, Kamil; Horníčková, Jitka; Havlíčková, Martina

2017-01-01

Ongoing evolution of viral pathogens is a significant issue in diagnostic virology employing TaqMan qPCR/RT-qPCR. Specific concerns are related to false negativity due to probe binding failure. One option for compensating for such deficiency is to integrate a second identically labelled probe in the assay. However, how this alteration influences the reaction parameters has not been comprehensively demonstrated. In the present study, we evaluate a TaqMan protocol using two identically labelled hydrolysis probes (simple, LNA (locked-nucleic-acid)) and MGB (minor-groove-binder) modified probes and combinations thereof in a single assay. Our results based on a synthetic amplicon suggest that the second probe does not compromise the TaqMan qPCR/RT-qPCR parameters, which repeatedly and reproducibly remained comparable to those of the corresponding single-probe assays, irrespective of the relative probe orientation, whether opposite or tandem, and probe modifications or combinations thereof. On the other hand, the second probe additively contributed to the overall fluorescence signal. The utility of the dual-probe approach was demonstrated on practical examples by using field specimens. We hope that the present study might serve as a theoretical basis for the development or improvement of TaqMan qPCR/RT-qPCR assays for the detection of highly variable nucleic acid templates. PMID:28120891

A label-free, fluorescence based assay for microarray

NASA Astrophysics Data System (ADS)

Niu, Sanjun

DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

The TUNEL assay suggests mandibular regression by programmed cell death during presoldier differentiation in the nasute termite Nasutitermes takasagoensis

NASA Astrophysics Data System (ADS)

Toga, Kouhei; Yoda, Shinichi; Maekawa, Kiyoto

2011-09-01

Termite soldiers are the most specialized caste of social insects in terms of their morphology and function. Soldier development requires increased juvenile hormone (JH) titer and the two molts via a presoldier stage. These molts are accompanied by dramatic morphological changes, including the exaggeration and regression of certain organs. Soldiers of the most apical termitid subfamily Nasutitermitinae possess not only a horn-like frontal tube, called the nasus, for the projection of defensive chemicals from the frontal gland reservoir but also regressed mandibles. Although candidate genes regulating soldier mandibular growth were reported in a relatively basal termite species, the regulatory mechanisms of mandibular regression remain unknown. To clarify these mechanisms, we performed morphological and histological examinations of the mandibles during soldier differentiation in Nasutitermes takasagoensis. Mandibular size reduced dramatically during soldier differentiation, and mandibular regression occurred just prior to the presoldier molt. Spotted TUNEL signals were observed in regressing mandibles of presoldiers, suggesting that the regression involved programmed cell death. Because soldiers of N. takasagoensis possess exaggerated organs (nasus and frontal gland), the present results suggest that JH-dependent regressive mechanisms exist in the mandibles without interfering with the formation of the exaggerated organs.

Technical note: The calibration of {sup 90}Y-labeled SIR-Spheresusing a nondestructive spectroscopic assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selwyn, R.; Micka, J.; DeWerd, L.

2008-04-15

{sup 90}Y-labeled SIR-Spheres are currently used to treat patients with hepatic metastases secondary to colorectal adenocarcinoma. In general, the prescribed activity is based on empirical data collected during clinical trials. The activity of the source vial is labeled by the manufacturer as 3.0 GBq{+-}10% and is not independently verified by the end user. This technical note shows that the results of a nondestructive spectroscopic assay of a SIR-Spheressample was 26% higher than the activity stated by the manufacturer. This difference should not impact the current empirical prescription method but may be problematic for patient-specific dosimetry applications, such as image-based dosimetry.

A novel colloidal gold labeled antigen for the detection of Deoxynivalenol using an immunochromatographic assay method

NASA Astrophysics Data System (ADS)

Jin, Yu; Liu, Renrong; Zhu, Lixin; Chen, Zhenzhen

2017-11-01

In this paper, an immunochromatographic assay card was developed for the detection of DON in feed and cereals using a novel colloidal gold labeling method. For the colloidal gold immunochromatographic rapid detection (GICD) card, a monoclonal antibody DON-mAb and a goat anti-chicken IgY were drawn on NC membrane as the test line (T line) and the control line (C line) respectively. A gold labeled DON-CBSA conjugate and a gold labeled chicken IgY were sprayed onto the conjugate pad. The GICD card has cut-off levels of 50ng/mL for DON, which is invulnerable to matrix interference, and applicable to a wide range of samples. The GICD detecting results of feed and grain samples were compared with the results of ELISA testing, which showed good consistency.

An in vivo, label-free quick assay for xylose transport in Escherichia coli.

PubMed

Chen, Tingjian; Zhang, Jingqing; Liang, Ling; Yang, Rong; Lin, Zhanglin

2009-07-01

Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-beta-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms.

Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

PubMed

Paradh, A D; Hill, A E; Mitchell, W J

2014-01-01

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories. © 2013 Elsevier B.V. All rights reserved.

Study of Extrinsic Apoptotic Pathway in Oral Pemphigus Vulgaris Using TNFR 1 and FasL Immunohistochemical Markers and TUNEL Technique

PubMed Central

Deyhimi, Parviz; Alishahi, Batoul

2018-01-01

Statement of the Problem: Pemphigus vulgaris is characterized by intraepithelial vesicles, but pathogenesis of vesicle formation in this disease has not been substantiated yet. Purpose: The present study investigate extrinsic apoptotic pathway in oral pemphigus vulgaris using TUNEL and important immunohistochemical markers of extrinsic pathway, TNFR1 and FasL. Materials and Method: In the present cross sectional study, 25 oral pemphigus vulgaris samples and 6 normal oral mucosa were analyzed for the presence of apoptosis by TUNEL and the staining of TNFR1 and FasL in basal and parabasal layers around vesicle, vesicle floor, vesicle roof and acantholytic cells. The staining expression and intensity were measured and the obtained data were analyzed by Wilcoxon and Mann-Whitney tests. Results: There was no or faint staining of TUNEL, FasL and TNFR1 in normal oral mucosa. In addition, there was no significant difference between the staining of TUNEL technique in different layers. The staining of TNFR1 marker was very high in all regions. FasL marker was not positive in the basal and parabasal layers around vesicle in 92% of samples but showed a varied and different staining in vesicle region. There was a significant difference between the each two markers in all layers (p <0.001). Conclusion: Apoptosis is probably is a preceding phenomenon to acantholysis in pemphigus vulgaris. It appears that the apoptosis occurs mostly by extrinsic pathway using proapototic mediators TNFR1 and FasL. PMID:29854887

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides.

PubMed

Hsieh, Yi-Wen; Alqadah, Amel; Chuang, Chiou-Fen

2016-11-29

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2 G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2 G73E proteins) as a biological example.

A novel label-free fluorescence assay for one-step sensitive detection of Hg2+ in environmental drinking water samples

NASA Astrophysics Data System (ADS)

Li, Ya; Liu, Nan; Liu, Hui; Wang, Yu; Hao, Yuwei; Ma, Xinhua; Li, Xiaoli; Huo, Yapeng; Lu, Jiahai; Tang, Shuge; Wang, Caiqin; Zhang, Yinhong; Gao, Zhixian

2017-04-01

A novel label-free fluorescence assay for detection of Hg2+ was developed based on the Hg2+-binding single-stranded DNA (ssDNA) and SYBR Green I (SG I). Differences from other assays, the designed rich-thymine (T) ssDNA probe without fluorescent labelling can be rapidly formed a T-Hg2+-T complex and folded into a stable hairpin structure in the presence of Hg2+ in environmental drinking water samples by facilitating fluorescence increase through intercalating with SG I in one-step. In the assay, the fluorescence signal can be directly obtained without additional incubation within 1 min. The dynamic quantitative working ranges was 5-1000 nM, the determination coefficients were satisfied by optimization of the reaction conditions. The lowest detection limit of Hg2+ was 3 nM which is well below the standard of U.S. Environmental Protection Agency. This method was highly specific for detecting of Hg2+ without being affected by other possible interfering ions from different background compositions of water samples. The recoveries of Hg2+ spiked in these samples were 95.05-103.51%. The proposed method is more viable, low-costing and simple for operation in field detection than the other methods with great potentials, such as emergency disposal, environmental monitoring, surveillance and supporting of ecological risk assessment and management.

Different Approaches for Assaying Melanosome Transfer

PubMed Central

Berens, Werner; Van Den Bossche, Karolien; Yoon, Tae-Jin; Westbroek, Wendy; Valencia, Julio C.; Out, Coby J.; Naeyaert, Jean Marie; Hearing, Vincent J.; Lambert, Jo

2006-01-01

Summary Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably due to incorrect sorting or deficient incorporation of the fluorescent protein and different localisation. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsination and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer. PMID:16162177

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

PubMed

Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

2003-02-01

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury.

PubMed

Matylevitch, N P; Schuschereba, S T; Mata, J R; Gilligan, G R; Lawlor, D F; Goodwin, C W; Bowman, P D

1998-08-01

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.

Label-free detection of protein molecules secreted from an organ-on-a-chip model for drug toxicity assays

NASA Astrophysics Data System (ADS)

Morales, Andres W.; Zhang, Yu S.; Aleman, Julio; Alerasool, Parissa; Dokmeci, Mehmet R.; Khademhosseini, Ali; Ye, Jing Yong

2016-03-01

Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.

Qdot Labeled Actin Super Resolution Motility Assay Measures Low Duty Cycle Muscle Myosin Step-Size

PubMed Central

Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P.

2013-01-01

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot’s were imaged in time with total internal reflection fluorescence microscopy then spatially localized to 1-3 nanometers using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full length β-cardiac myosin (MYH7). Average Qdot-actin velocity matches measurements with rhodamine-phalloidin labeled actin. The sHMM Qdot-actin velocity histogram contains low velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm, and, larger compliance than sHMM depending on MYH7 surface concentration. Low duty cycle skeletal and cardiac myosin present challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach provides quick, quantitative, and inexpensive step-size measurement for low duty cycle muscle myosin. PMID:23383646

Potency values from the local lymph node assay: application to classification, labelling and risk assessment.

PubMed

Loveless, S E; Api, A-M; Crevel, R W R; Debruyne, E; Gamer, A; Jowsey, I R; Kern, P; Kimber, I; Lea, L; Lloyd, P; Mehmood, Z; Steiling, W; Veenstra, G; Woolhiser, M; Hennes, C

2010-02-01

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment. 2009 Elsevier Inc. All rights reserved.

Apoptosis and Accidental Cell Death in Cultured Human Keratinocytes after Thermal Injury

PubMed Central

Matylevitch, Natalia P.; Schuschereba, Steven T.; Mata, Jennifer R.; Gilligan, George R.; Lawlor, David F.; Goodwin, Cleon W.; Bowman, Phillip D.

1998-01-01

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72°C for 1 second. After exposure to temperatures of 58 to 59°C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66°C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72°C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59°C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation. PMID:9708816

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

PubMed

Krasitskaya, V V; Korneeva, S I; Kudryavtsev, A N; Markova, S V; Stepanyuk, G A; Frank, L A

2011-11-01

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

Fluorescence Immunofiltration Assay of Brucella Melitensis.

DTIC Science & Technology

1995-01-01

second urease -labelled antibody directed against fluorescein. The assay system is useful for measuring protein, virus and bacteria in aqueous...binding site for the signal-generating urease -labelled antibody, it is a highly fluorescent molecule and has signal-generating capacity of its own

Shrink-induced graphene sensor for alpha-fetoprotein detection with low-cost self-assembly and label-free assay

NASA Astrophysics Data System (ADS)

Sando, Shota; Zhang, Bo; Cui, Tianhong

2017-12-01

Combination of shrink induced nano-composites technique and layer-by-layer (LbL) self-assembled graphene challenges controlling surface morphology. Adjusting shrink temperature achieves tunability on graphene surface morphology on shape memory polymers, and it promises to be an alternative in fields of high-surface-area conductors and molecular detection. In this study, self-assembled graphene on a shrink polymer substrate exhibits nanowrinkles after heating. Induced nanowrinkles on graphene with different shrink temperature shows distinct surface roughness and wettability. As a result, it becomes more hydrophilic with higher shrink temperatures. The tunable wettability promises to be utilized in, for example, microfluidic devices. The graphene on shrink polymer also exhibits capability of being used in sensing applications for pH and alpha-fetoprotein (AFP) detection with advantages of label free and low cost, due to self-assembly technique, easy functionalization, and antigen-antibody reaction on graphene surface. The detection limit of AFP detection is down to 1 pg/mL, and therefore the sensor also has a significant potential for biosensing as it relies on low-cost self-assembly and label-free assay.

Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.

PubMed

Chung, Heaseung Sophia; Murray, Christopher I; Venkatraman, Vidya; Crowgey, Erin L; Rainer, Peter P; Cole, Robert N; Bomgarden, Ryan D; Rogers, John C; Balkan, Wayne; Hare, Joshua M; Kass, David A; Van Eyk, Jennifer E

2015-10-23

S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations. © 2015 American Heart Association, Inc.

Reductive methods for isotopic labeling of antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Champney, W.S.

1989-08-15

Methods for the reductive methylation of the amino groups of eight different antibiotics using {sup 3}HCOH or H{sup 14}COH are presented. The reductive labeling of an additional seven antibiotics by NaB{sub 3}H{sub 4} is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Reactivity assay of surface carboxyl chain-ends of poly(ethylene terephthalate) (PET) film and track-etched microporous membranes using fluorine labelled- and/or 3H-labelled derivatization reagents: tandem analysis by X-ray photoelectron spectroscopy (XPS) and liquid scintillation counting (LSC)

NASA Astrophysics Data System (ADS)

Deldime, Michèle; Dewez, Jean-Luc; Schneider, Yves-Jacques; Marchand-Brynaert, Jacqueline

1995-09-01

Poly(ethylene terephthalate) (PET) films and track-etched microporous membranes of two different porosities were pretreated by hydrolysis and/or oxidation in order to enhance the amount of carboxyl chain-ends displayed on their surface. The reactivity of these carboxyl functions was determined by derivatization assays in which the reactions were carried out under conditions likely to be encountered in the coupling of water-soluble biochemical signals on the surface of biomaterials. Original reagents, fluorine-labelled and/or 3H-labelled aminoacid compounds, were used. The derivatized PET samples were examined by X-ray photoelectron spectroscopy (XPS) to characterize their apparent surfaces, and by liquid scintillation counting (LSC) to quantify the amount of tags fixed on their open surfaces. Using this dual assay technique, we analyzed the surface of microporous membranes which are currently used as substrates for cell culture systems.

Impedance-based cellular assay technologies: recent advances, future promise.

PubMed

McGuinness, Ryan

2007-10-01

Cell-based assays are continuing to grow in importance in the drug discovery workflow. Their early introduction holds the promise of limiting attrition in the later, more costly phases of the process. This article reviews recent advances in the development of impedance technologies for label-free cell-based assays. These systems are capable of monitoring endogenous receptor activation, and thus generate more physiologically relevant measures of pharmacological endpoints. Primary cells can be investigated as well, thus producing disease relevant information. Label-free assays significantly decrease assay development efforts and avoid many complications inherent in recombinant readout systems. Impedance-based systems have great potential to advance the utility of cell-based assays as they are applied to drug discovery and pharmacology.

Development of an upconverting chelate assay

NASA Astrophysics Data System (ADS)

Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

2005-04-01

We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments.

PubMed

Eng, Lars; Nygren-Babol, Linnéa; Hanning, Anders

2016-10-01

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system-the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors. Copyright © 2016 Elsevier Inc. All rights reserved.

Performance limitations of label-free sensors in molecular diagnosis using complex samples

NASA Astrophysics Data System (ADS)

Varma, Manoj

2016-03-01

Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

Development of binding assays in microfabricated picoliter vials: an assay for biotin.

PubMed

Grosvenor, A L; Feltus, A; Conover, R C; Daunert, S; Anderson, K W

2000-06-01

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.

An improved immobilized enzyme reactor-mass spectrometry-based label free assay for butyrylcholinesterase ligand screening.

PubMed

Vilela, Adriana Ferreira Lopes; Seidl, Cláudia; Lima, Juliana Maria; Cardoso, Carmen Lúcia

2018-05-15

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H] + m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC 50 values were 0.03 ± 0.006 μM and 0.88 ± 0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ± 0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results. Copyright © 2018. Published by Elsevier Inc.

Label-Enhanced Surface Plasmon Resonance: A New Concept for Improved Performance in Optical Biosensor Analysis

PubMed Central

Granqvist, Niko; Hanning, Anders; Eng, Lars; Tuppurainen, Jussi; Viitala, Tapani

2013-01-01

Surface plasmon resonance (SPR) is a well-established optical biosensor technology with many proven applications in the study of molecular interactions as well as in surface and material science. SPR is usually applied in the label-free mode which may be advantageous in cases where the presence of a label may potentially interfere with the studied interactions per se. However, the fundamental challenges of label-free SPR in terms of limited sensitivity and specificity are well known. Here we present a new concept called label-enhanced SPR, which is based on utilizing strongly absorbing dye molecules in combination with the evaluation of the full shape of the SPR curve, whereby the sensitivity as well as the specificity of SPR is significantly improved. The performance of the new label-enhanced SPR method was demonstrated by two simple model assays: a small molecule assay and a DNA hybridization assay. The small molecule assay was used to demonstrate the sensitivity enhancement of the method, and how competitive assays can be used for relative affinity determination. The DNA assay was used to demonstrate the selectivity of the assay, and the capabilities in eliminating noise from bulk liquid composition variations. PMID:24217357

Using label-free screening technology to improve efficiency in drug discovery.

PubMed

Halai, Reena; Cooper, Matthew A

2012-02-01

Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

Study and development of label-free optical biosensors for biomedical applications

NASA Astrophysics Data System (ADS)

Choi, Charles J.

For the majority of assays currently performed, fluorescent or colorimetric chemical labels are commonly attached to the molecules under study so that they may be readily visualized. The methods of using labels to track biomolecular binding events are very sensitive and effective, and are employed as standardized assay protocol across research labs worldwide. However, using labels induces experimental uncertainties due to the effect of the label on molecular conformation, active binding sites, or inability to find an appropriate label that functions equivalently for all molecules in an experiment. Therefore, the ability to perform highly sensitive biochemical detection without the use of fluorescent labels would further simplify assay protocols and would provide quantitative kinetic data, while removing experimental artifacts from fluorescent quenching, shelf-life, and background fluorescence phenomena. In view of the advantages mentioned above, the study and development of optical label-free sensor technologies have been undertaken here. In general, label-free photonic crystal (PC) biosensors and metal nanodome array surface-enhanced Raman scattering (SERS) substrates, both of which are fabricated by nanoreplica molding process, have been used as the method to attack the problem. Chapter 1 shows the work on PC label-free biosensor incorporated microfluidic network for bioassay performance enhancement and kinetic reaction rate constant determination. Chapter 2 describes the work on theoretical and experimental comparison of label-free biosensing in microplate, microfluidic, and spot-based affinity capture assays. Chapter 3 shows the work on integration of PC biosensor with actuate-to-open valve microfluidic chip for pL-volume combinatorial mixing and screening application. In Chapter 4, the development and characterization of SERS nanodome array is shown. Lastly, Chapter 5 describes SERS nanodome sensor incorporated tubing for point-of-care monitoring of

Direct Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The competitive enzyme-linked immunosorbent assay (ELISA) (cELISA; also called an inhibition ELISA) is designed so that purified antigen competes with antigen in the test sample for binding to an antibody that has been immobilized in microtiter plate wells. The same concept works if the immobilized molecule is antigen and the competing molecules are purified labeled antibody versus antibody in a test sample. Direct cELISAs incorporate labeled antigen or antibody, whereas indirect assay configurations use reporter-labeled secondary antibodies. The cELISA is very useful for determining the concentration of small-molecule antigens in complex sample mixtures. In the direct cELISA, antigen-specific capture antibody is adsorbed onto the microtiter plate before incubation with either known standards or unknown test samples. Enzyme-linked antigen (i.e., labeled antigen) is also added, which can bind to the capture antibody only when the antibody's binding site is not occupied by either the antigen standard or antigen in the test samples. Unbound labeled and unlabeled antigens are washed away and substrate is added. The amount of antigen in the standard or the test sample determines the amount of reporter-labeled antigen bound to antibody, yielding a signal that is inversely proportional to antigen concentration within the sample. Thus, the higher the antigen concentration in the test sample, the less labeled antigen is bound to the capture antibody, and hence the weaker is the resultant signal. © 2017 Cold Spring Harbor Laboratory Press.

Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase.

PubMed

King, J A; Ball, J K

1993-09-01

An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.

Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs.

PubMed

Hanko, Valoran P; Rohrer, Jeffrey S

2010-01-05

The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.

Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons

PubMed Central

Wang, Wei-Ping; Iyo, Abiye H.; Miguel-Hidalgo, Javier; Regunathan, Soundar; Zhu, Meng-Yang

2010-01-01

Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-d-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, β-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 µM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property. PMID:16546145

Methods and devices for protein assays

DOEpatents

Chhabra, Swapnil [San Jose, CA; Cintron, Jose M [Indianapolis, IN; Shediac, Renee [Oakland, CA

2009-11-03

Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

GaAlAs laser irradiation induces active tertiary dentin formation after pulpal apoptosis and cell proliferation in rat molars.

PubMed

Shigetani, Yoshimi; Sasa, Natsuki; Suzuki, Hironobu; Okiji, Takashi; Ohshima, Hayato

2011-08-01

This study aimed to clarify pulpal responses to gallium-aluminum-arsenide (GaAlAs) laser irradiation. Maxillary first molars of 8-week-old rats were irradiated at an output power of 0.5 or 1.5 W for 180 seconds, and the samples were collected at intervals of 0 to 14 days. The demineralized paraffin sections were processed for immunohistochemistry for heat-shock protein (HSP)-25 and nestin in addition to cell proliferation assay using bromodeoxyuridine (BrdU) labeling and apoptosis assay using deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL). Intense HSP-25 and nestin immunoreactivities in the odontoblast layer were weakened immediately after 0.5-W irradiation and recovered on day 1, resulting in slight tertiary dentin formation by day 14. On the contrary, 1.5-W irradiation immediately induced the loss of HSP-25 and nestin-immunoreactivities in the odontoblast layer. On day 1, numerous TUNEL-positive cells appeared in a degenerative zone that was surrounded by intense HSP-25 immunoreactivity. BrdU-positive cells occurred within the intensely HSP-25-immunopositive areas during days 2 through 5, whereas TUNEL-positive cells gradually decreased in number by day 5. HSP-25- and nestin-positive odontoblast-like cells were arranged along the pulp-dentin border by day 7, resulting in remarkable tertiary dentin formation on day 14. The output energy determined pulpal healing patterns after GaAlAs laser irradiation; the higher energy induced the apoptosis in the affected dental pulp including odontoblasts followed by active cell proliferation in the intense HSP-25-immunoreactive areas surrounding the degenerative tissue, resulting in abundant tertiary dentin formation. Thus, the optimal GaAlAs laser irradiation elicited intentional tertiary dentin formation in the dental pulp. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Toxin activity assays, devices, methods and systems therefor

DOEpatents

Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

2016-04-05

Embodiments of the present invention are directed toward devices, system and method for conducting toxin activity assay using sedimentation. The toxin activity assay may include generating complexes which bind to a plurality of beads in a fluid sample. The complexes may include a target toxin and a labeling agent, or may be generated due to presence of active target toxin and/or labeling agent designed to be incorporated into complexes responsive to the presence of target active toxin. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a lower density than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

PubMed

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Evaluation of a 99mTc-labeled AnnexinA5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate.

PubMed

Greupink, Rick; Sio, Charles F; Ederveen, Antwan; Orsel, Joke

2009-12-01

We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate. AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration. HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation. (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

Label-free DNA hybridization detection and single base-mismatch discrimination using CE-ICP-MS assay.

PubMed

Li, Yan; Sun, Shao-kai; Yang, Jia-lin; Jiang, Yan

2011-12-07

Detecting a specific DNA sequence and discriminating single base-mismatch is critical to clinical diagnosis, paternity testing, forensic sciences, food and drug industry, pathology, genetics, environmental monitoring, and anti-bioterrorism. To this end, capillary electrophoresis (CE) coupled with the inductively coupled plasma mass spectrometry (ICP-MS) method is developed using the displacing interaction between the target ssDNA and the competitor Hg(2+) for the first time. The thymine-rich capture ssDNA 1 is interacted with the competitor Hg(2+), forming an assembled complex in a hairpin-structure between the thymine bases arrangement at both sides of the capture ssDNA 1. In the presence of a target ssDNA with stronger affinity than that of the competitor Hg(2+), the energetically favorable hybridization between capture ssDNA 1 and the target ssDNA destroys the hairpin-structure and releases the competitor as free Hg(2+), which was then read out and accurately quantified by CE-ICP-MS assay. Under the optimal CE separation conditions, free Hg(2+) ions and its capture ssDNA 1 adduct were baseline separated and detected on-line by ICP-MS; the increased peak intensity of free Hg(2+) against the concentration of perfectly complementary target ssDNA was linear over the concentration range of 30-600 nmol L(-1) with a limit of detection of 8 nmol L(-1) (3s, n = 11) in the pre-incubated mixture containing 1 μmol L(-1) Hg(2+) and 0.2 μmol L(-1) capture ssDNA 1. This new assay method is simple in design since any target ssDNA binding can in principle result in free Hg(2+) release by 6-fold Hg(2+) signal amplification, avoiding oligonucleotide labeling or assistance by excess signal transducer and signal reporter to read out the target. Due to element-specific detection of ICP-MS in our assay procedure, the interference from the autofluorescence of substrata was eliminated.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

PubMed

Fonseca, M E; Marcellino, L H; Gander, E

1996-04-05

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

DNA Methyltransferase Activity Assays: Advances and Challenges

PubMed Central

Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

2016-01-01

DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

Label-free fluorescent enzymatic assay of citrate synthase by CoA-Au(I) co-ordination polymer and its application in a multi-enzyme logic gate cascade.

PubMed

Li, Yong; Wang, Huixia; Dai, Futao; Li, Pei; Jin, Xin; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

2016-12-15

Citrate synthase (CS) is one of the key metabolic enzymes in the Krebs tricarboxylic acid (TCA) cycle. It regulates energy generation in mitochondrial respiration by catalysing the reaction between oxaloacetic acid (OAA) and acetyl coenzyme A (Ac-CoA) to generate citrate and coenzyme A (CoA). CS has been shown to be a biomarker of neurological diseases and various kinds of cancers. Here, a label-free fluorescent assay has been developed for homogeneously detecting CS and its inhibitor based on the in situ generation of CoA-Au(I) co-ordination polymer (CP) and the fluorescence signal-on by SYBR Green II-stained CoA-Au(I) CP. Because of the unique property of the CoA-Au(I) CP, this CS activity assay method could achieve excellent selectivity and sensitivity, with a linear range from 0.0033 U/μL to 0.264 U/μL and a limit of detection to be 0.00165 U/μL. Meanwhile, this assay method has advantages of being facile and cost effective with quick detection. Moreover, based on this method, a biomimetic logic system was established by rationally exploiting the cascade enzymatic interactions in TCA cycle for chemical information processing. In the TCA cycle-derived logic system, an AND-AND-AND-cascaded gate was rigorously operated step by step in one pot, and is outputted by a label-free fluorescent signal with visualized readout. Copyright © 2016 Elsevier B.V. All rights reserved.

Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

PubMed

Marras, Salvatore A E

2008-03-01

The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

NASA Astrophysics Data System (ADS)

Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

2015-01-01

This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Assay for vitamin B12 absorption and method of making labeled vitamin B12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Peter J; Dueker, Stephen; Miller, Joshua

2012-06-19

The invention provides methods for labeling vitamin B12 with .sup.14C, .sup.13C, tritium, and deuterium. When radioisotopes are used, the invention provides for methods of labeling B12 with high specific activity. The invention also provides labeled vitamin B12 compositions made in accordance with the invention.

Expression of Cyt-c-Mediated Mitochondrial Apoptosis-Related Proteins in Rat Renal Proximal Tubules during Development.

PubMed

Song, Xiao-Feng; Tian, He; Zhang, Ping; Zhang, Zhen-Xing

2017-01-01

Apoptosis regulates embryogenesis, organ metamorphosis and tissue homeostasis. Mitochondrial signaling is an apoptotic pathway, in which Cyt-c and Apaf-1 are transformed into an apoptosome, which activates procaspase-9 and triggers apoptosis. This study evaluated Cyt-c, Apaf-1 and caspase-9 expression during renal development. Kidneys from embryonic (E) 16-, 18-, and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups were obtained. Immunohistochemical analysis, dual-labeled immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique assay and Western blot were performed in addition to histological analysis. Immunohistochemistry showed that Cyt-c was strongly expressed in proximal and distal tubules (DTs) at all time points. Caspase-9 and Apaf-1 were strongly expressed in proximal tubules (PTs) but only weakly expressed in DTs. Dual-labeled immunofluorescence showed that most tubules expressed both Cyt-c and Apaf-1, except for some tubules that only expressed Cyt-c. The TUNEL assay showed a greater percentage of apoptotic cells in PTs compared to DTs. Apaf-1 and cleaved caspase-9 protein expression gradually increased during the embryonic period and peaked during the early postnatal period but apparently declined from P7. Cyt-c protein expression was weak during the embryonic period but obviously increased after P1. This study showed that PTs are more sensitive to apoptosis than DTs during rat renal development, even though both tubule segments contain a large number of mitochondria. Furthermore, Cyt-c-mediated mitochondrial apoptosis-related proteins play an important role in PTs during the early postnatal kidney development. © 2016 S. Karger AG, Basel.

Thermometric enzyme linked immunosorbent assay: TELISA.

PubMed

Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

1977-08-11

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Base pair mismatch recognition using plasmon resonant particle labels.

PubMed

Oldenburg, Steven J; Genick, Christine C; Clark, Keith A; Schultz, David A

2002-10-01

We demonstrate the use of silver plasmon resonant particles (PRPs), as reporter labels, in a microarray-based DNA hybridization assay in which we screen for a known polymorphic site in the breast cancer gene BRCA1. PRPs (40-100 nm in diameter) image as diffraction-limited points of colored light in a standard microscope equipped with dark-field illumination, and can be individually identified and discriminated against background scatter. Rather than overall intensity, the number of PRPs counted in a CCD image by a software algorithm serves as the signal in these assays. In a typical PRP hybridization assay, we achieve a detection sensitivity that is approximately 60 x greater than that achieved by using fluorescent labels. We conclude that single particle counting is robust, generally applicable to a wide variety of assay platforms, and can be integrated into low-cost and quantitative detection systems for single nucleotide polymorphism analysis.

Y-27632, a Rho-associated protein kinase inhibitor, attenuates neuronal cell death after transient retinal ischemia.

PubMed

Hirata, Akira; Inatani, Masaru; Inomata, Yasuya; Yonemura, Naoko; Kawaji, Takahiro; Honjo, Megumi; Tanihara, Hidenobu

2008-01-01

Transient retinal ischemia induces the death of retinal neuronal cells. Postischemic damage is associated with the infiltration of leukocytes into the neural tissue through vascular endothelia. The current study aimed to investigate whether this damage was attenuated by the inhibition of Rho/ROCK (Rho kinases) signaling, recently shown to play a critical role in the transendothelial migration of leukocytes. Y-27632, a selective inhibitor of ROCK, was injected intravitreally into rat eyes with transient retinal ischemia. Cell loss of the ganglion cell layer (GCL) and thinning of the inner plexiform layer (IPL) with and without the administration of Y-27632 were evaluated by histological analysis, TUNEL assay and retrograde labeling of retinal ganglion cells (RGCs). To examine the attenuation of leukocyte infiltration in postischemic retinas with the administration of Y-27632, silver nitrate staining and immunohistochemistry using an anti-LCA antibody were performed. Cell loss of the GCL and thinning of the IPL were significantly attenuated when 100 nmol Y-27632 was administered within three hours of the induction of ischemia. TUNEL assay and retrograde labeling of RGCs showed a decreased number of apoptotic cells and an increased number of RGCs in Y-27632-injected retinas. Moreover, silver nitrate staining and immunohistochemical analysis using an anti-LCA antibody showed that Y-27632 injection dramatically inhibited leukocyte infiltration and endothelial disarrangement. Our data suggest that inhibition of Rho/ROCK signaling offers neuroprotective therapy against postischemic neural damage, by regulating leukocyte infiltration in the neural tissue.

Accuracy of a prey-specific DNA assay and a generic prey-immunomarking assay for detecting predation

USDA-ARS?s Scientific Manuscript database

1. Predator gut examinations are useful for detecting arthropod predation events. Here, the accuracy and reproducibility of two different types of gut assays are tested on various predator species that consumed an immature lacewing, Chrysoperla carnea (Stephens), that was externally labelled with ra...

The role of heat shock protein 70 (Hsp 70) in male infertility: is it a line of defense against sperm DNA fragmentation?

PubMed

Erata, Gül Ozdemirler; Koçak Toker, Necla; Durlanik, Ozgür; Kadioğlu, Ateş; Aktan, Gülşen; Aykaç Toker, Gülçin

2008-08-01

To clarify the role of heat shock protein 70 (Hsp 70) and its relation with DNA damage in male infertility. Prospective study. Andrology laboratory of Istanbul Medical Faculty. Semen samples from 37 infertile men and 13 fertile men (as controls). The percentage of DNA fragmentation was assayed with the use of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Sperm Hsp 70 expression was determined by using Western blot analysis. Both the percentages of sperm DNA fragmentation and Hsp 70 expression were correlated with semen analysis parameters. TUNEL-positive spermatozoa in the infertile group (18.7% for asthenospermics and 13.0% for oligoasthenospermics) were higher than the fertile group (4.9%). Significant inverse correlations were detected between percentage of TUNEL-positive cells and both concentration (r = -0.487) and motility (r = -0.377) of spermatozoa. No expression of Hsp 70 was observed in azospermic group, whereas Hsp 70 levels were found increased significantly in infertile group (U = 62 for asthenospermics and U = 38 for oligoasthenospermics) compared to fertile group as analyzed by using Mann-Whitney U Wilcoxon rank sum test. Furthermore, significant positive correlation was found between percentage of TUNEL-positive cells and Hsp 70 expression (r = 0.357). Hsp 70 expression may have been increased as a protective mechanism against apoptosis in spermatozoa of infertile men.

Effect of green light spectra on the reduction of retinal damage and stress in goldfish, Carassius auratus.

PubMed

Song, Jin Ah; Kim, Na Na; Choi, Young Jae; Choi, Cheol Young

2016-07-22

We investigated the effect of light spectra on retinal damage and stress in goldfish using green (530 nm) and red (620 nm) light emitting diodes (LEDs) at three intensities each (0.5, 1.0, and 1.5 W/m(2)). We measured the change in the levels of plasma cortisol and H2O2 and expression and levels of caspase-3. The apoptotic response of green and red LED spectra was assessed using the terminal transferase dUTP nick end labeling (TUNEL) assay. Stress indicator (cortisol and H2O2) and apoptosis-related genes (caspase-3) decreased in green light, but increased in red light with higher light intensities over time. The TUNEL assay revealed that more apoptotic cells were detected in outer nuclear layers after exposure to red LED over time with the increase in light intensity, than the other spectra. These results indicate that green light efficiently reduces retinal damage and stress, whereas red light induces it. Therefore, red light-induced retina damage may induce apoptosis in goldfish retina. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratcliffe, W.A.; Corrie, J.E.; Dalziel, A.H.

1982-06-01

Researchers compared two direct radioimmunoassays for progesterone in 50 microL of unextracted serum or plasma with assays involving extraction of serum. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11 alpha hemisuccinyl conjugate and the radioligand /sup 125/I-labeled progesterone 11 alpha-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r greatermore » than 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. Researchers conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum.« less

Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.

1982-06-01

Two direct radioimmunoassays for progesterone in 50 ..mu..L of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11..cap alpha..-hemisuccinyl conjugate and the radioligand /sup 125/I-labeled progesterone 11..cap alpha..-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96)more » with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum.« less

COX-2 regulation and TUNEL-positive cell death differ between genders in the secondary inflammatory response following experimental penetrating focal brain injury in rats.

PubMed

Günther, Mattias; Plantman, Stefan; Davidsson, Johan; Angéria, Maria; Mathiesen, Tiit; Risling, Mårten

2015-04-01

Traumatic brain injury is followed by secondary neuronal degeneration, largely dependent on an inflammatory response. This response is probably gender specific, since females are better protected than males in experimental models. The reasons are not fully known. We examined aspects of the inflammatory response following experimental TBI in male and female rats to explore possible gender differences at 24 h and 72 h after trauma, times of peak histological inflammation and neuronal degeneration. A penetrating brain injury model was used to produce penetrating focal TBI in 20 Sprague-Dawley rats, 5 males and 5 females for each time point. After 24 and 72 h the brains were removed and subjected to in situ hybridization and immunohistochemical analyses for COX-2, iNOS, osteopontin, glial fibrillary acidic protein, 3-nitrotyrosine, TUNEL and Fluoro-Jade. COX-2 mRNA and protein levels were increased in the perilesional area compared to the uninjured contralateral side and significantly higher in males at 24 h and 72 h (p < 0.05). iNOS mRNA was significantly increased in females at 24 h (p < 0.05) although protein was not. TUNEL was increased in male rats after 24 h (p < 0.05). Glial fibrillary acidic protein, osteopontin, 3-nitrotyrosine and Fluoro-Jade stained degenerating neurons were increased in the perilesional area, showing no difference between genders. COX-2 regulation differed between genders after TBI. The increased COX-2 expression in male rats correlated with increased apoptotic cell death detected by increased TUNEL staining at 24 h, but not with neuronal necrosis measured by Flouro-Jade. Astrogliosis and microgliosis did not differ, confirming a comparable level of trauma. The gender-specific trait of the secondary inflammatory response may be connected to prostaglandin regulation, which may partially explain gender variances in outcome after TBI.

Label-Free Aptasensors for the Detection of Mycotoxins

PubMed Central

Rhouati, Amina; Catanante, Gaelle; Nunes, Gilvanda; Hayat, Akhtar; Marty, Jean-Louis

2016-01-01

Various methodologies have been reported in the literature for the qualitative and quantitative monitoring of mycotoxins in food and feed samples. Based on their enhanced specificity, selectivity and versatility, bio-affinity assays have inspired many researchers to develop sensors by exploring bio-recognition phenomena. However, a significant problem in the fabrication of these devices is that most of the biomolecules do not generate an easily measurable signal upon binding to the target analytes, and signal-generating labels are required to perform the measurements. In this context, aptamers have been emerged as a potential and attractive bio-recognition element to design label-free aptasensors for various target analytes. Contrary to other bioreceptor-based approaches, the aptamer-based assays rely on antigen binding-induced conformational changes or oligomerization states rather than binding-assisted changes in adsorbed mass or charge. This review will focus on current designs in label-free conformational switchable design strategies, with a particular focus on applications in the detection of mycotoxins. PMID:27999353

Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

1988-07-01

A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis.

Allorecognition triggers autophagy and subsequent necrosis in the cnidarian Hydractinia symbiolongicarpus.

PubMed

Buss, Leo W; Anderson, Christopher; Westerman, Erica; Kritzberger, Chad; Poudyal, Monita; Moreno, Maria A; Lakkis, Fadi G

2012-01-01

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

PubMed

Evenson, Donald P

2016-06-01

Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of

GRP78 mediates the therapeutic efficacy of curcumin on colon cancer.

PubMed

Chang, Yu-Jia; Huang, Chien-Yu; Hung, Chin-Sheng; Chen, Wei-Yu; Wei, Po-Li

2015-02-01

Glucose-regulated protein 78 (GRP78) is the key regulator of endoplasmic reticular (ER) function. Expression of GRP78 was correlated with malignancy in different cancers. However, the role of GRP78 in the cytotoxic effect of curcumin on colon cancer cells is still unclear. A silencing RNA (siRNA) technique was used to knock down GRP78 expression. The anticancer effects of curcumin were assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometric cell cycle analysis, and a terminal dexynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. HT-29 cells expressed lower GRP78 compared with DLD-1 cells. The MTT assay revealed that HT-29 cells were more resistant to curcumin treatment than DLD-1 cells. GRP78KD cells showed more resistance to curcumin treatment compared with scrambled control cells. Overexpressed GRP78 in HT-29 cells increased the sensitivity to curcumin treatment. According to the cell cycle analysis and TUNEL assay, we found that apoptosis dramatically increased in scrambled control cells compared to GRP78KD DLD-1 cells after curcumin treatment. Finally, we evaluated levels of Bcl-2, BAX, and Bad and found that an increase of Bcl-2 level was observed in GRP78KD cells treated with curcumin. Those results were consistent with the increasing of resistance to curcumin after silencing of GRP78. The levels of GRP78 expression might determine the therapeutic efficacy of curcumin against colon cancer cells.

Measuring Sperm DNA Fragmentation and Clinical Outcomes of Medically Assisted Reproduction: A Systematic Review and Meta-Analysis.

PubMed

Cissen, Maartje; Wely, Madelon van; Scholten, Irma; Mansell, Steven; Bruin, Jan Peter de; Mol, Ben Willem; Braat, Didi; Repping, Sjoerd; Hamer, Geert

2016-01-01

Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the

Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

PubMed

Beck, P; Nicholas, H

1975-03-01

1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as

ENVIRONMENTAL TECHNOLOGY VERIFICATION REPORT, PCB DETECTION TECHNOLOGY, HYBRIZYME DELFIA TM ASSAY

EPA Science Inventory

The DELFIA PCB Assay is a solid-phase time-resolved fluoroimmunoassay based on the sequential addition of sample extract and europium-labeled PCB tracer to a monoclonal antibody reagent specific for PCBs. In this assay, the antibody reagent and sample extract are added to a strip...

Microbial identification by immunohybridization assay of artificial RNA labels

NASA Technical Reports Server (NTRS)

Kourentzi, Katerina D.; Fox, George E.; Willson, Richard C.

2002-01-01

Ribosomal RNA (rRNA) and engineered stable artificial RNAs (aRNAs) are frequently used to monitor bacteria in complex ecosystems. In this work, we describe a solid-phase immunocapture hybridization assay that can be used with low molecular weight RNA targets. A biotinylated DNA probe is efficiently hybridized in solution with the target RNA, and the DNA-RNA hybrids are captured on streptavidin-coated plates and quantified using a DNA-RNA heteroduplex-specific antibody conjugated to alkaline phosphatase. The assay was shown to be specific for both 5S rRNA and low molecular weight (LMW) artificial RNAs and highly sensitive, allowing detection of as little as 5.2 ng (0.15 pmol) in the case of 5S rRNA. Target RNAs were readily detected even in the presence of excess nontarget RNA. Detection using DNA probes as small as 17 bases targeting a repetitive artificial RNA sequence in an engineered RNA was more efficient than the detection of a unique sequence.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

PubMed

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-10-21

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

PubMed Central

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-01-01

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring. PMID:28335318

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

A mouse dry eye model induced by topical administration of benzalkonium chloride.

PubMed

Lin, Zhirong; Liu, Xiaochen; Zhou, Tong; Wang, Yihui; Bai, Li; He, Hui; Liu, Zuguo

2011-01-25

To develop a dry eye model of mouse induced by topical administration of benzalkonium chloride (BAC) and investigate the possible mechanisms. BAC at concentration of 0.2% was applied to the mouse ocular surface for 7 days. Phenol red thread tear test, tear break-up time (BUT) test, corneal inflammatory index scoring, fluorescein and rose bengal test were performed to evaluate the toxic effects of BAC on the ocular surface. Global specimens were collected on day (D) 7 and labeled with a series of antibodies including cytokeratin 10 (K10) and mucin 5AC (MUC5AC). Apoptosis of ocular surface epithelium was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Histologic analysis and transmission electron microscopy (TEM) were performed on D7. BAC at a concentration of 0.2% successfully induced a dry eye condition with decreased tear volume and BUTs, increased corneal fluorescein and rose bengal scores. The Inflammatory index was increased in accompaniment with higher tumor necrosis factor-α (TNF-α) expression and more inflammatory infiltration in the cornea. Immunolabeling revealed positive K10 expression in BAC-treated corneal epithelium and fewer MUC5AC-positive cells in the BAC-treated conjunctival fornix. TUNEL assay showed more apoptotic cells in the corneal basal epithelium. TEM showed that the size and intervals of the microvillis were both reduced in the corneal epithelium. Topical administration of 0.2% BAC in mouse induces changes resembling that of dry eye syndrome in humans, and thus, represents a novel model of dry eye.

A mouse dry eye model induced by topical administration of benzalkonium chloride

PubMed Central

Lin, Zhirong; Liu, Xiaochen; Zhou, Tong; Wang, Yihui; Bai, Li; He, Hui

2011-01-01

Purpose To develop a dry eye model of mouse induced by topical administration of benzalkonium chloride (BAC) and investigate the possible mechanisms. Methods BAC at concentration of 0.2% was applied to the mouse ocular surface for 7 days. Phenol red thread tear test, tear break-up time (BUT) test, corneal inflammatory index scoring, fluorescein and rose bengal test were performed to evaluate the toxic effects of BAC on the ocular surface. Global specimens were collected on day (D) 7 and labeled with a series of antibodies including cytokeratin 10 (K10) and mucin 5AC (MUC5AC). Apoptosis of ocular surface epithelium was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Histologic analysis and transmission electron microscopy (TEM) were performed on D7. Results BAC at a concentration of 0.2% successfully induced a dry eye condition with decreased tear volume and BUTs, increased corneal fluorescein and rose bengal scores. The Inflammatory index was increased in accompanyment with higher tumor necrosis factor-α (TNF-α) expression and more inflammatory infiltration in the cornea. Immunolabeling revealed positive K10 expression in BAC-treated corneal epithelium and fewer MUC5AC-positive cells in the BAC-treated conjunctival fornix. TUNEL assay showed more apoptotic cells in the corneal basal epithelium. TEM showed that the size and intervals of the microvillis were both reduced in the corneal epithelium. Conclusions Topical administration of 0.2% BAC in mouse induces changes resembling that of dry eye syndrome in humans, and thus, represents a novel model of dry eye. PMID:21283525

High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling.

PubMed

Ashby, Jonathan; Duan, Yaokai; Ligans, Erik; Tamsi, Michael; Zhong, Wenwan

2015-02-17

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

Latent Herpes Simplex Virus 1 Infection Does Not Induce Apoptosis in Human Trigeminal Ganglia

PubMed Central

Lindemann, Anja; Sinicina, Inga; Strupp, Michael; Brandt, Thomas; Hüfner, Katharina

2015-01-01

Herpes simplex virus 1 (HSV-1) can establish lifelong latency in human trigeminal ganglia. Latently infected ganglia contain CD8+ T cells, which secrete granzyme B and are thus capable of inducing neuronal apoptosis. Using immunohistochemistry and single-cell reverse transcription-quantitative PCR (RT-qPCR), higher frequency and transcript levels of caspase-3 were found in HSV-1-negative compared to HSV-1-positive ganglia and neurons, respectively. No terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-positive neurons were detected. The infiltrating T cells do not induce apoptosis in latently infected neurons. PMID:25762734

PET imaging of cardiomyocyte apoptosis in a rat myocardial infarction model.

PubMed

Ma, Hui; Liu, Shaoyu; Xiong, Ying; Zhang, Zhanwen; Sun, Aixia; Su, Shu; Liang, Hong; Yuan, Gongjun; Tang, Ganghua

2018-06-23

Cardiomyocyte apoptosis has been observed in several cardiovascular diseases and contributes to the subsequent cardiac remodeling processes and progression to heart failure. Consequently, apoptosis imaging is helpful for noninvasively detecting the disease progression and providing treatment guidance. Here, we tested 18 F-labeled 2-(5-fluoropentyl)-2-methyl-malonic acid ( 18 F-ML-10) and 18 F-labeled 2-(3-fluoropropyl)-2-methyl-malonic acid ( 18 F-ML-8) for apoptosis imaging in rat models of myocardial infarction (MI) and compared them with 18 F-fluorodeoxyglucose ( 18 F-FDG). MI was induced in Sprague-Dawley rats by permanent left coronary artery ligation. Procedural success was confirmed by echocardiography and positron emission tomography (PET) imaging with 18 F-FDG. In vivo PET imaging with 18 F-ML-10 and 18 F-ML-8 was performed in the MI models at different time points after operation. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays and immunohistochemical analyses were used to evaluate myocardial apoptosis. In vitro cell binding assays were performed to validate 18 F-ML-8 binding to apoptotic cardiomyocytes. PET imaging demonstrated high 18 F-ML-10 and 18 F-ML-8 uptake where 18 F-FDG uptake was absent. The focal accumulation of the two tracers was high on days 1 and 3 but was not notable on days 5 and 7 after surgery. The infarct-to-lung uptake ratio was 4.29 ± 0.30 for 18 F-ML-10 and 3.51 ± 0.18 for 18 F-ML-8 (n = 6, analyzed by averaging the uptake ratios on postoperative days 1 and 3, P < 0.05). The TUNEL results showed that myocardial cell apoptosis was closely related to the focal uptake of the apoptotic tracers in the infarct area. In addition, the apoptosis rates calculated from the TUNEL results were better correlated with 18 F-ML-8 uptake than with 18 F-ML-10 uptake. Ex vivo cell binding assays demonstrated that 18 F-ML-8 accumulated in apoptotic cells but not in necrotic or normal cells. PET imaging

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

PubMed

Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

PubMed

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

The ultrastructural research of liver in experimental obstructive jaundice and effect of honey.

PubMed

Kilicoglu, Bulent; Gencay, Cem; Kismet, Kemal; Serin Kilicoglu, Sibel; Erguder, Imge; Erel, Serap; Sunay, Asli Elif; Erdemli, Esra; Durak, Ilker; Akkus, Mehmet Ali

2008-02-01

To examine the effects of honey on oxidative stress and apoptosis in experimental obstructive jaundice model. Thirty rats were divided into 3 groups: group I, sham-operated; group II, ligation and division of the common bile duct (BDL); group III, BDL followed by oral supplementation of honey 10 g/kg/d. Liver samples were examined under light microscope and transmission electron microscope. Hepatocyte apoptosis was quantitated using the terminal deoxy-nucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Plasma and blood malondialdehyde (MDA) and glutation activities were measured for determining the oxidative stress. The liver levels of MDA and GSH were significantly different between the honey and BDL groups (P = .006 and .001, respectively). However, there was no significant difference between the plasma MDA and GSH levels of these groups (P > .05). In group III, significant reductions in the size of enlarged hepatocytes and the edema were demonstrated. The dilatation of the bile canaliculi dramatically turned to original dimention. By TUNEL assay, it was shown that administration of honey decreased the number of apoptotic cells. In the present study, we found that honey diminished the negative effects of BDL on the hepatic ultrastructure. We conclude that this effect might be due to its antioxidant and anti-inflammatory activities.

Activity-based assay for ricin-like toxins

DOEpatents

Keener, William K.; Ward, Thomas E.

2007-02-06

A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.

A novel label-free fluorescence strategy for methyltransferase activity assay based on dsDNA-templated copper nanoparticles coupled with an endonuclease-assisted signal transduction system.

PubMed

Lai, Q Q; Liu, M D; Gu, C C; Nie, H G; Xu, X J; Li, Z H; Yang, Z; Huang, S M

2016-02-21

Evaluating DNA methyltransferase (MTase) activity has received considerable attention due to its significance in the fields of early cancer clinical diagnostics and drug discovery. Herein, we proposed a novel label-free fluorescence method for MTase activity assay by coupling double-stranded DNA (dsDNA)-templated copper nanoparticles (CuNPs) with an endonuclease-assisted signal transduction system. In this strategy, dsDNA molecules were first methylated by DNA adenine methylation (Dam) MTase and then cleaved by the methylation-sensitive restriction endonuclease DpnI. The cleaved DNA fragments could not act as efficient templates for the formation of fluorescent CuNPs and thus no fluorescence signal was produced. Under optimized experimental conditions, the developed strategy exhibited a sensitive fluorescence response to Dam MTase activity. This strategy was also demonstrated to provide an excellent platform to the inhibitor screening for Dam MTase. These results demonstrated the great potential for the practical applications of the proposed strategy for Dam MTase activity assay.

Ocimum sanctum (L.) essential oil and its lead molecules induce apoptosis in Candida albicans.

PubMed

Khan, Amber; Ahmad, Aijaz; Khan, Luqman Ahmad; Manzoor, Nikhat

2014-01-01

Manipulation of endogenous responses during programmed cell death (PCD) in fungi can lead to development of effective therapeutic strategies. In the present study, we evaluated the physiology of cell death in Candida albicans in response to Ocimum sanctum essential oil (OSEO) and its two major constituents - methyl chavicol (MET CHAV) and linalool (LIN) at varying inhibitory concentrations. Apoptotic cell death was studied on the basis of externalization of membrane phosphatidylserine (PS) revealed by annexin-V-FITC labeling, morphological alterations revealed by transmission electron microscopy and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Exposure of fungal cells to MIC/4 of OSEO, MET CHAV and LIN resulted in morphological features characteristic of apoptosis, while necrosis was observed at higher concentrations. Necrotic cells displayed reduced TUNEL staining and an inability to exclude propidium iodide. In addition, they lacked a defined nucleus and an intact external morphology. Exposed cells were TUNEL-positive, showed chromatin condensation and margination, nuclear envelope separation, nuclear fragmentation, cytoplasmic shrinkage and plasma membrane blebbing. A dose-dependent decrease in cytochrome c oxidase activity was observed with each compound, but the decrease was not comparable to that elicited by H2O2, eliminating the primary involvement of cytochrome c release in apoptosis thus induced. Previously reported data revealed induction of apoptosis at low concentrations as a result of oxidative insult. Studies aimed at identifying other mitochondrial factors activated during this course to mediate apoptosis will further elucidate the mechanism of antifungal action of these natural products. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

PubMed

Schuller, Marion; Höfner, Georg; Wanner, Klaus T

2017-10-09

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deep Learning in Label-free Cell Classification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

Deep Learning in Label-free Cell Classification

PubMed Central

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-01-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells. PMID:26975219

Deep Learning in Label-free Cell Classification

DOE PAGES

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; ...

2016-03-15

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individualmore » cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. In conclusion, this system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.« less

Deep Learning in Label-free Cell Classification

NASA Astrophysics Data System (ADS)

Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

2016-03-01

Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate

PubMed Central

Levoye, Angélique; Zwier, Jurriaan M.; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z′-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization. PMID:26617570

Effects of Urtica dioica dichloromethane extract on cell apoptosis and related gene expression in human breast cancer cell line (MDA-MB-468).

PubMed

Mohammadi, A; Mansoori, B; Goldar, S; Shanehbandi, D; Khaze, V; Mohammadnejad, L; Baghbani, E; Baradaran, B

2016-02-29

Breast cancer is the most common cancer among women in worldwide, especially in developing countries. Therefore, a large number of anticancer agents with herbal origins have been reported against this deadly disease. This study is the first to examine the cytotoxic and apoptotic effects of Urtica dioica in MDA-MB-468, human breast adenocarcinoma cells. The 3-(4,5-dimethylethiazol-2 yl)-2,5- diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assay were performed in MDA-MB-468 cells as well as control cell line L929 to analyze the cytotoxic activity of the dichloromethane extract. In addition, Apoptosis induction of Urtica dioica on the MDA-MB-468 cells was assessed using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis and real-time polymerase chain reaction (PCR). The results showed that the extract significantly inhibited cell growth and viability without inducing damage to normal control cells. Nuclei Staining in TUNEL and DNA fragments in DNA fragmentation assay and increase in the mRNA expression levels of caspase-3, caspase-9, decrease in the bcl2 and no significant change in the caspase-8 mRNA expression level, showed that the induction of apoptosis was the main mechanism of cell death that induce by Urtica dioica extract. Our results suggest that urtica dioica dichloromethane extract may contain potential bioactive compound(s) for the treatment of breast adenocarcinoma.

Toxic effects of environmental pollutants: Comparative investigation using Allium cepa L. and Lactuca sativa L.

PubMed

Silveira, Graciele Lurdes; Lima, Maria Gabriela Franco; Reis, Gabriela Barreto Dos; Palmieri, Marcel José; Andrade-Vieria, Larissa Fonseca

2017-07-01

Studies that help understand the mechanisms of action of environmental pollutants are extremely important in environmental toxicology. In this context, assays using plants as models stand out for their simplicity and low performance cost. Among the plants used for this purpose, Allium cepa L. is the model most commonly applied for cytogenotoxic tests, while Lactuca sativa L., already widely used in phytotoxic investigations, has been gaining prominence in cytotoxic analyses. The present study aimed to compare the responses of A. cepa and L. sativa via macroscopic (root growth) and microscopic analyses (cell cycle and DNA fragmentation via TdT-mediated deoxy-uracil nick and labeling (TUNEL) and comet assays) after exposure of their roots to environmental pollutants with known cytogenotoxic mechanisms. Both species presented sensitive and efficient response to the applied tests after exposure to the DNA-alkylating agent Methyl Methanesulfonate (MMS), the heavy metal Cadmium, the aluminum industry waste Spent Potliner (SPL) and the herbicide Atrazine. However, they differed regarding the responses to the evaluated endpoints. Overall, A. cepa was more efficient in detecting clastogenic changes, arising from DNA breakage, while L. sativa rather detected aneugenic alterations, related to chromosome segregation in mitosis. In the tests applied to verify DNA fragmentation (comet and TUNEL assays), A. cepa presented higher sensitivity. In conclusion, both models are efficient to evaluate toxicological risks of environmental pollutants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities

PubMed Central

Fonnum, F.

1969-01-01

1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-14C]acetate or sodium [3H]acetate by incubation with CoA, ATP, Mg2+ and extract from acetone-dried pigeon liver were used. 3. [1-14C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [3H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [3H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively. PMID:4982085

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA.

PubMed

Arakawa, Hidetoshi; Nakabayashi, Shigeo; Ohno, Ken-Ichi; Maeda, Masako

2012-04-01

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA). The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm. The measurable range of HRP was 1.0×10 -18 to 1.0×10 -15  mol/assay, with a detection limit of 1.0×10 -18  mol/assay. The coefficient of variation (CV, n =8) was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements

PubMed Central

Cushman-Vokoun, Allison M.; Connealy, Solomon; Greiner, Timothy C.

2010-01-01

Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. PMID:20959612

G protein-coupled receptor internalization assays in the high-content screening format.

PubMed

Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

2006-01-01

High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

Clinical Application of an Innovative Multiplex-Fluorescent-Labeled STRs Assay for Prader-Willi Syndrome and Angelman Syndrome.

PubMed

Zhang, Kaihui; Liu, Shu; Feng, Bing; Yang, Yali; Zhang, Haiyan; Dong, Rui; Liu, Yi; Gai, Zhongtao

2016-01-01

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by absence of paternally or maternally expressed imprinted genes on chr15q11.2-q13.3. Three mechanisms are known to be involved in the pathogenesis: microdeletions, uniparental disomy (UPD) and imprinting defects. Both disorders are difficult to be definitely diagnosed at early age if no available molecular cytogenetic tests. In this study, we identified 5 AS patients with the maternal deletion and 26 PWS patients with paternal deletion on chr15q11-q13 by using an innovative multiplex-fluorescent-labeled short tandem repeats (STRs) assay based on linkage analysis, and validated by the methylation-specific PCR and array comparative genomic hybridization techniques. More interesting, one of these PWS patients was confirmed as maternal uniparental isodisomy by the STR linkage analysis. The phenotypic and genotypic characteristics of these individuals were also presented. Our results indicate that the new linkage analysis is much faster and easier for large-scale screening deletion and uniparental disomy, thus providing a valuable method for early diagnosis of PWS/AS patients, which is critical for genetic diagnosis, management and improvement of prognosis.

Clinical Application of an Innovative Multiplex-Fluorescent-Labeled STRs Assay for Prader-Willi Syndrome and Angelman Syndrome

PubMed Central

Feng, Bing; Yang, Yali; Zhang, Haiyan; Dong, Rui; Liu, Yi; Gai, Zhongtao

2016-01-01

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by absence of paternally or maternally expressed imprinted genes on chr15q11.2-q13.3. Three mechanisms are known to be involved in the pathogenesis: microdeletions, uniparental disomy (UPD) and imprinting defects. Both disorders are difficult to be definitely diagnosed at early age if no available molecular cytogenetic tests. In this study, we identified 5 AS patients with the maternal deletion and 26 PWS patients with paternal deletion on chr15q11-q13 by using an innovative multiplex-fluorescent-labeled short tandem repeats (STRs) assay based on linkage analysis, and validated by the methylation-specific PCR and array comparative genomic hybridization techniques. More interesting, one of these PWS patients was confirmed as maternal uniparental isodisomy by the STR linkage analysis. The phenotypic and genotypic characteristics of these individuals were also presented. Our results indicate that the new linkage analysis is much faster and easier for large-scale screening deletion and uniparental disomy, thus providing a valuable method for early diagnosis of PWS/AS patients, which is critical for genetic diagnosis, management and improvement of prognosis. PMID:26841067

Development of the NBT assay as a marker of sperm oxidative stress.

PubMed

Tunc, Ozlem; Thompson, Jeremy; Tremellen, Kelton

2010-02-01

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 microg formazan/10(7) sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.

Impedance-based cellular assays for regenerative medicine.

PubMed

Gamal, W; Wu, H; Underwood, I; Jia, J; Smith, S; Bagnaninchi, P O

2018-07-05

Therapies based on regenerative techniques have the potential to radically improve healthcare in the coming years. As a result, there is an emerging need for non-destructive and label-free technologies to assess the quality of engineered tissues and cell-based products prior to their use in the clinic. In parallel, the emerging regenerative medicine industry that aims to produce stem cells and their progeny on a large scale will benefit from moving away from existing destructive biochemical assays towards data-driven automation and control at the industrial scale. Impedance-based cellular assays (IBCA) have emerged as an alternative approach to study stem-cell properties and cumulative studies, reviewed here, have shown their potential to monitor stem-cell renewal, differentiation and maturation. They offer a novel method to non-destructively assess and quality-control stem-cell cultures. In addition, when combined with in vitro disease models they provide complementary insights as label-free phenotypic assays. IBCA provide quantitative and very sensitive results that can easily be automated and up-scaled in multi-well format. When facing the emerging challenge of real-time monitoring of three-dimensional cell culture dielectric spectroscopy and electrical impedance tomography represent viable alternatives to two-dimensional impedance sensing.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

Rapid detection of trace amounts of surfactants using nanoparticles in fluorometric assays

NASA Astrophysics Data System (ADS)

Härmä, Harri; Laakso, Susana; Pihlasalo, Sari; Hänninen, Pekka; Faure, Bertrand; Rana, Subhasis; Bergström, Lennart

2010-01-01

Rapid microtiter assays that utilize the time-resolved fluorescence resonance energy transfer or quenching of dye-labeled proteins adsorbed onto the surfaces of polystyrene or maghemite nanoparticles have been developed for the detection and quantification of trace amounts of surfactants at concentrations down to 10 nM.Rapid microtiter assays that utilize the time-resolved fluorescence resonance energy transfer or quenching of dye-labeled proteins adsorbed onto the surfaces of polystyrene or maghemite nanoparticles have been developed for the detection and quantification of trace amounts of surfactants at concentrations down to 10 nM. Electronic supplementary information (ESI) available: Experimental details and Fig. S1 and S2. See DOI: 10.1039/b9nr00172g

Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds.

PubMed

Lema, Carolina; Varela-Ramirez, Armando; Aguilera, Renato J

As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z' factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC 50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity.

Differential nuclear staining assay for high-throughput screening to identify cytotoxic compounds

PubMed Central

LEMA, Carolina; VARELA-RAMIREZ, Armando; AGUILERA, Renato J.

2016-01-01

As large quantities of novel synthetic molecules continue to be generated there is a challenge to identify therapeutic agents with cytotoxic activity. Here we introduce a Differential Nuclear Staining (DNS) assay adapted to live-cell imaging for high throughput screening (HTS) that utilizes two fluorescent DNA intercalators, Hoechst 33342 and Propidium iodide (PI). Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells. The DNS assay was successfully validated by utilizing well known cytotoxic agents with fast or slow cytotoxic activities. The assay was found to be suitable for HTS with Z′ factors ranging from 0.86 to 0.60 for 96 and 384-well formats, respectively. Furthermore, besides plate-to-plate reproducibility, assay quality performance was evaluated by determining ratios of signal-to-noise and signal-to-background, as well as coefficient of variation, which resulted in adequate values and validated the assay for HTS initiatives. As proof of concept, eighty structurally diverse compounds from a small molecule library were screened in a 96-well plate format using the DNS assay. Using this DNS assay, six hits with cytotoxic properties were identified and all of them were also successfully identified by using the commercially available MTS assay (CellTiter 96® Cell Proliferation Assay). In addition, the DNS and a flow cytometry assay were used to validate the activity of the cytotoxic compounds. The DNS assay was also used to generate dose-response curves and to obtain CC50 values. The results indicate that the DNS assay is reliable and robust and suitable for primary and secondary screens of compounds with potential cytotoxic activity. PMID:27042697

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

PubMed

Aitichou, Mohamed; Saleh, Sharron; Kyusung, Park; Huggins, John; O'Guinn, Monica; Jahrling, Peter; Ibrahim, Sofi

2008-11-01

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

A novel assay to identify the trafficking proteins that bind to specific vesicle populations

PubMed Central

Bentley, Marvin; Banker, Gary

2016-01-01

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin–binding domain (FRB)–tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. PMID:26621371

Fluorescence lifetime assays: current advances and applications in drug discovery.

PubMed

Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

2011-06-01

Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

PubMed

Shabanpoor, Fazel; Gait, Michael J

2013-11-11

We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

Latent herpes simplex virus 1 infection does not induce apoptosis in human trigeminal Ganglia.

PubMed

Himmelein, Susanne; Lindemann, Anja; Sinicina, Inga; Strupp, Michael; Brandt, Thomas; Hüfner, Katharina

2015-05-01

Herpes simplex virus 1 (HSV-1) can establish lifelong latency in human trigeminal ganglia. Latently infected ganglia contain CD8(+) T cells, which secrete granzyme B and are thus capable of inducing neuronal apoptosis. Using immunohistochemistry and single-cell reverse transcription-quantitative PCR (RT-qPCR), higher frequency and transcript levels of caspase-3 were found in HSV-1-negative compared to HSV-1-positive ganglia and neurons, respectively. No terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay-positive neurons were detected. The infiltrating T cells do not induce apoptosis in latently infected neurons. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads.

PubMed

He, Hong-qiu; Ma, Xiao-hui; Liu, Bin; Chen, Wei-zu; Wang, Cun-xin; Cheng, Shao-hui

2008-03-01

To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization.

PubMed

Zhang, Qing; Zhu, Liang; Feng, Hanhua; Ang, Simon; Chau, Fook Siong; Liu, Wen-Tso

2006-01-18

This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap=1-2 microm) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 microL/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.

Decrease of peritoneal inflammatory CD4(+), CD8(+), CD19(+) lymphocytes and apoptosis of eosinophils in a murine Taenia crassiceps infection.

PubMed

Zepeda, Nadia; Solano, Sandra; Copitin, Natalia; Fernández, Ana María; Hernández, Lilián; Tato, Patricia; Molinari, José L

2010-10-01

After an intraperitoneal infection of mice with Taenia crassiceps metacestodes, peritoneal inflammatory cells labeled with fluoresceinated MoAb anti-mouse were analyzed by flow cytometry. Apoptosis was studied by annexin A/PI, TUNEL assays, DNA laddering, caspase-3 activity, and electron microscopy. An important continuous decrease of CD4+, CD8+ and CD19+ lymphocytes, and an increase of eosinophils and macrophages throughout the observation time were found. Apoptosis of eosinophils was quantified during the observation period with a peak at 6 days post-infection (67.27%). In an additional experiment at 12 days post-infection using TUNEL staining, a high level of apoptosis of eosinophil (92.3%) and a significant decrease of CD4+, CD8+, and CD19+ lymphocytes were confirmed. Caspase-3 activity in peritoneal fluid, peritoneal cells' DNA fragmentation, and apoptosis of eosinophils and monocytes were found. The dramatic decrease of peritoneal inflammatory T and B cells and the high level of apoptosis of inflammatory eosinophils induced in mice by infection with T. crassiceps cysticerci may be important factors of the immunosuppression observed in cysticercosis.

An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.

PubMed

Liu, Xingfen; Ouyang, Lan; Cai, Xiaohui; Huang, Yanqin; Feng, Xiaomiao; Fan, Quli; Huang, Wei

2013-03-15

Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and "turn-on" detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1 pg/μL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Hierarchical Nanogold Labels to Improve the Sensitivity of Lateral Flow Immunoassay

NASA Astrophysics Data System (ADS)

Serebrennikova, Kseniya; Samsonova, Jeanne; Osipov, Alexander

2018-06-01

Lateral flow immunoassay (LFIA) is a widely used express method and offers advantages such as a short analysis time, simplicity of testing and result evaluation. However, an LFIA based on gold nanospheres lacks the desired sensitivity, thereby limiting its wide applications. In this study, spherical nanogold labels along with new types of nanogold labels such as gold nanopopcorns and nanostars were prepared, characterized, and applied for LFIA of model protein antigen procalcitonin. It was found that the label with a structure close to spherical provided more uniform distribution of specific antibodies on its surface, indicative of its suitability for this type of analysis. LFIA using gold nanopopcorns as a label allowed procalcitonin detection over a linear range of 0.5-10 ng mL-1 with the limit of detection of 0.1 ng mL-1, which was fivefold higher than the sensitivity of the assay with gold nanospheres. Another approach to improve the sensitivity of the assay included the silver enhancement method, which was used to compare the amplification of LFIA for procalcitonin detection. The sensitivity of procalcitonin determination by this method was 10 times better the sensitivity of the conventional LFIA with gold nanosphere as a label. The proposed approach of LFIA based on gold nanopopcorns improved the detection sensitivity without additional steps and prevented the increased consumption of specific reagents (antibodies).

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

NASA Astrophysics Data System (ADS)

Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

2013-06-01

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Specific labeling of zinc finger proteins using noncanonical amino acids and copper-free click chemistry.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A; Schroeder, Charles M

2012-09-19

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays, and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry.

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takamiya, Mari; Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama; Sakurai, Masaaki

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed amore » RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive {sup 14}C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial

Sensitive electrochemical immunoassay for 2,4,6-trinitrotoluene based on functionalized silica nanoparticle labels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jun; Liu, Guodong; Wu, Hong

2008-03-03

We present a poly(guanine)-functionalized silica nanoparticle (NP) label-based electrochemical immunoassay for sensitively detecting 2,4,6-trinitrotoluene (TNT). This immunoassay takes advantage of magnetic bead–based platform for competitive displacement immunoreactions and separation, and use electroactive nanoparticles as labels for signal amplification. For this assay, anti-TNT-coated magnetic beads interacted with TNT analog-conjugated poly(guanine)-silica NPs and formed analog-anti-TNT immunocomplexes on magnetic beads. The immunocomplexes coated magnetic beads were exposed to TNT samples, which resulted in displacing the analog conjugated poly(guanine) silica NPs into solution by TNT. In contrast, there are no guanine residues releasing into the solution in the absence of TNT. The reaction solutionmore » was then separated from the magnetic beads and transferred to the electrode surface for electrochemical measurements of guanine oxidation with Ru(bpy)32+ as mediator. The sensitivity of this TNT assay was greatly enhanced through dual signal amplifications: 1) a large amount of guanine residues on silica nanoparticles is introduced into the test solution by displacement immunoreactions and 2) a Ru(bpy)32+-induced guanine catalytic oxidation further enhances the electrochemical signal. Some experimental parameters for the nanoparticle label-based electrochemical immunoassay were studied and the performance of this assay was evaluated. The method is found to be very sensitive and the detection limit of this assay is ~ 0.1 ng mL-1 TNT. The electrochemical immunoassay based on the poly[guanine]-functionalized silica NP label offers a new approach for sensitive detection of explosives.« less

Non-Animal Testing Approach to EPA Labeling for Eye Irritation

EPA Pesticide Factsheets

This document is an update to EPA’s 2013 published alternative testing approach (using in vitro/ex vivo assays) for determination of eye irritation potential in the pesticide program under EPA's classification and labeling system.

Electrochemical detection of leukemia oncogenes using enzyme-loaded carbon nanotube labels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ai Cheng; Du, Dan; Chen, Baowei

2014-09-07

Here we describe an ultrasensitive electrochemical nucleic acids assay amplified by carbon nanotubes (CNTs)-based labels for the detection of human acute lymphocytic leukemia (ALL) related p185 BCR-ABL fusion transcript. The carboxylated CNTs were functionalized with horseradish peroxidase (HRP) molecules and target-specific detection probes (DP) via diimide-activated amidation, and used to label and amplify target hybridization signal. The activity of captured HRP was monitored by square-wave voltammetry measuring the electroactive enzymatic product in the presence of 2-aminophenol and hydrogen peroxide substrate solution. The effect of DP and HRP loading of the CNT-based labels on its signal-to-noise ratio of electrochemical detection wasmore » studied systematically for the first time. Under optimized conditions, the signal-amplified assay achieved a detection limit of 83 fM targets oligonuecleotides and a 4-order wide dynamic range of target concentration. The resulting assay allowed a robust discrimination between the perfect match and a three-base mismatch sequence. When subjected to full-length (491 bp) DNA oncogene, the approach demonstrated a detection limit of approximately 33 pg of the target gene. The high sensitivity and specificity of assay enabled PCR-free detection of target transcripts in as little as 65 ng of mRNA extracted from positive ALL cell lines SUP-B15, in comparison to those obtained from negative cell lines HL-60. The approach holds promise for simple, low cost and ultrasensitive electrochemical nucleic acids detection in portable devices, point-of-care and early disease diagnostic applications.« less

Coupling liquid chromatography/mass spectrometry detection with microfluidic droplet array for label-free enzyme inhibition assay.

PubMed

Wang, Xiu-Li; Zhu, Ying; Fang, Qun

2014-01-07

In this work, the combination of droplet-based microfluidics with liquid chromatography/mass spectrometry (LC/MS) was achieved, for providing a fast separation and high-information-content detection method for the analysis of nanoliter-scale droplets with complex compositions. A novel interface method was developed using an oil-covered droplet array chip to couple with an LC/MS system via a capillary sampling probe and a 4 nL injection valve without the need of a droplet extraction device. The present system can perform multistep operations including parallel enzyme inhibition reactions in nanoliter droplets, 4 nL sample injection, fast separation with capillary LC, and label-free detection with ESI-MS, and has significant flexibility in the accurate addressing and sampling of droplets of interest on demand. The system performance was evaluated using angiotensin I and angiotensin II as model samples, and the repeatabilities of peak area for angiotensin I and angiotensin II were 2.7% and 7.5% (RSD, n = 4), respectively. The present system was further applied to the screening for inhibitors of cytochrome P450 (CYP1A2) and measurement of the IC50 value of the inhibitor. The sample consumption for each droplet assay was 100 nL, which is reduced 10-100 times compared with conventional 384-multi-well plate systems usually used in high-throughput drug screening.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening.

PubMed

Brengdahl, Johan; Fowler, Christopher J

2006-12-01

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.

Label-free integrative pharmacology on-target of drugs at the β2-adrenergic receptor

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Fang, Ye

2011-07-01

We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the β2-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

A Novel ImageJ Macro for Automated Cell Death Quantitation in the Retina

PubMed Central

Maidana, Daniel E.; Tsoka, Pavlina; Tian, Bo; Dib, Bernard; Matsumoto, Hidetaka; Kataoka, Keiko; Lin, Haijiang; Miller, Joan W.; Vavvas, Demetrios G.

2015-01-01

Purpose TUNEL assay is widely used to evaluate cell death. Quantification of TUNEL-positive (TUNEL+) cells in tissue sections is usually performed manually, ideally by two masked observers. This process is time consuming, prone to measurement errors, and not entirely reproducible. In this paper, we describe an automated quantification approach to address these difficulties. Methods We developed an ImageJ macro to quantitate cell death by TUNEL assay in retinal cross-section images. The script was coded using IJ1 programming language. To validate this tool, we selected a dataset of TUNEL assay digital images, calculated layer area and cell count manually (done by two observers), and compared measurements between observers and macro results. Results The automated macro segmented outer nuclear layer (ONL) and inner nuclear layer (INL) successfully. Automated TUNEL+ cell counts were in-between counts of inexperienced and experienced observers. The intraobserver coefficient of variation (COV) ranged from 13.09% to 25.20%. The COV between both observers was 51.11 ± 25.83% for the ONL and 56.07 ± 24.03% for the INL. Comparing observers' results with macro results, COV was 23.37 ± 15.97% for the ONL and 23.44 ± 18.56% for the INL. Conclusions We developed and validated an ImageJ macro that can be used as an accurate and precise quantitative tool for retina researchers to achieve repeatable, unbiased, fast, and accurate cell death quantitation. We believe that this standardized measurement tool could be advantageous to compare results across different research groups, as it is freely available as open source. PMID:26469755

Infrared microspectroscopy of live cells in microfluidic devices (MD-IRMS): toward a powerful label-free cell-based assay.

PubMed

Vaccari, L; Birarda, G; Businaro, L; Pacor, S; Grenci, G

2012-06-05

Until nowadays most infrared microspectroscopy (IRMS) experiments on biological specimens (i.e., tissues or cells) have been routinely carried out on fixed or dried samples in order to circumvent water absorption problems. In this paper, we demonstrate the possibility to widen the range of in-vitro IRMS experiments to vibrational analysis of live cellular samples, thanks to the development of novel biocompatible IR-visible transparent microfluidic devices (MD). In order to highlight the biological relevance of IRMS in MD (MD-IRMS), we performed a systematic exploration of the biochemical alterations induced by different fixation protocols, ethanol 70% and formaldehyde solution 4%, as well as air-drying on U937 leukemic monocytes by comparing their IR vibrational features with the live U937 counterpart. Both fixation and air-drying procedures affected lipid composition and order as well as protein structure at a different extent while they both induced structural alterations in nucleic acids. Therefore, only IRMS of live cells can provide reliable information on both DNA and RNA structure and on their cellular dynamic. In summary, we show that MD-IRMS of live cells is feasible, reliable, and biologically relevant to be recognized as a label-free cell-based assay.

Urtica dioica dichloromethane extract induce apoptosis from intrinsic pathway on human prostate cancer cells (PC3).

PubMed

Mohammadi, A; Mansoori, B; Aghapour, M; Baradaran, B

2016-03-31

Prostate cancer is considered as the major cause of death among men around the world. There are a number of medicinal plants triggering apoptosis response in cancer cells, thus have a therapeutic potential. Therefore, further studies to characterize beneficial properties of these plants in order to introduce novel anti-cancer drugs are the interest of recent researches on the alternative medicine. On the other hand, due to traditional uses and availability of Urtica dioica extract, we decided to evaluate the efficacy of this medicinal herb on pc3 prostate cancer cell line. In the present study the cytotoxic effects of Urtica dioica extract were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and trypan blue viability dye. Then, DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were exploited to measure cell death and apoptosis stage. The expression levels of caspase 3, caspase 9 and Bcl-2 genes were quantified by Real-Time PCR. Finally, Cell cycle was analyzed by flow cytometry. MTT assay showed that dichloromethanolic extract of Urtica dioica significantly inhibited the cell growth. According to the DNA fragmentation and TUNEL assay results, the herbal extract was able to induce apoptosis in prostate cancer cells. Our findings also demonstrated that the plant extract substantially increases the caspase 3 and 9 mRNA expression, while decreases Bcl-2. Cell cycle arrest was occurred in G2 stage, due to the results of flow cytometry. These results indicate that dichloromethanolic extract of Urtica dioica can successfully induce apoptosis in PC3 cells. Therefore, it could be used as a novel therapeutic candidate for prostate tumor treatment.

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice.

PubMed

Wang, Hai-Jun; Liu, Yu; Zhou, Bao-Jun; Zhang, Zhan-Xue; Li, Ai-Ying; An, Ran; Yue, Bin; Fan, Li-Qiao; Li, Yong

2018-05-01

Objective To investigate the potential antitumour effects of [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonic acid (CP) against gastric adenocarcinoma. Methods Human BGC-823 xenotransplants were established in nude mice. Animals were randomly divided into control and CP groups, which were administered NaHCO 3 vehicle alone or CP dissolved in NaHCO 3 (200 µg/kg body weight) daily, respectively. Tumour volume was measured weekly for 6 weeks. Resected tumours were assayed for proliferative activity with anti-Ki-67 or anti-proliferating cell nuclear antigen (PCNA) antibodies. Cell apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays and with caspase-3 immunostaining. Proteins were measured by Western blotting. Results There was a significant reduction in tumour volume and a reduced percentage of Ki-67-positive or PCNA-positive cells in the CP group compared with the control group. The percentage of TUNEL-positive or caspase 3-positive cells significantly increased following CP treatment compared with the control group. Tumours from the CP group had higher levels of phosphorylated-extracellular signal-regulated kinase (p-ERK) and phosphorylated-AKT (p-AKT) compared with control tumours. Conclusion CP treatment inhibited tumour growth and induced tumour cell apoptosis in a nude mouse model of BGC-823 gastric adenocarcinoma. Activation of the AKT and ERK signalling pathways may mediate this antitumour activity.

DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome.

PubMed

Avendaño, Conrado; Franchi, Anahí; Duran, Hakan; Oehninger, Sergio

2010-07-01

To evaluate DNA fragmentation in morphologically normal sperm recovered from the same sample used for intracytoplasmic sperm injection (ICSI) and to correlate DNA damage with embryo quality and pregnancy outcome. Prospective study. Academic center. 36 infertile men participating in the ICSI program. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL) assay and morphologic assessment by phase contrast. Simultaneous assessment of sperm morphology and DNA fragmentation by TUNEL assay was performed in the same cell, then the percentage of normal sperm with fragmented DNA (normal SFD) was correlated with embryo quality and pregnancy outcomes. A highly statistically significant negative correlation was found between the percentage of normal SFD and embryo quality. This association was confirmed for the transferred embryos and for the total embryo cohort. The receiver operating characteristics curve analysis demonstrated that the percentage of normal SFD and embryo quality were statistically significant predictors of pregnancy. When the percentage of normal SFD was

Apoptosis in human unfertilized oocytes after intracytoplasmic sperm injection.

PubMed

Bosco, Liana; Ruvolo, Giovanni; Morici, Giovanni; Manno, Maurizio; Cittadini, Ettore; Roccheri, Maria C

2005-11-01

To investigate the presence of programmed cell death in unfertilized oocytes after intracytoplasmic sperm injection (ICSI), assuming that previous apoptotic events could be correlated with the fertilization failure. Comparison of the rate of DNA fragmentation in human oocytes at different stages of maturation soon after pick-up (control) and in unfertilized oocytes after ICSI treatment. In vitro fertilization (IVF) laboratory with extensive ICSI experience. Sixty-three patients undergoing assisted fertilization by ICSI. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay and anticaspase-3 cleaved immunoassay to detect apoptosis in control and ICSI-treated oocytes. Differences in the percentage of oocytes demonstrating DNA fragmentation between control oocytes and unfertilized ICSI treated oocytes at different stages of maturation. The DNA fragmentation, by TUNEL assay, appeared in all the immature control oocytes, but only 37% of mature oocytes showed DNA fragmentation. This DNA fragmentation was observed in 88.8% of the oocytes unfertilized after ICSI; furthermore, DNA fragmentation appeared as well in the sperm injected into the cytoplasm. The study has shown DNA fragmentation in human oocytes unfertilized after ICSI. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. Caspase-3 immunoassay suggests the presence of apoptosis. The high percentage of oocytes demonstrating DNA fragmentation in the unfertilized oocytes could be correlated with fertilization failure.

Effect of green light spectra on the reduction of retinal damage and stress in goldfish, Carassius auratus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Jin Ah; Kim, Na Na; Choi, Young Jae

We investigated the effect of light spectra on retinal damage and stress in goldfish using green (530 nm) and red (620 nm) light emitting diodes (LEDs) at three intensities each (0.5, 1.0, and 1.5 W/m{sup 2}). We measured the change in the levels of plasma cortisol and H{sub 2}O{sub 2} and expression and levels of caspase-3. The apoptotic response of green and red LED spectra was assessed using the terminal transferase dUTP nick end labeling (TUNEL) assay. Stress indicator (cortisol and H{sub 2}O{sub 2}) and apoptosis-related genes (caspase-3) decreased in green light, but increased in red light with higher light intensities over time.more » The TUNEL assay revealed that more apoptotic cells were detected in outer nuclear layers after exposure to red LED over time with the increase in light intensity, than the other spectra. These results indicate that green light efficiently reduces retinal damage and stress, whereas red light induces it. Therefore, red light-induced retina damage may induce apoptosis in goldfish retina. -- Highlights: •Green light efficiently reduces retinal damage and stress. •Green spectra reduce caspase production and apoptosis. •Red light-induced retina damage may induce apoptosis in goldfish retina. •The retina of goldfish recognizes green spectra as a stable environment.« less

Ultrasensitive biomolecular assays with amplifying nanowire FET biosensors

NASA Astrophysics Data System (ADS)

Chui, Chi On; Shin, Kyeong-Sik; Mao, Yufei

2013-09-01

In this paper, we review our recent development and validation of the ultrasensitive electronic biomolecular assays enabled by our novel amplifying nanowire field-effect transistor (nwFET) biosensors. Our semiconductor nwFET biosensor platform technology performs extreme proximity signal amplification in the electrical domain that requires neither labeling nor enzymes nor optics. We have designed and fabricated the biomolecular assay prototypes and developed the corresponding analytical procedures. We have also confirmed their analytical performance in quantitating key protein biomarker in human serum, demonstrating an ultralow limit of detection and concurrently high output current level for the first time.

Polo-like kinase 1, a new therapeutic target in hepatocellular carcinoma

PubMed Central

Mok, Wei Chuen; Wasser, Shanthi; Tan, Theresa; Lim, Seng Gee

2012-01-01

AIM: To investigate the role of polo-like kinase 1 (PLK1) as a therapeutic target for hepatocellular carcinoma (HCC). METHODS: PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA (siRNA) was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) and bromodeoxyuridine (BrdU) assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progression was compared with controls. RESULTS: RT-PCR showed that PLK1 was overexpressed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells. siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLK1-treated mice, but not in controls. CONCLUSION: Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G pathway. PMID:22826617

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

In vitro and in vivo comparison of binding of 99m-Tc-labeled anti-CEA MAb F33-104 with 99m-Tc-labeled anti-CEA MAb BW431/26.

PubMed

Watanabe, N; Oriuchi, N; Sugiyama, S; Kuroki, M; Matsuoka, Y; Tanada, S; Murata, H; Inoue, T; Sasaki, Y

1999-01-01

The purpose of this study was to assess the potential for radio-immunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tc-labeled anti-CEA MAb BW431/261 (31.4 +/- 0.95% vs. 11.9 +/- 0.55% at 100 ng/mL of soluble CEA, 83.5 +/- 2.84% vs. 54.0 +/- 2.54% at 10(7) of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 +/- 3.50% ID/g vs. 14.4 +/- 3.30% ID/g). 99m-Tc-activity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 +/- 2.10% ID/g vs. 8.01 +/- 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 +/- 1.70% ID/g vs. 8.10 +/- 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Antibody labeling with Remazol Brilliant Violet 5R, a vinylsulphonic reactive dye.

PubMed

Ferrari, Alejandro; Friedrich, Adrián; Weill, Federico; Wolman, Federico; Leoni, Juliana

2013-01-01

Colloidal gold is the first choice for labeling antibodies to be used in Point Of Care Testing. However, there are some recent reports on a family of textile dyes-named "reactive dyes"-being suitable for protein labeling. In the present article, protein labeling conditions were optimized for Remazol Brilliant Violet 5R, and the sensitivity of the labeled antibodies was assessed and compared with that of colloidal-gold labeled antibodies. Also, the accelerated stability was explored. Optimal conditions were pH 10.95, dye:Ab molar ratio of 264 and an incubation time of 132 min. Labeled antibodies were stable, and could be successfully used in a slot blot assay, detecting as low as 400 ng/mL. Therefore, the present work demonstrates that vinylsulphonic reactive dyes can be successfully used to label antibodies, and are excellent candidates for the construction of a new generation of Point of Care Testing kits.

Specific Labeling of Zinc Finger Proteins using Non-canonical Amino Acids and Copper-free Click Chemistry

PubMed Central

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A.

2012-01-01

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry. PMID:22871171

An exonuclease III and graphene oxide-aided assay for DNA detection.

PubMed

Peng, Lu; Zhu, Zhi; Chen, Yan; Han, Da; Tan, Weihong

2012-05-15

We have developed a novel DNA assay based on exonuclease III (ExoIII)-induced target recycling and the fluorescence quenching ability of graphene oxide (GO). This assay consists of a linear DNA probe labeled with a fluorophore in the middle. Introduction of target sequence induces the exonuclease III catalyzed probe digestion and generation of single nucleotides. After each cycle of digestion, the target is recycled to realize the amplification. Finally, graphene oxide is added to quench the remaining probes and the signal from the resulting fluorophore labeled single nucleotides is detected. With this approach, a sub-picomolar detection limit can be achieved within 40 min at 37°C. The method was successfully applied to multicolor DNA detection and the analysis of telomerase activity in extracts from cancer cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Fluorescent Filter-Trap Assay for Amyloid Fibril Formation Kinetics in Complex Solutions

PubMed Central

2015-01-01

Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration. PMID:25946560

Adaptation of the TdT assay for semi-quantitative flow cytometric detection of DNA strand breaks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bromidge, T.J.; Howe, D.J.; Johnson, S.A.

The enzyme Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase which can be used to label DNA strand breaks by the incorporation of a labelled nucleotide followed by a fluorescent detection step. The amount of label incorporated can then be assessed by flow cytometry. The mechanism of action of TdT, however, will allow the addition of varying numbers of nucleotides to the free 3{prime} termini produced by DNA strand breaks. The substitution of Digoxigenin (DIG){trademark} labelled dideoxynucleotides for labelled deoxy-nucleotides in the TdT assay will limit the addition of label to a DNA break to a single nucleotide, thus ensuringmore » a direct relationship between an increase in DNA strand breaks and an increase in fluorescence. We have used this adaptation of the TdT assay to evaluate DNA damage incurred in lymphocytes, from patients with Chronic Lymphocytic Leukemia (CLL), on exposure to UV irradiation and apoptosis-inducing drugs, fludarabine and 2-Chloro-2{prime}-deoxyadenosine (2-CdA). This technique may give a good indication of the susceptibility of CLL patients to apoptosis inducing drugs, and hence an indication of the likely response to these therapies. 7 refs., 2 figs., 2 tabs.« less

FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

EPA Science Inventory

A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

Development and utilization of an ex vivo bromodeoxyuridine local lymph node assay protocol for assessing potential chemical sensitizers.

PubMed

Williams, W C; Copeland, C; Boykin, E; Quell, S J; Lehmann, D M

2015-01-01

The murine local lymph node assay (LLNA) is widely used to identify chemicals that may cause allergic contact dermatitis. Exposure to a dermal sensitizer results in proliferation of local lymph node T cells, which has traditionally been measured by in vivo incorporation of [(3) H]methyl thymidine. A more recent non-isotopic variation of the assay utilizes bromodeoxyuridine (BrdU) incorporation in vivo. To further improve the utility of this assay, we developed an ex vivo BrdU labeling procedure eliminating the need for in vivo injections. The results of this assay correctly identified a strong sensitizer (i.e., trimellitic anhydride) as well as weak/moderate sensitizers (i.e., eugenol, cinnamaldehyde and hexylcinnaminic aldehyde). As anticipated, neither non-sensitizers isopropanol and lactic acid nor the false negative chemical nickel II sulfate hexahydrate induced a positive threshold response in the assay. The results of this assay are in close agreement with those of the in vivo LLNA:BrdU-enzyme-linked immunosorbent assay labeling procedure. We also used the ex vivo BrdU LLNA procedure to evaluate ammonium hexachloroplatinate, ammonium tetrachloroplatinate and cis-diamminedichloroplatinum(II) and the assay correctly identified them as sensitizers based on the calculation of EC2 values. We conclude that this ex vivo BrdU labeling method offers predictive capacity comparable to previously established LLNA protocols while eliminating animal injections and the use of radioisotope. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Ocular Safety of Intravitreal Connective Tissue Growth Factor Neutralizing Antibody.

PubMed

Motevasseli, Tahmineh; Daftarian, Narsis; Kanavi, Mozhgan Rezaei; Ahmadieh, Hamid; Bagheri, Abouzar; Hosseini, Seyed Bagher; Ansari, Shabnam; Soheili, Zahra-Soheila

2017-08-01

To detect the safety of intravitreal injection of anti-connective tissue growth factor (CTGF) (IVAC) in rat eyes in order to apply this neutralizing antibody for experimental animal studies. Forty-five Lister Hooded male pigmented rats were divided into five groups that received IVAC (2 μl) corresponding to the doses of 10 (B), 20 (C), 50 (D), and 100 μg/ml (E), equal to 1.25, 2.5, 6.25, and 12.5 µg/ml of antibody concentration in rat vitreous, respectively. The sham group (A) received 2 μl of normal saline. Full field electroretinography (ERG) was performed at baseline and on days 7 and 28 after IVAC. The animals were euthanized and the corresponding eyes were subjected to routine histopathology, immunohistochemistry for glial fibrillary acidic protein (GFAP), and terminal transferase dUTP nick end-labeling (TUNEL) assay. Scotopic rod b-wave amplitude and maximal combined b-wave amplitude were 111.89 ± 71.2 and 178.57 ± 55.58 μV, respectively, at baseline which significantly reduced to 79.31 ± 52.59 and 128.73 ± 41.61 μV, respectively, after 28 days in group E (p < 0.05). There was no significant reduction of amplitudes in other groups with lower doses of anti-CTGF antibody. Retinal ganglion cells were significantly decreased in group E as compared to other groups. GFAP immune reactivity was not significant in any of the groups. TUNEL test showed inner retinal neural cell apoptosis only in group E. ERG, histopathologic, and apoptotic assays revealed no toxic effects of 10-50 μg/ml of IVAC in rat eyes. Using 100 μg/ml IVAC led to a significant toxic effect in terms of functional, histopathologic, and TUNEL findings.

Non-radioactive TRF assay modifications to improve telomeric DNA detection efficiency in plants

PubMed Central

Nigmatullina, Liliia R.; Sharipova, Margarita R.; Shakirov, Eugene V.

2016-01-01

The length of telomeric DNA is often considered a cellular biomarker of aging and general health status. Several telomere length measuring assays have been developed, of which the most common is the Telomere Restriction Fragment (TRF) analysis, which typically involves the use of radioactively labeled oligonucleotide probes. While highly effective, this method potentially poses substantial health concerns and generates radioactive waste. Digoxigenin (DIG) alternatives to radioactive probes have been developed and used successfully in a number of assays. Here we optimize the DIG protocol to measure telomere length in the model plant Arabidopsis thaliana and present evidence that this approach can be used successfully to efficiently and accurately measure telomere length in plants. Specifically, hybridization temperature of 42 °C instead of the typical 55 °C appears to generate stronger signals. In addition, DIG incorporation at 5′-end instead of 3′-end of the labeled oligonucleotide greatly enhances signal. We conclude that non-radioactive TRF assays can be as efficient as radioactive methods in detecting and measuring telomere length in plants, making this assay suitable for medical and research laboratories unable to utilize radioactivity due to hazardous waste disposal and safety concerns. PMID:28133587

Apoptosis-mediated seasonal testicular regression in the Japanese Jungle Crow (Corvus macrorhynchos).

PubMed

Islam, M Nazrul; Tsukahara, N; Sugita, S

2012-06-01

The present study investigated effects of apoptosis observed during seasonal testicular regression in Japanese Jungle Crows. The study was conducted during January to June 2008, 2009. Testes from adults captured during non-breeding (January), prebreeding (February to mid-March), main-breeding (late March to early May), transition (mid-May to late May), and post-breeding (June) seasons were analyzed. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Paired-testis volume increased 95-fold from the non-breeding to the main-breeding season (P < 0.05), and subsequently decreased 26-fold from the main breeding to the post-breeding season (P < 0.05). Testicular activity was evaluated from the total germ cell count and sperm index, which increased 42- and 5-fold, respectively, in the main-breeding season, and subsequently decreased 33- and 5-fold in the post-breeding season. In testes, TUNEL-positive germ cells were at low levels in the non-breeding season, absent in the prebreeding and the main-breeding seasons, and highest in mid-May (P < 0.05). In contrast, TUNEL-positive Sertoli cells occurred only in late-April. In addition, TUNEL-positive fibroblast-like cells were observed in the outer zone of the tunica albuginea in the post-breeding season. Collectively, these data suggested that the seasonal rise in the testicular competence occurred slowly in Japanese Jungle Crows; however, testis function was terminated rapidly after the breeding season. Furthermore, we concluded, similar to other avian species, Sertoli cell apoptosis followed by massive germ cell death was responsible for rapid testicular regression in Jungle Crows. Copyright © 2012 Elsevier Inc. All rights reserved.

Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

NASA Astrophysics Data System (ADS)

Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

2016-03-01

In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

PCR-enzyme-linked immunosorbent assay and partial rRNA gene sequencing: a rational approach to identifying mycobacteria.

PubMed Central

Patel, S; Yates, M; Saunders, N A

1997-01-01

A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of mycobacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified product with a 5' fluorescein-labelled primer. The identity of the labelled strand was subsequently determined by hybridization to a panel of mycobacterial species-specific capture probes, which were immobilized via their 5' biotin ends to a streptavidin-coated microtiter plate. Specific hybridization of a 5' fluorescein-labelled strand to a species probe was detected colorimetrically with an anti-fluorescein enzyme conjugate. The assay was able to identify 10 Mycobacterium spp. A probe able to hybridize to all Mycobacterium species (All1) was also included. By a heminested PCR, the assay was sensitive enough to detect as little as 10 fg of DNA, which is equivalent to approximately three bacilli. The assay was able to detect and identify mycobacteria directly from sputa. The specificities of the capture probes were assessed by analysis of 60 mycobacterial strains corresponding to 18 species. Probes Avi1, Int1, Kan1, Xen1, Che1, For1, Mal1, Ter1, and Gor1 were specific. The probe Tbc1 cross-hybridized with the Mycobacterium terrae amplicon. Analysis of 35 strains tested blind resulted in 34 strains being correctly identified. This method could be used for rapid identification of early cultures and may be suitable for the detection and concurrent identification of mycobacteria within clinical specimens. PMID:9276419

Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples.

PubMed

Büttner, Barbara E; Öhrvik, Veronica E; Witthöft, Cornelia M; Rychlik, Michael

2011-01-01

New stable isotope dilution assays were developed for the simultaneous quantitation of [(13)C(5)]-labelled and unlabelled 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, folic acid along with unlabelled tetrahydrofolic acid and 10-formylfolic acid in clinical samples deriving from human bioavailability studies, i.e. plasma, ileostomy samples, and food. The methods were based on clean-up by strong anion exchange followed by LC-MS/MS detection. Deuterated analogues of the folates were applied as the internal standards in the stable isotope dilution assays. Assay sensitivity was sufficient to detect all relevant folates in the respective samples as their limits of detection were below 0.62 nmol/L in plasma and below 0.73 μg/100 g in food or ileostomy samples. Quantification of the [(13)C(5)]-label in clinical samples offers the possibility to differentiate between folate from endogenous body pools and the administered dose when executing bioavailability trials.

Sensitive, label-free protein assay using 1-ethyl-3-methylimidazolium tetrafluoroborate-supported microchip electrophoresis with laser-induced fluorescence detection.

PubMed

Xu, Yuanhong; Li, Jing; Wang, Erkang

2008-05-01

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) instead of PBS was applied as running buffers in microchip electrophoresis. Due to the excellent properties of EMImBF4, not only nonspecific protein adsorption was more efficiently suppressed, but also approximately ten-fold higher fluorescence intensity enhancement was obtained than that using PBS. Under the optimal conditions, detection limits for BSA, bovine hemoglobin, cytochrome c, and trypsin were 1.00x10(-6), 2x10(-6), 7x10(-7), and 5x10(-7) mg/mL, respectively. Thus, without covalent modification of the protein, a protein assay method with high sensitivity was achieved on microchips.

Detection of chitinase activity by 2-aminobenzoic acid labeling of chito-oligosaccharides.

PubMed

Ghauharali-van der Vlugt, Karen; Bussink, Anton P; Groener, Johanna E M; Boot, Rolf G; Aerts, Johannes M F G

2009-01-01

Chitinases are hydrolases capable of hydrolyzing the abundant natural polysaccharide chitin. Next to artificial fluorescent substrates, more physiological chito-oligomers are commonly used in chitinase assays. Analysis of chito-oligosaccharides products is generally accomplished by UV detection. However, the relatively poor sensitivity poses a serious limitation. Here we report on a novel, much more sensitive assay for the detection of chito-oligosaccharide reaction products released by chitinases, based on fluorescent detection, following chemical labeling by 2-aminobenzoic acid. Comparison with existing UV-based assays, shows that the novel assay offers the same advantages yet allows detection of chito-oligosaccharides in the low picomolar range.

Engineered biomarkers for leprosy diagnosis using labeled and label-free analysis.

PubMed

de Santana, Juliana F; da Silva, Mariângela R B; Picheth, Guilherme F; Yamanaka, Isabel B; Fogaça, Rafaela L; Thomaz-Soccol, Vanete; Machado-de-Avila, Ricardo A; Chávez-Olórtegui, Carlos; Sierakowski, Maria Rita; de Freitas, Rilton Alves; Alvarenga, Larissa M; de Moura, Juliana

2018-09-01

The biotechnological evolution towards the development of antigens to detect leprosy has been progressing. However, the identification of leprosy in paucibacillary patients, based solely on the antigen-antibody interaction still remains a challenge. The complexity of clinical manifestations requires innovative approaches to improve the sensitivity of assays to detect leprosy before the onset of symptoms, thus avoiding disabilities and contributing, indirectly, to reduce transmission. In this study, the strategies employed for early leprosy diagnosis were: i. using a phage-displayed mimotope (APDDPAWQNIFNLRR) which mimics an immunodominant sequence (PPNDPAWQRNDPILQ) of an antigen of Mycobacterium leprae known as Ag85B; ii. engineering the mimotope by adding a C-terminal flexible spacer (SGSG-C); iii. conjugating the mimotope to a carrier protein to provide better exposure to antibodies; iv. amplifying the signal using biotin-streptavidin detection system in an ELISA; and v. coating the optimized mimotope on a quartz crystal microbalance (QCM) sensor for label-free biosensing. The ELISA sensitivity increased up to 91.7% irrespective of the immunological profile of the 132 patients assayed. By using comparative modeling, the M. tuberculosis Ag85B was employed as a template to ascertain which features make the mimotope a good antigen in terms of its specificity. For the first time, a sensitive QCM-based immunosensor to detect anti M. leprae antibodies in human serum was used. M. leprae antibodies could also be detected in the sera of paucibacillary patients; thus, the use of a mimotope-derived synthetic peptide as bait for antibodies in a novel analytical label-free immunoassay for leprosy diagnosis exhibits great potential. Copyright © 2018 Elsevier B.V. All rights reserved.

Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation

DOEpatents

Schaff, Ulrich Y.; Koh, Chung-Yan; Sommer, Gregory J.

2016-04-05

Embodiments of the present invention are directed toward devices, systems, and method for conducting assays using sedimentation. In one example, a method includes layering a mixture on a density medium, subjecting sedimentation particles in the mixture to sedimentation forces to cause the sedimentation particles to move to a detection area through a density medium, and detecting a target analyte in a detection region of the sedimentation channel. In some examples, the sedimentation particles and labeling agent may have like charges to reduce non-specific binding of labeling agent and sedimentation particles. In some examples, the density medium is provided with a separation layer for stabilizing the assay during storage and operation. In some examples, the sedimentation channel may be provided with a generally flat sedimentation chamber for dispersing the particle pellet over a larger surface area.

Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation

DOEpatents

Schaff, Ulrich Y; Koh, Chung-Yan; Sommer, Gregory J

2015-02-24

Embodiments of the present invention are directed toward devices, systems, and method for conducting assays using sedimentation. In one example, a method includes layering a mixture on a density medium, subjecting sedimentation particles in the mixture to sedimentation forces to cause the sedimentation particles to move to a detection area through a density medium, and detecting a target analyte in a detection region of the sedimentation channel. In some examples, the sedimentation particles and labeling agent may have like charges to reduce non-specific binding of labeling agent and sedimentation particles. In some examples, the density medium is provided with a separation layer for stabilizing the assay during storage and operation. In some examples, the sedimentation channel may be provided with a generally flat sedimentation chamber for dispersing the particle pellet over a larger surface area.

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

PubMed

Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (Passay.

Carbon nanotube-based labels for highly sensitive colorimetric and aggregation-based visual detection of nucleic acids

NASA Astrophysics Data System (ADS)

Lee, Ai Cheng; Ye, Jian-Shan; Ngin Tan, Swee; Poenar, Daniel P.; Sheu, Fwu-Shan; Kiat Heng, Chew; Meng Lim, Tit

2007-11-01

A novel carbon nanotube (CNT) derived label capable of dramatic signal amplification of nucleic acid detection and direct visual detection of target hybridization has been developed. Highly sensitive colorimetric detection of human acute lymphocytic leukemia (ALL) related oncogene sequences amplified by the novel CNT-based label was demonstrated. Atomic force microscope (AFM) images confirmed that a monolayer of horseradish peroxidase and detection probe molecules was immobilized along the carboxylated CNT carrier. The resulting CNT labels significantly enhanced the nucleic acid assay sensitivity by at least 1000 times compared to that of conventional labels used in enzyme-linked oligosorbent assay (ELOSA). An excellent detection limit of 1 × 10-12 M (60 × 10-18 mol in 60 µl) and a four-order wide dynamic range of target concentration were achieved. Hybridizations using these labels were coupled to a concentration-dependent formation of visible dark aggregates. Targets can thus be detected simply with visual inspection, eliminating the need for expensive and sophisticated detection systems. The approach holds promise for ultrasensitive and low cost visual inspection and colorimetric nucleic acid detection in point-of-care and early disease diagnostic application.

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry.

PubMed

Soulard, Patricia; McLaughlin, Meg; Stevens, Jessica; Connolly, Brendan; Coli, Rocco; Wang, Leyu; Moore, Jennifer; Kuo, Ming-Shang T; LaMarr, William A; Ozbal, Can C; Bhat, B Ganesh

2008-10-03

Several recent reports suggest that stearoyl-CoA desaturase 1 (SCD1), the rate-limiting enzyme in monounsaturated fatty acid synthesis, plays an important role in regulating lipid homeostasis and lipid oxidation in metabolically active tissues. As several manifestations of type 2 diabetes and related metabolic disorders are associated with alterations in intracellular lipid partitioning, pharmacological manipulation of SCD1 activity might be of benefit in the treatment of these disease states. In an effort to identify small molecule inhibitors of SCD1, we have developed a mass spectrometry based high-throughput screening (HTS) assay using deuterium labeled stearoyl-CoA substrate and induced rat liver microsomes. The methodology developed allows the use of a nonradioactive substrate which avoids interference by the endogenous SCD1 substrate and/or product that exist in the non-purified enzyme source. Throughput of the assay was up to twenty 384-well assay plates per day. The assay was linear with protein concentration and time, and was saturable for stearoyl-CoA substrate (K(m)=10.5 microM). The assay was highly reproducible with an average Z' value=0.6. Conjugated linoleic acid and sterculic acid, known inhibitors of SCD1, exhibited IC(50) values of 0.88 and 0.12 microM, respectively. High-throughput mass spectrometry screening of over 1.7 million compounds in compressed format demonstrated that the enzyme target is druggable. A total of 2515 hits were identified (0.1% hit rate), and 346 were confirmed active (>40% inhibition of total SCD activity at 20 microM--14% conformation rate). Of the confirmed hits 172 had IC(50) values of <10 microM, including 111 <1 microM and 48 <100 nM. A large number of potent drug-like (MW<450) hits representing six different chemical series were identified. The application of mass spectrometry to high-throughput screening permitted the development of a high-quality screening protocol for an otherwise intractable target, SCD1. Further

Epigenetic Mechanisms of Folate Nutrition in Breast Cancer

DTIC Science & Technology

2012-04-01

made we will study the effects of depletion of protein expression on breast cancer cell growth, apoptosis , replication, migration, ability to...AHCY or DNMT. Measured Endpoint Assay Cell proliferation Trypan blue exclusion assay and MTT assay Cell apoptosis TUNEL assay and caspase 3 staining

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

PubMed Central

Bentley, Marvin; Decker, Helena; Luisi, Julie

2015-01-01

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. PMID:25624392

Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

PubMed

Koh, Hye Ran; Wang, Xinlei; Myong, Sua

2016-08-01

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.

Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.

PubMed

Boomer, Laura A; Bellister, Seth A; Stephenson, Linda L; Hillyard, Stanley D; Khoury, Joseph D; Youson, John H; Gosche, John R

2010-01-01

Research in biliary atresia has been hindered by lack of a suitable animal model. Lampreys are primitive vertebrates with distinct larval and adult life cycle stages. During metamorphosis the biliary system of the larval lamprey disappears. Lamprey metamorphosis has been proposed as a model for biliary atresia. We have begun to explore cellular events during lamprey metamorphosis by assessing for cholangiocyte apoptosis. Sea lamprey larvae were housed under controlled environmental conditions. Premetamorphic larvae were induced to undergo metamorphosis by exposure to 0.01% KClO(4). Animals were photographed weekly, and the stage of metamorphosis was assigned based upon external features. Livers were harvested and processed for routine histology and immunohistochemistry. DNA fragmentation was detected using deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays and cholangiocytes were identified with antibodies to cytokeratin-19. Percent TUNEL+ cholangiocytes at different stages of metamorphosis was determined. The percentage of TUNEL+ cholangiocytes was 10% in premetamorphic (stage 0) lamprey (n = 6), 51% at stage 1 (n = 6), 40% at stage 2 (n = 5), 18% at stage 3 (n = 5), and 9% stage 4 (n = 4). Routine hemotoxylin and eosin stained paraffin-embedded tissue sections revealed frequent apoptotic bodies at stages 3 and 4 of metamorphosis without histologic evidence of necrosis. DNA fragmentation is identified at the earliest stages of metamorphosis during induced metamorphosis in lampreys. Additional studies are necessary to validate this potentially valuable animal model. Copyright 2010 Elsevier Inc. All rights reserved.

Standard addition with internal standardisation as an alternative to using stable isotope labelled internal standards to correct for matrix effects-Comparison and validation using liquid chromatography-​tandem mass spectrometric assay of vitamin D.

PubMed

Hewavitharana, Amitha K; Abu Kassim, Nur Sofiah; Shaw, Paul Nicholas

2018-06-08

With mass spectrometric detection in liquid chromatography, co-eluting impurities affect the analyte response due to ion suppression/enhancement. Internal standard calibration method, using co-eluting stable isotope labelled analogue of each analyte as the internal standard, is the most appropriate technique available to correct for these matrix effects. However, this technique is not without drawbacks, proved to be expensive because separate internal standard for each analyte is required, and the labelled compounds are expensive or require synthesising. Traditionally, standard addition method has been used to overcome the matrix effects in atomic spectroscopy and was a well-established method. This paper proposes the same for mass spectrometric detection, and demonstrates that the results are comparable to those with the internal standard method using labelled analogues, for vitamin D assay. As conventional standard addition procedure does not address procedural errors, we propose the inclusion of an additional internal standard (not co-eluting). Recoveries determined on human serum samples show that the proposed method of standard addition yields more accurate results than the internal standardisation using stable isotope labelled analogues. The precision of the proposed method of standard addition is superior to the conventional standard addition method. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

PubMed

Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S

2008-07-01

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.

Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

PubMed Central

Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

2015-01-01

PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

Comparative studies of nucleic acid hybridization assay for Listeria in foods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klinger, J.D.; Johnson, A.; Croan, D.

A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a /sup 32/P-labeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in amore » filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results.« less

Category labels versus feature labels: category labels polarize inferential predictions.

PubMed

Yamauchi, Takashi; Yu, Na-Yung

2008-04-01

What makes category labels different from feature labels in predictive inference? This study suggests that category labels tend to make inductive reasoning polarized and homogeneous. In two experiments, participants were shown two schematic pictures of insects side by side and predicted the value of a hidden feature of one insect on the basis of the other insect. Arbitrary verbal labels were shown above the two pictures, and the meanings of the labels were manipulated in the instructions. In one condition, the labels represented the category membership of the insects, and in the other conditions, the same labels represented attributes of the insects. When the labels represented category membership, participants' responses became substantially polarized and homogeneous, indicating that the mere reference to category membership can modify reasoning processes.

Nanoplasmonic biochips for rapid label-free detection of imidacloprid pesticides with a smartphone.

PubMed

Lee, Kuang-Li; You, Meng-Lin; Tsai, Chia-Hsin; Lin, En-Hung; Hsieh, Shu-Yi; Ho, Ming-Hsun; Hsu, Ju-Chun; Wei, Pei-Kuen

2016-01-15

The widespread and intensive use of neonicotinoid insecticides induces negative cascading effects on ecosystems. It is desirable to develop a portable sensitive sensing platform for on-site screening of high-risk pesticides. We combined an indirect competitive immunoassay, highly sensitive surface plasmon resonance (SPR) biochip and a simple portable imaging setup for label-free detection of imidacloprid pesticides. The SPR biochip consists of several capped nanoslit arrays with different periods which form a spectral image on the chip. The qualitative and semiquantitative analyses of pesticides can be directly observed from the spot shift on the chip. The precise semiquantitative analyses can be further completed by using image processing in a smartphone. We demonstrate simultaneous detection of four different concentrations of imidacloprid pesticides. The visual detection limit is about 1ppb, which is well below the maximum residue concentration permitted by law (20ppb). Compared to the one-step strip assay, the proposed chip is capable of performing semiquantitative analyses and multiple detection. Compared to the enzyme-linked immunosorbent assay, our method is label-free and requires simple washing steps and short reaction time. In addition, the label-free chip has a comparable sensitivity but wider working range than those labeling techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels

NASA Astrophysics Data System (ADS)

Malý, J.; Lampová, H.; Semerádtová, A.; Štofik, M.; Kováčik, L.

2009-09-01

This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH4 silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

PubMed

Urusov, Alexandr E; Petrakova, Alina V; Gubaydullina, Milyausha K; Zherdev, Anatoly V; Eremin, Sergei A; Kong, Dezhao; Liu, Liqiang; Xu, Chuanlai; Dzantiev, Boris B

2017-05-01

To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml -1 at 15 min of assay duration. The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

Mitomycin C-induced apoptosis in cultured human Tenon's capsule fibroblasts.

PubMed

Kim, J W; Kim, S K; Song, I H; Kim, I T

1999-06-01

To investigate the mitomycin C-induced apoptotic cell death of fibroblasts, the primarily cultured human Tenon's capsule fibroblasts were exposed to a clinically used dosage of 0.4 mg/ml of mitomycin C for 5 minutes. TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay and electron microscopic studies were performed to determine the extent of mitomycin C-induced apoptosis. A flow cytometric study was performed to quantify the apoptotic cell population over time. The TUNEL stains were positive and electron microscopy showed features of apoptotic cell death in some fibroblasts 3 and 5 days after treatment. Flow cytometric analysis using Annexin V-propidium iodide double staining detected apoptotic cells 3 days after treatment. These apoptotic cell populations increased at 4 days and were sustained for one week. This study revealed that the clinical effects of mitomycin C on fibroblasts may be mediated not only by antiproliferative but also apoptotic cell death to some degree. Therefore, the apoptotic cell death of fibroblasts induced by mitomycin C should be considered to properly understand the mechanism of wound healing after trabeculectomy with adjunctive mitomycin C.

Macrophage apoptosis in rat skeletal muscle treated with bupivacaine hydrochloride: possible role of MCP-1.

PubMed

Horiguchi, Taisuke; Shibata, Masa-Aki; Ito, Yuko; Eid, Nabil A S; Abe, Muneaki; Otsuki, Yoshinori

2002-07-01

The fate of macrophages infiltrating damaged rat skeletal muscle fibers after intramuscular injection of the anesthetic bupivacaine hydrochloride (BPVC) and the possible roles of monocyte chemoattractant protein-1 (MCP-1) were investigated. The number of macrophages reached a maximum level at 2 days after the injection and then gradually decreased. The number of apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was elevated at 2-4 days and decreased thereafter. In serial sections, TUNEL-positive cells were also immunopositive for RM-4, an antibody specific for identification of macrophages. Electron microscopy further confirmed that it was the macrophages themselves that were undergoing apoptosis. As assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), high levels of MCP-1 mRNA in BPVC-treated muscles were observed and positively correlated with maximum macrophage infiltration. However, the levels of MCP-1 mRNA returned to normal low values coincident with decrease of inflammation and healing of damaged muscle fiber. Local apoptosis of macrophages, under the control of MCP-1, may be involved in healing of BPVC-treated muscles. Copyright 2002 Wiley Periodicals, Inc.

The role of hexon in egg drop syndrome virus (EDSV) inducing apoptosis in duck embryo fibroblast cells.

PubMed

Qi, Xuefeng; Xu, Jiamin; Wang, Zugui; Wang, Xueping; Wang, Jingyu

2017-10-01

Although extensive efforts have been made to understand adenovirus infection in human cells, little is known for egg drop syndrome virus (EDSV) infection in the avian-derived cells. In this study, the effects of EDSV infection as well as the possible role hexon protein, the main building block of the EDSV capsid, on apoptosis induction in duck embryo fibroblast (DEF) cells was examined. Flow cytometry analysis and TUNEL assay revealed that EDSV infection induced significant apoptosis in DEF cells compared with mock infected cells. Interestingly, the increase of the apoptosis rate detected in EDSV infected DEF cells were accompanied by an increased virus load in cells in a time-dependent manner. Furthermore, a time-dependent decrease in hexon protein expression levels in hexon transfected DEF cells in parallel with a gradual decrease in TUNEL-labeling cells was also observed in the current study. In addition, caspase activity detection and western blot analysis indicates that either EDSV infection or EDSV hexon transfection both induced apoptosis of DEF cells via activating both the exogenous and the mitochondrial pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

The use of complimentary assays to evaluate the enrichment of human sperm quality in asthenoteratozoospermic and teratozoospermic samples processed with Annexin-V magnetic activated cell sorting.

PubMed

Delbes, G; Herrero, M B; Troeung, E-T; Chan, P T K

2013-09-01

Sperm chromatin integrity may affect the outcomes of assisted reproductive technology (ART). Developing a clinically reliable strategy to enrich sperm samples with high chromatin quality spermatozoa prior to sperm banking or use in ART would thus be advantageous. The objectives of this study were to: (i) assess the sperm chromatin quality in men with different categories of semen parameters; and (ii) evaluate the extents of Annexin-V magnetic-activated cell sorting (MACS) technology coupled with differential density gradient centrifugation (DGC) in improving sperm chromatin quality. Three categories of men from couples attending a university-based fertility clinic were recruited based on their semen parameters: normozoospermic (n = 13), asthenoteratozoospermic (n = 17) and teratozoospermic (n = 12). For each patient, spermatozoa in semen samples were processed first by DGC to enrich the motility and further by MACS to remove spermatozoa showing apoptotic features. The yield and enrichment of sperm quality was evaluated at each step with conventional semen parameters in conjunction with a combination of five complementary assays, to assess sperm maturity, chromatin structure, compaction and DNA integrity (Hyaluronic Binding Assay, SCSA, chromomycine A3 staining and TUNEL and COMET assays). Our results demonstrated that, compared with normozoospermic samples, raw asthenoteratozoospermic and teratozoospermic samples had a higher proportion of spermatozoa containing DNA breaks, but only asthenoteratozoospermic exhibited altered chromatin structure and decreased binding to hyaluronic acid. Interestingly, the DGC appeared to select for more mature spermatozoa with high DNA compaction. More importantly, in all categories of semen samples, Annexin-V MACS allows enrichment of spermatozoa with good chromatin quality as measured by the TUNEL and SCSA. Because effective treatment modalities to improve sperm DNA damage are limited, our results suggest a potential clinical

Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay.

PubMed

Mahboubi, Arash; Sadjady, Seyyed Kazem; Mirzaei Saleh Abadi, Mohammad; Azadi, Saeed; Solaimanian, Roya

2012-01-01

DETERMINATION OF STREPTOKINASE ACTIVITY IS USUALLY ACCOMPLISHED THROUGH TWO ASSAY METHODS: a) Clot lysis, b) Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase®, which is a native product and Heberkinasa®, which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase® was found to be 101 ± 4% and 97 ± 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa® the potency values obtained were 42 ± 5% and 92.5 ± 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein.

Receptor-based screening assays for the detection of antibiotics residues - A review.

PubMed

Ahmed, Saeed; Ning, Jianan; Cheng, Guyue; Ahmad, Ijaz; Li, Jun; Mingyue, Liu; Qu, Wei; Iqbal, Mujahid; Shabbir, M A B; Yuan, Zonghui

2017-05-01

Consumer and regulatory agencies have a high concern to antibiotic residues in food producing animals, so appropriate screening assays of fast, sensitive, low cost, and easy sample preparation for the identification of these residues are essential for the food-safety insurance. Great efforts in the development of a high-throughput antibiotic screening assay have been made in recent years. Concerning the screening of antibiotic residue, this review elaborate an overview on the availability, advancement and applicability of antibiotic receptor based screening assays for the safety assessment of antibiotics usage (i.e. radio receptor assay, enzyme labeling assays, colloidal gold receptor assay, enzyme colorimetry assay and biosensor assay). This manuscript also tries to shed a light on the selection, preparation and future perspective of receptor protein for antibiotic residue detection. These assays have been introduced for the screening of numerous food samples. Receptor based screening technology for antibiotic detection has high accuracy. It has been concluded that at the same time, it can detect a class of drugs for certain receptor, and realize the multi-residue detection. These assays offer fast, easy and precise detection of antibiotics. Copyright © 2017 Elsevier B.V. All rights reserved.

Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

PubMed

Basu, Sankha S; Mesaros, Clementina; Gelhaus, Stacy L; Blair, Ian A

2011-02-15

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

Quantification of DNA using the luminescent oxygen channeling assay.

PubMed

Patel, R; Pollner, R; de Keczer, S; Pease, J; Pirio, M; DeChene, N; Dafforn, A; Rose, S

2000-09-01

Simplified and cost-effective methods for the detection and quantification of nucleic acid targets are still a challenge in molecular diagnostics. Luminescent oxygen channeling assay (LOCI(TM)) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linking probes, to different DNA targets. These oligomer-conjugated LOCI particles survive thermocycling in a PCR reaction and allow quantified detection of DNA targets in both real-time and endpoint formats. The endpoint DNA quantification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annealed to target or control amplicons, and separate illuminations generated time-resolved chemiluminescence, which distinguished the two amplicon types. In the endpoint method, ratios of the two signals allowed determination of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. This is the first report of the use of an oligomer-labeled latex particle assay capable of producing DNA quantification and sequence-specific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods described here have been shown to be easily adaptable to new DNA targets because of the generic nature of the oligomer-labeled LOCI particles.

In Vitro and In Vivo Efficacy Studies of Lavender angustifolia Essential Oil and Its Active Constituents on the Proliferation of Human Prostate Cancer.

PubMed

Zhao, Yunqi; Chen, Ran; Wang, Yun; Qing, Chen; Wang, Wei; Yang, Yixin

2017-06-01

Lavandula angustifolia is the most widely cultivated Lavandula species. The extraction of its flower and leaves has been used as herbal medicine. In this study, the in vitro antitumor activities were tested on human prostate cancer PC-3 and DU145 cell lines. Flow cytometry technology was applied to study apoptosis induction and cell cycle arrest. The PC-3 cell line was used to establish subcutaneous xenograft tumors in nude mice. Paraffin sections from xenograft tumor specimens were used in the TUNEL (terminal deocynucleotide transferase dUTP nick end labeling) assay and an immunohistochemistry assay to detect cell proliferation markers Ki67 and PCNA. Lavender essential oil, linalool, and linalyl acetate showed stronger inhibitory effect on PC-3 cells than on DU145 cells. The apoptotic cell populations observed in PC-3 cells treated with lavender essential oil, linalool, and linalyl acetate were 74.76%, 67.11%, and 56.14%, respectively. The PC-3 cells were mainly arrested in the G 2 /M phase. In the xenograft model with PC-3 cell transplantation, essential oil and linalool significantly suppressed tumor growth. The immunosignals of Ki67 and PCNA in the essential oil, linalool, and linalyl acetate treatment groups were significantly lower than that of the control group in xenograft tumor sections. The TUNEL assay indicated that each of the 3 phytochemicals significantly induced apoptosis compared to the control group. This study provides novel insight and evidence on the antiproliferative effect of L angustifolia essential oil and its major constituents on human prostate cancer. The antitumor effect was associated with cell proliferation inhibition and apoptosis induction in xenograft tumors.

In Vitro and In Vivo Efficacy Studies of Lavender angustifolia Essential Oil and Its Active Constituents on the Proliferation of Human Prostate Cancer

PubMed Central

Zhao, Yunqi; Chen, Ran; Wang, Yun; Qing, Chen; Wang, Wei; Yang, Yixin

2016-01-01

Lavandula angustifolia is the most widely cultivated Lavandula species. The extraction of its flower and leaves has been used as herbal medicine. In this study, the in vitro antitumor activities were tested on human prostate cancer PC-3 and DU145 cell lines. Flow cytometry technology was applied to study apoptosis induction and cell cycle arrest. The PC-3 cell line was used to establish subcutaneous xenograft tumors in nude mice. Paraffin sections from xenograft tumor specimens were used in the TUNEL (terminal deocynucleotide transferase dUTP nick end labeling) assay and an immunohistochemistry assay to detect cell proliferation markers Ki67 and PCNA. Lavender essential oil, linalool, and linalyl acetate showed stronger inhibitory effect on PC-3 cells than on DU145 cells. The apoptotic cell populations observed in PC-3 cells treated with lavender essential oil, linalool, and linalyl acetate were 74.76%, 67.11%, and 56.14%, respectively. The PC-3 cells were mainly arrested in the G2/M phase. In the xenograft model with PC-3 cell transplantation, essential oil and linalool significantly suppressed tumor growth. The immunosignals of Ki67 and PCNA in the essential oil, linalool, and linalyl acetate treatment groups were significantly lower than that of the control group in xenograft tumor sections. The TUNEL assay indicated that each of the 3 phytochemicals significantly induced apoptosis compared to the control group. This study provides novel insight and evidence on the antiproliferative effect of L angustifolia essential oil and its major constituents on human prostate cancer. The antitumor effect was associated with cell proliferation inhibition and apoptosis induction in xenograft tumors. PMID:27151584

Devices, systems, and methods for conducting sandwich assays using sedimentation

DOEpatents

Schaff, Ulrich Y; Sommer, Gregory J; Singh, Anup K; Hatch, Anson V

2015-02-03

Embodiments of the present invention are directed toward devices, systems, and method for conducting sandwich assays using sedimentation. In one example, a method includes generating complexes on a plurality of beads in a fluid sample, individual ones of the complexes comprising a capture agent, a target analyte, and a labeling agent. The plurality of beads including the complexes may be transported through a density media, wherein the density media has a density lower than a density of the beads and higher than a density of the fluid sample, and wherein the transporting occurs, at least in part, by sedimentation. Signal may be detected from the labeling agents of the complexes.

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

PubMed

Anumula, Kalyan Rao

2012-07-01

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

Label-free protein assay based on a nanomechanical cantilever array

NASA Astrophysics Data System (ADS)

Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, Ch

2003-01-01

We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen-antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml-1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen-antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

Microvolume, kinetic-dependent enzyme-linked immunosorbent assay for amoeba antibodies.

PubMed Central

Mathews, H M; Walls, K W; Huong, A Y

1984-01-01

We describe a microvolume enzyme-linked immunosorbent assay based on enzyme rate kinetics. Antigens from Entamoeba histolytica were adsorbed in wells of disposable polystyrene strips containing 12 flat-bottom wells. After exposure to the serum of a patient and peroxidase-labeled anti-human immunoglobulin G, the rate of color change in specific substrate was determined by eight sequential readings of individual wells over a 2-min period with a microcomputer-controlled model MR-600 automated plate reader. The changes in absorbance readings were converted to slope values for each well by the microcomputer. Thus, 12 samples were read, and results were printed in ca. 3.5 min. Assay conditions are described and data are presented to show that this assay is quantitative for antibody and antigen concentration with a single-tube (well) dilution. PMID:6321547

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

PubMed

LeBlanc, André; Michaud, Sarah A; Percy, Andrew J; Hardie, Darryl B; Yang, Juncong; Sinclair, Nicholas J; Proudfoot, Jillaine I; Pistawka, Adam; Smith, Derek S; Borchers, Christoph H

2017-07-07

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens.

PubMed

Cheng, Fang; Wu, Jiajie; Zhang, Jin; Pan, Aihu; Quan, Sheng; Zhang, Dabing; Kim, HaeYeong; Li, Xiang; Zhou, Shan; Yang, Litao

2016-05-15

Food allergies cause health risks to susceptible consumers and regulations on labeling of food allergen contents have been implemented in many countries and regions. To achieve timely and accurate food allergen labeling, the development of fast and effective allergen detection methods is very important. Herein, a decaplex polymerase chain reaction (PCR) assay combined with capillary electrophoresis was developed to detect simultaneously 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan. The absolute limit of detection (LODa) of this system is between 2 and 20 copies of haploid genome, and the relative LOD (LODr) is as low as 0.005% (w/w) in simulated food mixtures. The developed assay was subsequently applied to 20 commercial food products and verified the allergen ingredients stated on the labels. Furthermore, results using this decaplex PCR assay was successfully replicated in three other laboratories, demonstrating the repeatability and applicability of this assay in routine analysis of the 10 food allergens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Exploring blocking assays using Octet, ProteOn, and Biacore biosensors.

PubMed

Abdiche, Yasmina N; Malashock, Dan S; Pinkerton, Alanna; Pons, Jaume

2009-03-15

We demonstrate the use of label-free real-time optical biosensors in competitive binding assays by epitope binning a panel of antibodies. We describe three assay orientations that we term in tandem, premix, and classical sandwich blocking, and we perform each of them on three platforms: ForteBio's Octet QK, Bio-Rad's ProteOn XPR36, and GE Healthcare's Biacore 3000. By testing whether antibodies block one another's binding to their antigen in a pairwise fashion, we establish a blocking profile for each antibody relative to the others in the panel. The blocking information is then used to create "bins" of antibodies with similar epitopes. The advantages and disadvantages of each biosensor, factors to consider when deciding on the most appropriate blocking assay orientation for a particular interaction system, and tips for dealing with ambiguous data are discussed. The data from our different assay orientations and biosensors agree very well, establishing these machines as valuable tools for characterizing antibody epitopes and multiprotein complexes of biological significance.

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.

PubMed

Zhang, Hua; Zhao, Zhen; Lei, Zhen; Wang, Zhenxin

2016-12-06

The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL -1 and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

Novel Multiplexed Assay for Identifying SH2 Domain Antagonists of STAT Family Proteins

PubMed Central

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors. PMID:23977103

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

PubMed

Takakuma, Kazuyuki; Ogo, Naohisa; Uehara, Yutaka; Takahashi, Susumu; Miyoshi, Nao; Asai, Akira

2013-01-01

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Antelo, Alvaro; Vieytes, Mercedes R; Botana, Luis M

2011-08-01

The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.

Label-free and amplified quantitation of proteins in complex mixtures using diffractive optics technology.

PubMed

Cleverley, Steve; Chen, Irene; Houle, Jean-François

2010-01-15

Immunoaffinity approaches remain invaluable tools for characterization and quantitation of biopolymers. Their application in separation science is often limited due to the challenges of immunoassay development. Typical end-point immunoassays require time consuming and labor-intensive approaches for optimization. Real-time label-free analysis using diffractive optics technology (dot) helps guide a very effective iterative process for rapid immunoassay development. Both label-free and amplified approaches can be used throughout feasibility testing and ultimately in the final assay, providing a robust platform for biopolymer analysis over a very broad dynamic range. We demonstrate the use of dot in rapidly developing assays for quantitating (1) human IgG in complex media, (2) a fusion protein in production media and (3) protein A contamination in purified immunoglobulin preparations. 2009 Elsevier B.V. All rights reserved.

Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

PubMed

Kováčová, Kristína; Farkaš, Vladimír

2016-04-01

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.

Estradiol Valerate and Remifemin ameliorate ovariectomy-induced decrease in a serotonin dorsal raphe-preoptic hypothalamus pathway in rats.

PubMed

Wang, Wenjuan; Cui, Guangxia; Jin, Biao; Wang, Ke; Chen, Xing; Sun, Yu; Qin, Lihua; Bai, Wenpei

2016-11-01

Perimenopausal syndromes begin as ovarian function ceases and the most common symptoms are hot flushes. Data indicate that the projections of serotonin to hypothalamus may be involved in the mechanism of hot flushes. Therefore, the aim of this study is to investigate the potential role of the serotonin dorsal raphe-preoptic hypothalamus pathway for hot flushes in an animal model of menopause. We determined the changes in serotonin expression in the dorsal raphe (DR) and preoptic anterior hypothalamus (POAH) in ovariectomized rats. We also explored the therapeutical effects of estradiol valerate and Remifemin in this model. Eighty female Sprague-Dawley rats were randomly assigned to sham-operated (SHAM) group, ovariectomy (OVX) group with vehicle, ovariectomy with estradiol valerate treatment (OVX+E) group and ovariectomy with Remifemin (OVX+ICR) group. Serotonin expression was evaluated in the DR and POAH using immunofluorescence and quantified in the DR using an enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed in the DR by TUNEL assay. The number of serotonin immunoreactive neurons and the level of serotonin expression in the DR decreased significantly following OVX compared to the SHAM group. No TUNEL-positive cells were detected in the DR in any group. In addition, following OVX, the number of serotonin-positive fibers decreased significantly in the ventromedial preoptic nucleus (VMPO), especially in the ventrolateral preoptic nucleus (VLPO). Treatment with either estradiol or Remifemin for 4 weeks countered the OVX-induced decreases in serotonin levels in both the DR and the hypothalamus, with levels in the treated rats similar to those in the SHAM group. A fluorescently labeled retrograde tracer was injected into the VLPO at the 4-week time point. A significantly lower percentage of serotonin with CTB double-labeled neurons in CTB-labeled neurons was demonstrated after ovariectomy, and both estradiol and Remifemin countered this OVX

Short communication: use of the BetaStar Plus assay for detection of ceftiofur antimicrobial residues in milk from individual cows following intramammary treatment for mastitis.

PubMed

Grooms, D L; Norby, B; Grooms, K E; Jagodzinski, E N; Erskine, R J; Halbert, L W; Coetzee, J F; Wulf, L; Rice, J A

2015-09-01

Development and use of on-farm assays to detect antimicrobial residues in milk is important to reduce the risk of violative residues in marketed milk. The objective of this study was to evaluate the effectiveness of a lateral-flow immunodiagnostic assay (BetaStar Plus, Neogen Corp., Lansing, MI) in detecting ceftiofur residues in milk from individual cows treated for mastitis. This assay is currently approved by the US Federal Drug Administration (FDA) for detecting β-lactam residues in commingled milk. Forty-five dairy cows with clinical mastitis from 4 dairy farms were enrolled and treated intramammary with 125 mg of ceftiofur hydrochloride (Spectramast LC, Zoetis, Madison, NJ) according to the manufacturer's label recommendation. Composite milk samples were collected (A) before first intramammary antimicrobial treatment, (B) before the last intramammary antimicrobial treatment, (C) the last milking of the product-labeled milk withhold, (D) the first milking after the product-labeled milk withhold had been met, and (E) 72 h after the product-labeled milk withhold had been met. Samples were tested using the BetaStar Plus assay within 48 h of collection. Parallel samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and for somatic cell count and milk components. The BetaStar Plus assay identified 6.7, 60.0, 46.7, 22.2, and 6.7% positive samples at each of the respective time points. The assay had sensitivity and specificity of 100 and 84.7%, respectively, compared with liquid chromatography-tandem mass spectrometry analysis using FDA published residue tolerance levels for ceftiofur (or ceftiofur metabolites) as a threshold. The BetaStar Plus assay could be useful for detecting ceftiofur residues in milk from individual cows following intramammary treatment for mastitis before the milk is shipped for processing. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay.

PubMed

Lazarovici, Philip; Lahiani, Adi; Gincberg, Galit; Haham, Dikla; Fluksman, Arnon; Benny, Ofra; Marcinkiewicz, Cezary; Lelkes, Peter I

2018-01-01

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation.

PubMed

Dittmer, W U; de Kievit, P; Prins, M W J; Vissers, J L M; Mersch, M E C; Martens, M F W C

2008-09-30

A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions as well as the bound/free separation are entirely controlled by magnetic forces induced by electromagnets above and below the sensor chip. During the assay, particles conjugated with tracer antibodies are actuated through the sample for target capture, and rapidly brought to the sensor surface where they bind to immobilized capture antibodies. Weakly or unbound labels are removed with a magnetic force oriented away from the GMR sensor surface. For the measurement of parathyroid hormone (PTH), a detection limit in the 10 pM range is obtained with a total assay time of 15 min when 300 nm particles are used. The same sensitivity can be achieved in 5 min when 500 nm particles are used. If 500 nm particles are employed in a 15-minute assay, then 0.8 pM of PTH is detectable. The low sample volume, high analytical performance and high speed of the test coupled with the compact GMR biosensor make the system especially suitable for sensitive testing outside of laboratory environments.

A single-vial biphasic liquid extraction assay for choline acetyltransferease using (/sup 3/H)choline

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rand, J.B.; Johnson, C.D.

1981-09-15

A single-vial liquid extraction assay for choline acetyltransferase that uses (/sup 3/H)choline as the labeled substrate has been devised. (/sup 3/H)Choline is incubated with an excess of acetyl-CoA in a small reaction vial which also serves as a scintillation vial. After a suitable reaction period, unreacted (/sup 3/H)choline is quickly and quantitatively converted to phosphoryl-(/sup 3/H)choline by the addition of an excess of choline kinase. This treatment is followed by the addition of scintillation fluid containing sodium tetraphenylboron after which the vial is capped, shaken, and counted. A two-phase system is produced in which product (/sup 3/H)choline is selectively extractedmore » into the scintillation fluid, where is is counted. Phosphoryl-(/sup 3/H)choline remains in the aqueous phase and is not counted. This assay is rapid, simple, and quite sensitive. In comparison to assays using acetyl-CoA as the labeled substrate, it is less sensitive to interference by other enzymes and thus more suitable for measuring choline acetyltransferase in crude extracts and in the initial stages of purificaton. Similar single-vial radiometric assays are described for choline kinase and acetyl-CoA hydrolases.« less

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

PubMed

Tan, Joshua; Bull, Peter C

2015-01-01

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Involvement of ciliary neurotrophic factor in early diabetic retinal neuropathy in streptozotocin-induced diabetic rats.

PubMed

Ma, Mingming; Xu, Yupeng; Xiong, Shuyu; Zhang, Jian; Gu, Qing; Ke, Bilian; Xu, Xun

2018-05-23

Ciliary neurotrophic factor (CNTF) has been evaluated as a candidate therapeutic agent for diabetes and its neural complications. However, its role in diabetic retinopathy has not been fully elucidated. This is a randomized unblinded animal experiment. Wistar rats with streptozocin (STZ)-induced diabetes were regularly injected with CNTF or vehicle control in their vitreous bodies beginning at 2 weeks after STZ injection. A total of five injections were used. In diabetic rats, the levels of CNTF and neurotrophin-3 (NT-3) were evaluated by enzyme-linked immunosorbent assays (ELISA) and real-time PCR. The abundance of tyrosine hydroxylase (TH) and β-III tubulin was detected by western blot. Transferase-mediated dUTP nick-end labeling staining (TUNEL) was used to detect cell apoptosis in the retinal tissue. The activation of caspase-3 was also measured. The protein and mRNA levels of CNTF in diabetic rat retinas were reduced compared to control rats. In addition, retinal ganglion cells (RGCs) and dopaminergic amacrine cells appeared to undergo degeneration in diabetic rat retinas, as revealed by transferase-mediated dUTP nick-end labeling staining (TUNEL). Tyrosine hydroxylase (TH) and β-III tubulin protein levels also decreased significantly. Intraocular administration of CNTF rescued RGCs and dopaminergic amacrine cells from neurodegeneration and counteracted the downregulation of β-III tubulin and TH expression, thus demonstrating its therapeutic potential. Our study suggests that early diabetic retinal neuropathy involves the reduced expression of CNTF and can be ameliorated by an exogenous supply of this neurotrophin.

Rapid colorimetric assay for gentamicin injection.

PubMed

Tarbutton, P

1987-01-01

A rapid colorimetric method for determining gentamicin concentration in commercial preparations of gentamicin sulfate injection was developed. Methods currently available for measuring gentamicin concentration via its colored complex with cupric ions in alkaline solution were modified to reduce the time required for a single analysis. The alkaline copper tartrate (ACT) reagent solution was prepared such that each milliliter contained 100 mumol cupric sulfate, 210 mumol potassium sodium tartrate, and 1.25 mmol sodium hydroxide. The assay involves mixing 0.3 mL gentamicin sulfate injection 40 mg/mL (of gentamicin), 1.0 mL ACT reagent, and 0.7 mL water; the absorbance of the resulting solution at 560 nm was used to calculate the gentamicin concentration in the sample. For injections containing 10 mg/mL of gentamicin, the amount of the injection was increased to 0.5 mL and water decreased to 0.5 mL. The concentration of gentamicin in samples representing 11 lots of gentamicin sulfate injection 40 mg/mL and 8 lots of gentamicin sulfate injection 10 mg/mL was determined. The specificity, reproducibility, and accuracy of the assay were assessed. The colored complex was stable for at least two hours. Gentamicin concentration ranged from 93.7 to 108% and from 95 to 109% of the stated label value of the 40 mg/mL and the 10 mg/mL injections, respectively. No components of the preservative system present in the injections interfered with the assay. Since other aminoglycosides produced a colored complex, the assay is not specific for gentamicin. The assay was accurate and reproducible over the range of 4-20 mg of gentamicin. This rapid and accurate assay can be easily applied in the hospital pharmacy setting.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

NASA Astrophysics Data System (ADS)

Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

Magneto-optical labeling of fetal neural stem cells for in vivo MRI tracking.

PubMed

Flexman, J A; Minoshima, S; Kim, Y; Cross, D J

2006-01-01

Neural stem cell therapy for neurological pathologies, such as Alzheimer's and Parkinson's disease, may delay the onset of symptoms, replace damaged neurons and/or support the survival of endogenous cells. Magnetic resonance imaging (MRI) can be used to track magnetically labeled cells in vivo to observe migration. Prior to transplantation, labeled cells must be characterized to show that they retain their intrinsic properties, such as cell proliferation into neurospheres in a supplemented environment. In vivo images must also be correlated to sensitive, histological markers. In this study, we show that fetus-derived neural stem cells can be co-labeled with superparamagnetic iron oxide and PKH26, a fluorescent dye. Labeled cells retain the ability to proliferate into neurospheres in culture, but labeling prevents neurospheres from merging in a non-adherent culture environment. After labeled NSCs were transplantation into the rat brain, their location and subsequent migration along the corpus callosum was detected using MRI. This study demonstrates an imaging paradigm with which to develop an in vivo assay for quantitatively evaluating fetal neural stem cell migration.

Revised device labeling for the Cepheid Xpert MTB/RIF assay for detecting Mycobacterium tuberculosis.

PubMed

2015-02-27

The Food and Drug Administration (FDA) has cleared the Xpert MTB/RIF Assay (Cepheid; Sunnyvale, California) with an expanded intended use that includes testing of either one or two sputum specimens as an alternative to examination of serial acid-fast stained sputum smears to aid in the decision of whether continued airborne infection isolation (AII) is warranted for patients with suspected pulmonary tuberculosis. This change reflects the outcome of a recent multicenter international study demonstrating that negative Xpert MTB/RIF Assay results from either one or two sputum specimens are highly predictive of the results of two or three negative acid-fast sputum smears.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Label-Free Fluorescence Assay of S1 Nuclease and Hydroxyl Radicals Based on Water-Soluble Conjugated Polymers and WS₂ Nanosheets.

PubMed

Li, Junting; Zhao, Qi; Tang, Yanli

2016-06-13

We developed a new method for detecting S1 nuclease and hydroxyl radicals based on the use of water-soluble conjugated poly[9,9-bis(6,6-(N,N,N-trimethylammonium)-fluorene)-2,7-ylenevinylene-co-alt-2,5-dicyano-1,4-phenylene)] (PFVCN) and tungsten disulfide (WS₂) nanosheets. Cationic PFVCN is used as a signal reporter, and single-layer WS₂ is used as a quencher with a negatively charged surface. The ssDNA forms complexes with PFVCN due to much stronger electrostatic interactions between cationic PFVCN and anionic ssDNA, whereas PFVCN emits yellow fluorescence. When ssDNA is hydrolyzed by S1 nuclease or hydroxyl radicals into small fragments, the interactions between the fragmented DNA and PFVCN become weaker, resulting in PFVCN being adsorbed on the surface of WS₂ and the fluorescence being quenched through fluorescence resonance energy transfer. The new method based on PFVCN and WS₂ can sense S1 nuclease with a low detection limit of 5 × 10(-6) U/mL. Additionally, this method is cost-effective by using affordable WS₂ as an energy acceptor without the need for dye-labeled ssDNA. Furthermore, the method provides a new platform for the nuclease assay and reactive oxygen species, and provides promising applications for drug screening.

A biolayer interferometry-based assay for rapid and highly sensitive detection of biowarfare agents.

PubMed

Mechaly, Adva; Cohen, Hila; Cohen, Ofer; Mazor, Ohad

2016-08-01

Biolayer interferometry (BLI) is an optical technique that uses fiber-optic biosensors for label-free real-time monitoring of protein-protein interactions. In this study, we coupled the advantages of the Octet Red BLI system (automation, fluidics-free, and on-line monitoring) with a signal enhancement step and developed a rapid and sensitive immunological-based method for detection of biowarfare agents. As a proof of concept, we chose to demonstrate the efficacy of this novel assay for the detection of agents representing two classes of biothreats, proteinaceous toxins, and bacterial pathogens: ricin, a lethal plant toxin, and the gram-negative bacterium Francisella tularensis, the causative agent of tularemia. The assay setup consisted of biotinylated antibodies immobilized to the biosensor coupled with alkaline phosphatase-labeled antibodies as the detection moiety to create nonsoluble substrate crystals that precipitate on the sensor surface, thereby inducing a significant wavelength interference. It was found that this BLI-based assay enables sensitive detection of these pathogens (detection limits of 10 pg/ml and 1 × 10(4) pfu/ml ricin and F. tularensis, respectively) within a very short time frame (17 min). Owing to its simplicity, this assay can be easily adapted to detect other analytes in general, and biowarfare agents in particular, in a rapid and sensitive manner. Copyright © 2016 Elsevier Inc. All rights reserved.

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

PubMed Central

Gupta, R C; Randerath, E; Randerath, K

1976-01-01

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units. Images PMID:826884

Application of a Homogenous Assay for the Detection of 2,4,6-Trinitrotoluene to Environmental Water Samples

PubMed Central

Goldman, Ellen R.; Egge, Adrienne L.; Medintz, Igor L.; Lassman, Michael E.; Anderson, George P.

2005-01-01

A homogeneous assay was used to detect 2,4,6-trinitrotoluene (TNT) spiked into environmental water samples. This assay is based on changes in fluorescence emission intensity when TNT competitively displaces a fluorescently labeled, TNT analog bound to an anti-TNT antibody. The effectiveness of the assay was highly dependent on the source of the sample being tested. As no correlation between pH and assay performance was observed, ionic strength was assumed to be the reason for variation in assay results. Addition of 10x phosphate-buffered saline to samples to increase their ionic strength to that of our standard laboratory buffer (about 0.17 M) significantly improved the range over which the assay functioned in several river water samples. PMID:15915298

Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format.

PubMed

Kornegay, J R; Shepard, A P; Hankins, C; Franco, E; Lapointe, N; Richardson, H; Coutleé, F

2001-10-01

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses

Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.

PubMed

Gregorius, Barbara; Jakoby, Thomas; Schaumlöffel, Dirk; Tholey, Andreas

2013-05-21

Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1.

PubMed

Sun, Linlin; Zhao, Qiang

2018-03-01

Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination. Copyright © 2017 Elsevier B.V. All rights reserved.

Reliability of three bulk-tank antimicrobial residue detection assays used to test individual milk samples from cows with mild clinical mastitis.

PubMed

Gibbons-Burgener, S N; Kaneene, J B; Lloyd, J W; Leykam, J F; Erskine, R J

2001-11-01

To determine the likelihood of false-positive results when testing milk samples from individual cows by use of 3 commercially available assays (Penzyme MilkTest and the SNAP beta-lactam and Delvo-SP assays) labeled for use with commingled milk. Milk samples from 111 cows with mild clinical mastitis. Cows were randomly assigned to the control (no antimicrobials) or intramammary treatment group. Posttreatment milk samples were collected at the first milking after the labeled withholding period or an equivalent time for controls, randomly ordered, and tested twice by use of each assay and once by use of high-performance liquid chromatography. Sensitivity, specificity, and positive and negative predictive values were determined for each assay. Concordance of results for the same sample was assessed for each assay by calculating kappa. Sensitivities of the Delvo-SP and SNAP lactam assays were > 90%, whereas the sensitivity of the Penzyme Milk Test was 60%. Positive predictive values (range, 39.29 to 73.68%) were poor for all 3 assays. Concordance of test results was excellent for the SNAP beta-lactam and Delvo-SP assays (kappa = 0.846 and 0.813, respectively) but was less for the Penzyme MilkTest (kappa = 0.545). Because of the low positive predictive values, these 3 assays may not be useful for detecting violative antimicrobial residues in individual milk samples from cows treated for mild clinical mastitis. However, repeatability of each assay was considered good to excellent.

Magneto immunofluorescence assay for diagnosis of celiac disease.

PubMed

Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Fabiano, Silvia N; Pividori, Maria Isabel; Hernandez, Silvia R

2013-10-10

A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit. Copyright © 2013 Elsevier B.V. All rights reserved.

[131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity.

PubMed

Zanzonico, Pat; Koehne, Guenther; Gallardo, Humilidad F; Doubrovin, Mikhail; Doubrovina, Ekaterina; Finn, Ronald; Blasberg, Ronald G; Riviere, Isabelle; O'Reilly, Richard J; Sadelain, Michel; Larson, Steven M

2006-09-01

Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium. Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

PubMed Central

Hyslop, P A; Kuhn, C E; Sauerheber, R D

1984-01-01

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells. PMID:6324752

Surface plasmon resonance imaging for label-free detection of foodborne pathogens and toxins

USDA-ARS?s Scientific Manuscript database

More rapid and efficient detection methods for foodborne pathogenic bacteria and toxins are needed to address the long assay time and limitations in multiplex capacity. Surface plasmon resonance imaging (SPRi) is an emerging optical technique, which allows for rapid and label-free screening of multi...

Fluorescent cellular assay for screening agents inhibiting Pseudomonas aeruginosa adherence.

PubMed

Nosková, Libuše; Kubíčková, Božena; Vašková, Lucie; Bláhová, Barbora; Wimmerová, Michaela; Stiborová, Marie; Hodek, Petr

2015-01-16

Antibodies against Pseudomonas aeruginosa (PA) lectin, PAIIL, which is a virulence factor mediating the bacteria binding to epithelium cells, were prepared in chickens and purified from egg yolks. To examine these antibodies as a prophylactic agent preventing the adhesion of PA we developed a well plate assay based on fluorescently labeled bacteria and immortalized epithelium cell lines derived from normal and cystic fibrosis (CF) human lungs. The antibodies significantly inhibited bacteria adhesion (up to 50%) in both cell lines. In agreement with in vivo data, our plate assay showed higher susceptibility of CF cells towards the PA adhesion as compared to normal epithelium. This finding proved the reliability of the developed experimental system.

A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams

NASA Astrophysics Data System (ADS)

Chen, Yanni; Wang, Yongwei; Liu, Liqiang; Wu, Xiaoling; Xu, Liguang; Kuang, Hua; Li, Aike; Xu, Chuanlai

2015-10-01

A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples.A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively

A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation.

PubMed

Eisenhardt, Nathalie; Chaugule, Viduth K; Pichler, Andrea

2016-01-01

Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.

Gold Nanoparticle Labels and Heterogeneous Immunoassays: The Case for the Inverted Substrate.

PubMed

Crawford, Alexis C; Young, Colin C; Porter, Marc D

2018-06-15

This paper examines how the difference in the spatial orientation of the capture substrate influences the analytical sensitivity and limits of detection for immunoassays that use gold nanoparticle labels (AuNPs) and rely on diffusion in quiet solution in the antigen capture and labeling steps. Ideally, the accumulation of both reactants should follow a dependence governed by the rate in which diffusion delivers reactants to the capture surface. In other words, the accumulation of reactants should increase with the square root of the incubation time, i.e., t1/2. The work herein shows, however, that this expectation is only obeyed when the capture substrate is oriented to direct the gravity-induced sedimentation of the AuNP labels away from the substrate. Using an assay for human IgG, the results show that circumventing the sedimentation of the gold nanoparticle labels by substrate inversion enables the dependence of the labeling step on diffusion, reduces nonspecific label adsorption, and improves the estimated detection limit by ~30×. High-density maps of the signal across the two types of substrates also demonstrate that inversion in the labeling step results in a more uniform distribution of AuNP labels across the surface, which translates to a greater measurement reproducibility. These results, which are supported by model simulations via the Mason-Weaver sedimentation-diffusion equation, and their potential implications when using other nanoparticle labels and related materials in diagnostic tests and other applications, are briefly discussed.

DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

PubMed

Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

2014-10-15

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. Copyright © 2014 Elsevier Inc. All rights reserved.

Protective effect of hexane and ethanol extract of piper longum L. On gentamicin-induced hair cell loss in neonatal cultures.

PubMed

Yadav, Mukesh Kumar; Choi, June; Song, Jae-Jun

2014-03-01

Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures. The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. SPL extract at a concentration of 1 µg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 µg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay. An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.

Combination of anginex gene therapy and radiation decelerates the growth and pulmonary metastasis of human osteosarcoma xenografts.

PubMed

Zhao, Kai; Yang, Shang-You; Geng, Jun; Gong, Xuan; Gong, Weiming; Shen, Lin; Ning, Bin

2018-06-01

Investigate whether rAAV-anginex gene therapy combined with radiotherapy could decrease growth and pulmonary metastasis of osteosarcoma in mice and examine the mechanisms involved in this therapeutic strategy. During in vitro experiment, multiple treatment regimes (rAAV-eGFP, radiotherapy, rAAV-anginex, combination therapy) were applied to determine effects on proliferation of endothelial cells (ECs) and G-292 osteosarcoma cells. During in vivo analysis, the same multiple treatment regimes were applied to osteosarcoma tumor-bearing mice. Use microcomputed tomography to evaluate tumor size. Eight weeks after tumor cell inoculation, immunohistochemistry was used to assess the therapeutic efficacy according to microvessel density (MVD), proliferating cell nuclear antigen (PCNA), and terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. Metastasis of lungs was also evaluated by measuring number of metastatic nodules and wet weight of metastases. The proliferation of ECs and the tumor volumes in combination therapy group were inhibited more effectively than the other three groups at end point (P < 0.05). Cell clone assay showed anginex had radiosensitization effect on ECs. Immunohistochemistry showed tumors from mice treated with combination therapy exhibited the lowest MVD and proliferation rate, with highest apoptosis rate, as confirmed by IHC staining for CD34 and PCNA and TUNEL assays (P < 0.05). Combination therapy also induced the fewest metastatic nodules and lowest wet weights of the lungs (P < 0.05). rAAV-anginex combined with radiotherapy induced apoptosis of osteosarcoma cells and inhibited tumor growth and pulmonary metastasis on the experimental osteosarcoma models. We conclude that the primary mechanism of this process may be due to sensitizing effect of anginex to radiotherapy. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

PubMed

Kitchen, S; Kershaw, G; Tiefenbacher, S

2016-07-01

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. © 2016 John Wiley & Sons Ltd.

In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

PubMed

Kanje, Sara; Hober, Sophia

2015-04-01

Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spaceflight Associated Apoptosis

NASA Technical Reports Server (NTRS)

Ichiki, Albert T.; Gibson, Linda A.; Allebban, Zuhair

1996-01-01

Lymphoid tissues have been shown to atrophy in rats flown on Russian spaceflights. Histological examination indicated evidence for cell degradation. Lymphoid tissues from rats flown on Spacelab Life Sciences-2 mission were analyzed for apoptosis by evidence of fragmented lymphocytes, which could be engulfed by macrophages, or DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Apoptosis was not detected in the thymus and spleen collected inflight or from the synchronous ground rats but was detected in the thymus, spleen and inguinal lymph node of the flight animals on recovery. These results indicate that the apoptosis observed in the lymphatic tissues of the rats on recovery could have been induced by the gravitational stress of reentry, corroborating the findings from the early space-flight observations.

Age-related effects of dexamethasone administration in adrenal zona reticularis.

PubMed

Almeida, Henrique; Matos, Liliana; Ferreira, Jorge; Neves, Delminda

2006-05-01

Suppression of adrenocorticotropic hormone results in reduced adrenal steroid output, adrenocortical cell atrophy, and apoptosis in young rats. To verify such effects during aging, dexamethasone was injected into rats for 3 days at five different ages; at day 4, adrenals and blood were collected for morphologic and corticosterone assay. Adrenal structure was similar at all ages, but in dexamethasone-injected animals there were ultrastructural features of apoptosis and a higher percentage of TUNEL and caspase-3-labeled nuclei and cytoplasm; their corticosterone decreased significantly. In both groups, there was age-related decrease in the percentage of apoptotic cells, significant only in dexamethasone-injected rats. The data suggest that aged adrenocortical cells are less susceptible to the lack of adrenocorticotropic hormone (ACTH), possibly as a result of their decreased functional ability.

Nanocolloidal gold-based immuno-dip strip assay for rapid detection of Sudan red I in food samples.

PubMed

Wang, Jia; Wang, Zhanhui; Liu, Jing; Li, Hao; Li, Qing X; Li, Ji; Xu, Ting

2013-02-15

A semiquantitative dip strip assay was developed using nanocolloidal gold-labelled monoclonal antibody (Mab) 8A10 for the rapid detection of Sudan red I in food samples. A protein-Sudan red I conjugate was coated on a nitro cellulose membrane strip in a defined test line. In flow of the complex of nanocolloidal gold labelled-Mab and Sudan red I along the strip, intensive red colour that was formed in the test line reflected the Sudan red I concentration. The test required 10 min and had a visual limit of detection of 10 ng/g Sudan red I in tomato sauce and chilli powder samples. The results of the strip assay agreed well with those of a high performance liquid chromatography method for both spiked and real commercial samples. The strip was stable for at least 2 months at 4°C. The strip assay offers the potential as a useful rapid and simple method for screening of Sudan red I in food samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

PubMed

Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H

2015-09-22

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Direct fluorescent labeling for efficient biological assessment of inhalable particles.

PubMed

Poudel, Bijay Kumar; Park, Jae Hong; Lim, Jiseok; Byeon, Jeong Hoon

2017-10-01

Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

PubMed

Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa

2010-03-01

Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

PubMed Central

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-01-01

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. PMID:28835375

A proteomics assay to detect eight CBRN-relevant toxins in food.

PubMed

Gilquin, Benoit; Jaquinod, Michel; Louwagie, Mathilde; Kieffer-Jaquinod, Sylvie; Kraut, Alexandra; Ferro, Myriam; Becher, François; Brun, Virginie

2017-01-01

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Behaviour of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labelling for magnetic resonance imaging.

PubMed

Kolecka, Malgorzata Anna; Arnhold, Stefan; Schmidt, Martin; Reich, Christine; Kramer, Martin; Failing, Klaus; von Pückler, Kerstin

2017-02-24

Therapy with mesenchymal stem cells (MSCs) has been reported to provide beneficial effects in the treatment of neurological and orthopaedic disorders in dogs. The exact mechanism of action is poorly understood. Magnetic resonance imaging (MRI) gives the opportunity to observe MSCs after clinical administration. To visualise MSCs with the help of MRI, labelling with an MRI contrast agent is necessary. However, it must be clarified whether there is any negative influence on cell function and viability after labelling prior to clinical administration. For the purpose of the study, seven samples with canine adipose-derived stem cells were incubated with superparamagnetic iron oxide nanoparticles (SPIO: 319.2 μg/mL Fe) for 24 h. The internalisation of the iron particles occurred via endocytosis. SPIO particles were localized as free clusters in the cytoplasm or within lysosomes depending on the time of investigation. The efficiency of the labelling was investigated using Prussian blue staining and MACS assay. After 3 weeks the percentage of SPIO labelled canine stem cells decreased. Phalloidin staining showed no negative effect on the cytoskeleton. Labelled cells underwent osteogenic and adipogenic differentiation. Chondrogenic differentiation occurred to a lesser extent compared with a control sample. MTT-Test and wound healing assay showed no influence of labelling on the proliferation. The duration of SPIO labelling was assessed using a 1 Tesla clinical MRI scanner and T2 weighted turbo spin echo and T2 weighted gradient echo MRI sequences 1, 2 and 3 weeks after labelling. The hypointensity caused by SPIO lasted for 3 weeks in both sequences. An Endorem labelling concentration of 319.2 μg/mL Fe (448 μg/mL SPIO) had no adverse effects on the viability of canine ASCs. Therefore, this contrast agent could be used as a model for iron oxide labelling agents. However, the tracking ability in vivo has to be evaluated in further studies.

Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

PubMed Central

2013-01-01

Background Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. Results In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. Conclusions Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process. PMID:23835323

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

Flow-aggregated traffic-driven label mapping in label-switching networks

NASA Astrophysics Data System (ADS)

Nagami, Kenichi; Katsube, Yasuhiro; Esaki, Hiroshi; Nakamura, Osamu

1998-12-01

Label switching technology enables high performance, flexible, layer-3 packet forwarding based on the fixed length label information mapped to the layer-3 packet stream. A Label Switching Router (LSR) forwards layer-3 packets based on their label information mapped to the layer-3 address information as well as their layer-3 address information. This paper evaluates the required number of labels under traffic-driven label mapping policy using the real backbone traffic traces. The evaluation shows that the label mapping policy requires a large number of labels. In order to reduce the required number of labels, we propose a label mapping policy which is a traffic-driven label mapping for the traffic toward the same destination network. The evaluation shows that the proposed label mapping policy requires only about one tenth as many labels compared with the traffic-driven label mapping for the host-pair packet stream,and the topology-driven label mapping for the destination network packet stream.

The effects of Crataegus aronia var. dentata Browicz extract on biochemical indices and apoptosis in partially hepatectomized liver in rats.

PubMed

Keskin, Nazan; Mammadov, Ramazan; Ili, Pinar

2012-08-01

Crataegus species have been widely used in herbal medicine, especially for the hearth diseases. In the present study, the effect of Crataegus aronia var. dentata Browicz extract on partially hepatectomized rats was investigated with biochemical and TUNEL apoptosis assays. The extracts of the plant at the concentrations of 0.5 and 1 ml/100 g body weight/day were administered orally to the two experimental groups including partially hepatectomized rats for 42 days. At the end of the experimental period, animals were sacrificed, blood was collected for the assessment of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT), and the liver tissue was used for TUNEL assay. In biochemical assay, it was found a significant decrease in the levels of serum ALT and AST in the experimental groups. On the other hand, the plant extract did not cause any significant changes in the level of GGT in these groups. In apoptosis assay, TUNEL positive hepatocytes could not be detected in both experimental groups. The present findings can suggest that Crataegus aronia var. dentata Browicz extract can decrease the levels of serum ALT and AST and play a role in apoptosis of hepatocytes in the liver of partially hepatectomized rats. However, further studies are required to confirm the effects of the plant extract on hepatoprotection and apoptosis in the regenerating liver after partial hepatectomy in animal models.

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Guitot, Karine; Scarabelli, Silvia; Drujon, Thierry; Bolbach, Gérard; Amoura, Mehdi; Burlina, Fabienne; Jeltsch, Albert; Sagan, Sandrine; Guianvarc'h, Dominique

2014-07-01

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

Nutrition Label Viewing during a Food-Selection Task: Front-of-Package Labels vs Nutrition Facts Labels.

PubMed

Graham, Dan J; Heidrick, Charles; Hodgin, Katie

2015-10-01

Earlier research has identified consumer characteristics associated with viewing Nutrition Facts labels; however, little is known about those who view front-of-package nutrition labels. Front-of-package nutrition labels might appeal to more consumers than do Nutrition Facts labels, but it might be necessary to provide consumers with information about how to locate and use these labels. This study quantifies Nutrition Facts and front-of-package nutrition label viewing among American adult consumers. Attention to nutrition information was measured during a food-selection task. One hundred and twenty-three parents (mean age=38 years, mean body mass index [calculated as kg/m(2)]=28) and one of their children (aged 6 to 9 years) selected six foods from a university laboratory-turned-grocery aisle. Participants were randomized to conditions in which front-of-package nutrition labels were present or absent, and signage explaining front-of-package nutrition labels was present or absent. Adults' visual attention to Nutrition Facts labels and front-of-package nutrition labels was objectively measured via eye-tracking glasses. To examine whether there were significant differences in the percentages of participants who viewed Nutrition Facts labels vs front-of-package nutrition labels, McNemar's tests were conducted across all participants, as well as within various sociodemographic categories. To determine whether hypothesized factors, such as health literacy and education, had stronger relationships with front-of-package nutrition label vs Nutrition Facts label viewing, linear regression assessed the magnitude of relationships between theoretically and empirically derived factors and each type of label viewing. Overall, front-of-package nutrition labels were more likely to be viewed than Nutrition Facts labels; however, for all subgroups, higher rates of front-of-package nutrition label viewership occurred only when signage was present drawing attention to the presence and

Influence of Light Emitting Diode-Derived Blue Light Overexposure on Mouse Ocular Surface.

PubMed

Lee, Hyo Seok; Cui, Lian; Li, Ying; Choi, Ji Suk; Choi, Joo-Hee; Li, Zhengri; Kim, Ga Eon; Choi, Won; Yoon, Kyung Chul

2016-01-01

To investigate the influence of overexposure to light emitting diode (LED)-derived light with various wavelengths on mouse ocular surface. LEDs with various wavelengths were used to irradiate C57BL/6 mice at an energy dose of 50 J/cm2, twice a day, for 10 consecutive days. The red, green, and blue groups represented wavelengths of 630 nm, 525 nm, and 410 nm, respectively. The untouched group (UT) was not exposed to LED light and served as the untreated control. Tear volume, tear film break-up time (TBUT), and corneal fluorescein staining scores were measured on days 1, 3, 5, 7, and 10. Levels of interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were measured in the cornea and conjunctiva using a multiplex immunobead assay at day 10. Levels of malondialdehyde (MDA) were measured with an enzyme-linked immunosorbent assay. Flow cytometry, 2'7'-dichlorofluorescein diacetate (DCF-DA) assay, histologic analysis, immunohistochemistry with 4-hydroxynonenal, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining were also performed. TBUT of the blue group showed significant decreases at days 7 and 10, compared with the UT and red groups. Corneal fluorescein staining scores significantly increased in the blue group when compared with UT, red, and green groups at days 5, 7, and 10. A significant increase in the corneal levels of IL-1β and IL-6 was observed in the blue group, compared with the other groups. The blue group showed significantly increased reactive oxygen species production in the DCF-DA assay and increased inflammatory T cells in the flow cytometry. A significantly increased TUNEL positive cells was identified in the blue group. Overexposure to blue light with short wavelengths can induce oxidative damage and apoptosis to the cornea, which may manifest as increased ocular surface inflammation and resultant dry eye.

Development of a miRNA surface-enhanced Raman scattering assay using benchtop and handheld Raman systems

NASA Astrophysics Data System (ADS)

Schechinger, Monika; Marks, Haley; Locke, Andrea; Choudhury, Mahua; Cote, Gerard

2018-01-01

DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

PubMed

Magiati, Maria; Sevastou, Areti; Kalogianni, Despina P

2018-06-04

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

Co-Labeling for Multi-View Weakly Labeled Learning.

PubMed

Xu, Xinxing; Li, Wen; Xu, Dong; Tsang, Ivor W

2016-06-01

It is often expensive and time consuming to collect labeled training samples in many real-world applications. To reduce human effort on annotating training samples, many machine learning techniques (e.g., semi-supervised learning (SSL), multi-instance learning (MIL), etc.) have been studied to exploit weakly labeled training samples. Meanwhile, when the training data is represented with multiple types of features, many multi-view learning methods have shown that classifiers trained on different views can help each other to better utilize the unlabeled training samples for the SSL task. In this paper, we study a new learning problem called multi-view weakly labeled learning, in which we aim to develop a unified approach to learn robust classifiers by effectively utilizing different types of weakly labeled multi-view data from a broad range of tasks including SSL, MIL and relative outlier detection (ROD). We propose an effective approach called co-labeling to solve the multi-view weakly labeled learning problem. Specifically, we model the learning problem on each view as a weakly labeled learning problem, which aims to learn an optimal classifier from a set of pseudo-label vectors generated by using the classifiers trained from other views. Unlike traditional co-training approaches using a single pseudo-label vector for training each classifier, our co-labeling approach explores different strategies to utilize the predictions from different views, biases and iterations for generating the pseudo-label vectors, making our approach more robust for real-world applications. Moreover, to further improve the weakly labeled learning on each view, we also exploit the inherent group structure in the pseudo-label vectors generated from different strategies, which leads to a new multi-layer multiple kernel learning problem. Promising results for text-based image retrieval on the NUS-WIDE dataset as well as news classification and text categorization on several real-world multi

Development of a Highly Automated and Multiplexed Targeted Proteome Pipeline and Assay for 112 Rat Brain Synaptic Proteins

PubMed Central

Colangelo, Christopher M.; Ivosev, Gordana; Chung, Lisa; Abbott, Thomas; Shifman, Mark; Sakaue, Fumika; Cox, David; Kitchen, Rob R.; Burton, Lyle; Tate, Stephen A; Gulcicek, Erol; Bonner, Ron; Rinehart, Jesse; Nairn, Angus C.; Williams, Kenneth R.

2015-01-01

We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay. PMID:25476245

Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification

PubMed Central

Zhi, Wenbo; Wang, Meiyao

2014-01-01

The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrated the feasibility of using the selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG) have good linear ranges, generally 103 – 104. There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R2 is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins at 0.2 – 2 fmol level, which are comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R2 = 0.928 and 0.851 respectively). The correlation between SRM/MRM and ELISA for CP is less desirable (R2 = 0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides (R2 = 0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or Luminex beads are not available. PMID:21594933

Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells.

PubMed

Park, Ki-Soo; Tae, Jinsung; Choi, Bongkum; Kim, Young-Seok; Moon, Cheol; Kim, Sa-Hyun; Lee, Han-Sin; Kim, Jinhyun; Kim, Junsung; Park, Jaeberm; Lee, Jung-Hee; Lee, Jong Eun; Joh, Jae-Won; Kim, Sungjoo

2010-04-01

Live imaging is a powerful technique that can be used to characterize the fate and location of stem cells in animal models. Here we investigated the characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, MNPs@SiO2(RITC). We also conducted various in vivo-uptake tests with nanoparticle-labeled human MSCs. MNPs@SiO2(RITC) showed photostability against ultraviolet light exposure and were nontoxic to human MSCs, based on the MTT, apoptosis, and cell cycle arrest assays. In addition, MNPs@SiO2(RITC) did not affect the surface phenotype or morphology of human MSCs. We also demonstrated that MNPs@SiO2(RITC) have stable retention properties in MSCs in vitro. Furthermore, using optical and magnetic resonance imaging, we successfully detected a visible signal from labeled human MSCs that were transplanted into NOD.CB17-Prkdc(SCID) (NOD-SCID) mice. These results demonstrate that MNPs@SiO2(RITC) are biocompatible and useful tools for human MSC labeling and bioimaging. The characteristics and in vitro cytotoxicity of human mesenchymal stem cells (MSCs) labeled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate, RITC were investigated in this study. RITC showed photostability against ultraviolet light exposure and was nontoxic to human MSCs. Using both optical and magnetic resonance imaging, successful detection of signal from labeled human MSCs transplanted into mice is demonstrated. Copyright 2010 Elsevier Inc. All rights reserved.

Cytotoxic effect of commercially available methylprednisolone acetate with and without reduced preservatives on dorsal root ganglion sensory neurons in rats.

PubMed

Knezevic, Nebojsa Nick; Candido, Kenneth D; Cokic, Ivan; Krbanjevic, Aleksandar; Berth, Sarah L; Knezevic, Ivana

2014-01-01

Epidural and intrathecal injections of methylprednisolone acetate (MPA) have become the most commonly performed interventional procedures in the United States and worldwide in the last 2 decades. However neuraxial MPA injection has been dogged by controversy regarding the presence of different additives used in commercially prepared glucocorticoids. We previously showed that MPA could be rendered 85% free of polyethylene glycol (PEG) by a simple physical separation of elements in the suspension. The objective of the present study was to explore a possible cytotoxic effect of commercially available MPA (with intact or reduced preservatives) on rat sensory neurons. We exposed primary dissociated rat dorsal root ganglia (DRG) sensory neurons to commercially available MPA for 24 hours with either the standard (commercial) concentration of preservatives or to different fractions following separation (MPA suspension whose preservative concentration had been reduced, or fractions containing higher concentrations of preservatives). Cells were stained with the TUNEL assay kit to detect apoptotic cells and images were taken on the Bio-Rad Laser Sharp-2000 system. We also detected expression of caspase-3, as an indicator of apoptosis in cell lysates. We exposed sensory neurons from rat DRG to different concentrations of MPA from the original commercially prepared vial. TUNEL assay showed dose-related responses and increased percentages of apoptotic cells with increasing concentrations of MPA. Increased concentrations of MPA caused 1.5 - 2 times higher caspase-3 expression in DRG sensory neurons than in control cells (ANOVA, P = 0.001). Our results showed that MPA with reduced preservatives caused significantly less apoptosis observed with TUNEL assay labeling (P < 0.001) and caspase-3 immunoblotting (P = 0.001) than in neurons exposed to MPA from a commercially prepared vial or "clear phase" that contained higher concentrations of preservatives. Even though MPA with reduced

17alpha-methyltestosterone: 28-day oral toxicity study in the rat based on the "Enhanced OECD Test Guideline 407" to detect endocrine effects.

PubMed

Wason, Sheila; Pohlmeyer-Esch, Gabriele; Pallen, Catherine; Palazzi, Xavier; Espuña, Gemma; Bars, Remi

2003-11-05

A 28-day oral gavage toxicity study in the rat with 17alpha-methyltestosterone was conducted as part of the international validation exercise on the modified Enhanced OECD Test Guideline 407 (Organisation for Economic Co-operation and Development, Paris). Special emphasis was placed on the endocrine mediated effects exerted by 17alpha-methyltestosterone, a potent androgen agonist. The test compound was administered daily by oral gavage for at least 28 days to groups of 7-week-old-Wistar rats. Dose levels were 0, 10, 40 and 200 mg/kg body weight per day for males and 0, 10, 100 and 600 mg/kg body weight per day for females. In addition, and outside the remit of the enhanced protocol, testosterone levels in males, oestradiol levels in females and luteinizing hormone (LH) levels in both sexes were measured, to provide a broader profile on the hormonally mediated effects of 17alpha-methyltestosterone. Furthermore, stage-specific quantification of Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL)-labeled germ cells (apoptotic germ cells) in the seminiferous tubules was also performed, in an effort to demonstrate the precise stages in the spermatogenic cycle 17alpha-methyltestosterone exerts its effect. In this study, the most critical additional parameters contained in the Enhanced OECD Test Guideline 407 for the detection of endocrine disruption were considered to be the histopathological assessment and organ weight data of endocrine-related tissues. Beyond the scope of this validation exercise, an increase in apoptosis in specific germ cell types was detected using the TUNEL assay in male rats treated at 200 and 40 mg/kg.

Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

NASA Astrophysics Data System (ADS)

Zhou, Huijuan; Wu, Baoyan

2008-12-01

The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample.

PubMed

Sharma, Atul; Catanante, Gaëlle; Hayat, Akhtar; Istamboulie, Georges; Ben Rejeb, Ines; Bhand, Sunil; Marty, Jean Louis

2016-09-01

The discovery of in-vitro systematic evolution of ligands by exponential enrichment (SELEX) process has considerably broaden the utility of aptamer as bio-recognition element, providing the high binding affinity and specificity against the target analytes. Recent research has focused on the development of structure switching signaling aptamer assay, transducing the aptamer- target recognition event into an easily detectable signal. In this paper, we demonstrate the development of structure switching aptamer assay for determination of aflatoxin M1 (AFM1) employing the quenching-dequenching mechanism. Hybridization of fluorescein labelled anti-AFM1 aptamer (F-aptamer) with TAMRA labelled complementary sequences (Q-aptamer) brings the fluorophore and the quencher into close proximity, which results in maximum fluorescence quenching. On addition of AFM1, the target induced conformational formation of antiparallel G-quadruplex aptamer-AFM1 complex results in fluorescence recovery. Under optimized experimental conditions, the developed method showed the good linearity with limit of detection (LOD) at 5.0ngkg(-1) for AFM1. The specificity of the sensing platform was carefully investigated against aflatoxin B1 (AFB1) and ochratoxin A (OTA). The developed assay platform showed the high specificity towards AFM1. The practical application of the developed aptamer assay was verified for detection of AFM1 in spiked milk samples. Good recoveries were obtained in the range from 94.40% to 95.28% (n=3) from AFM1 spiked milk sample. Copyright © 2016. Published by Elsevier B.V.

Insulin-like growth factor-1 (IGF-1) promotes primordial follicle growth and reduces DNA fragmentation through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signalling pathway.

PubMed

Bezerra, Maria É S; Barberino, Ricássio S; Menezes, Vanúzia G; Gouveia, Bruna B; Macedo, Taís J S; Santos, Jamile M S; Monte, Alane P O; Barros, Vanessa R P; Matos, Maria H T

2018-05-30

We investigated the effects of insulin-like growth factor 1 (IGF-1) on the morphology and follicular activation of ovine preantral follicles cultured in situ and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway is involved in IGF-1 action in the sheep ovary. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) analyses (fresh control) or cultured in supplemented alpha-minimum essential medium (α-MEM+; control) or α-MEM+ with IGF-1 (1, 10, 50, 100 or 200ngmL-1) for 7 days. Follicles were classified as normal or atretic, primordial or growing and the oocyte and follicle diameters were measured. DNA fragmentation was evaluated by TUNEL assay. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed on the fresh control, α-MEM+ and 100ngmL-1 IGF-1 samples. Inhibition of PI3K activity was performed through pretreatment with the PI3K inhibitor LY294002 and phosphorylated AKT (pAKT) expression was analysed after culture in the absence or presence of LY294002. IGF-1 at 100ngmL-1 increased (P<0.05) follicular activation compared with α-MEM+ and decreased TUNEL-positive cells (P<0.05) compared with other treatments. PCNA-positive cells also increased (P<0.05) in 100ngmL-1 IGF-1. LY294002 significantly inhibited follicular activation stimulated by α-MEM+ and 100ngmL-1 IGF-1 and reduced pAKT expression in follicles. Overall, IGF-1 at 100ngmL-1 promoted primordial follicle activation, cell proliferation and reduced DNA fragmentation after in situ culture through the PI3K/AKT pathway.

Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine

PubMed Central

Vyas, Dinesh; Robertson, Charles M; Stromberg, Paul E; Martin, James R.; Dunne, W. Michael; Houchen, Courtney W; Barrett, Terrence A; Ayala, Alfred; Perl, Mario; Buchman, Timothy G; Coopersmith, Craig M

2007-01-01

Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death. PMID:17357092

Increased Dickkopf-1 expression accelerates bone cell apoptosis in femoral head osteonecrosis.

PubMed

Ko, Jih-Yang; Wang, Feng-Sheng; Wang, Ching-Jen; Wong, To; Chou, Wen-Yi; Tseng, Shin-Ling

2010-03-01

Intensive bone cell apoptosis contributes to osteonecrosis of femoral head (ONFH). Dickkopf-1 (DKK1) reportedly mediates various types of skeletal disorders. This study investigated whether DKK1 was linked to the occurrence of ONFH. Thirty-nine patients with various stages of ONFH were recruited. Bone specimens were harvested from 34 ONFH patients underwent hip arthroplasty, and from 10 femoral neck fracture patients. Bad, Bcl2 TNFalpha, DKK1, Wnt3a, LRP5, and Axin1 expressions were analyzed by quantitative RT-PCR and ELISA. Apoptotic cells were assayed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL). Primary bone-marrow mesenchymal cells were treated with DKK1 RNA interference and recombinant DKK1 protein. ONFH patients with the histories of being administrated corticosteroids and excessive alcohol consumption had significantly higher Bad and DKK1 mRNA expressions in bone tissue and DKK1 abundances in serum than femoral neck fracture patients. Bone cells adjacent to osteonecrotic bone displayed strong DKK1 immunoreactivity and TUNEL staining. Increased DKK1 expression in bone tissue and serum correlated with Bad expression and TUNEL staining. Serum DKK1 abundance correlated with the severity of ONFH. The DKK1 RNA interference and recombinant DKK1 protein regulated Bad expression and apoptosis of primary bone-marrow mesenchymal cells. Knock down of DKK1 reduced dexamethasone-induced apoptosis of mesenchymal cells. Taken together, promoted DKK1 expression was associated with bone cell apoptosis in the occurrence of ONFH patients with the histories of corticosteroid and alcohol intake and progression of ONFH. DKK1 expression in injured tissue provides new insight into ONFH pathogenesis.

A Quick and Parallel Analytical Method Based on Quantum Dots Labeling for ToRCH-Related Antibodies

NASA Astrophysics Data System (ADS)

Yang, Hao; Guo, Qing; He, Rong; Li, Ding; Zhang, Xueqing; Bao, Chenchen; Hu, Hengyao; Cui, Daxiang

2009-12-01

Quantum dot is a special kind of nanomaterial composed of periodic groups of II-VI, III-V or IV-VI materials. Their high quantum yield, broad absorption with narrow photoluminescence spectra and high resistance to photobleaching, make them become a promising labeling substance in biological analysis. Here, we report a quick and parallel analytical method based on quantum dots for ToRCH-related antibodies including Toxoplasma gondii, Rubella virus, Cytomegalovirus and Herpes simplex virus type 1 (HSV1) and 2 (HSV2). Firstly, we fabricated the microarrays with the five kinds of ToRCH-related antigens and used CdTe quantum dots to label secondary antibody and then analyzed 100 specimens of randomly selected clinical sera from obstetric outpatients. The currently prevalent enzyme-linked immunosorbent assay (ELISA) kits were considered as “golden standard” for comparison. The results show that the quantum dots labeling-based ToRCH microarrays have comparable sensitivity and specificity with ELISA. Besides, the microarrays hold distinct advantages over ELISA test format in detection time, cost, operation and signal stability. Validated by the clinical assay, our quantum dots-based ToRCH microarrays have great potential in the detection of ToRCH-related pathogens.

Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

PubMed

Cappione, Amedeo; Mabuchi, Masaharu; Briggs, David; Nadler, Timothy

2015-04-01

Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. Copyright © 2015 Elsevier B.V. All rights reserved.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

PubMed

Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

2009-01-01

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

PubMed Central

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-01-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

Growth inhibition of malignant melanoma by intermediate frequency alternating electric fields, and the underlying mechanisms.

PubMed

Chen, H; Liu, R; Liu, J; Tang, J

2012-01-01

This study investigated the antitumour effects of intermediate frequency alternating electric fields (IF-AEF) in a murine melanoma cell line (B16F10) and a mouse tumour model. IF-AEF was applied at 100 kHz. Proliferation of B16F10 cells in vitro was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. IF-AEF was applied in vivo to mice bearing B16F10 tumours. Terminal deoxy nucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay for apoptosis, and immunohistochemical detection of CD34 and vascular endothelial growth factor (VEGF), were performed. IF-AEF inhibited the proliferation of B16F10 cells in an electrical intensity and time-dependent manner. Treatment with IF-AEF for 7 days significantly inhibited the growth of tumours compared with untreated controls. IF-AEF induced apoptosis in vivo and reduced CD34-positive cell numbers; CD34 is a special marker of microvessel density. IF-AEF reduced microvessel density related to tumour growth and may serve as a therapeutic strategy for cancer treatment.

Decursin from Angelica gigas Nakai Inhibits B16F10 Melanoma Growth Through Induction of Apoptosis

PubMed Central

Kim, Byung Soo; Seo, Hyobin; Kim, Ha-Jeong; Bae, Sang Mun; Son, Hye-Nam; Lee, Yoon Jeong; Ryu, Sungpil; Park, Rang-Woon; Nam, Ju-Ock

2015-01-01

Abstract Decursin, a bioactive phytochemical isolated from Angelica gigas Nakai (danggwi), has shown preclinical anticancer efficacy in various cancer models. However, the antitumor effect of decursin in melanoma models remains undefined. The antitumor activities of decursin were investigated in B16F10 cells in vitro and in vivo. In this study, we show that treatment with decursin inhibited cell proliferation in a dose-dependent manner in B16F10 cells, but not in normal cells. Decursin also induced apoptosis in B16F10 cells, as determined by annexin V-staining assay and transferase-mediated nick-end labeling (TUNEL) staining assay. Decursin increased the phosphorylation of p38 as well as the expression of Bax while decreasing the phosphorylation of extracellular signaling-regulated kinase (ERK) and the expression of Bcl-2 in B16F10 cells. Moreover, decursin activated caspase-3 in B16F10 cells and xenograft tumor tissue. Together, these findings support further investigations into the potential use of decursin in the treatment of melanoma cells. PMID:26336081

Decursin from Angelica gigas Nakai Inhibits B16F10 Melanoma Growth Through Induction of Apoptosis.

PubMed

Kim, Byung Soo; Seo, Hyobin; Kim, Ha-Jeong; Bae, Sang Mun; Son, Hye-Nam; Lee, Yoon Jeong; Ryu, Sungpil; Park, Rang-Woon; Nam, Ju-Ock

2015-10-01

Decursin, a bioactive phytochemical isolated from Angelica gigas Nakai (danggwi), has shown preclinical anticancer efficacy in various cancer models. However, the antitumor effect of decursin in melanoma models remains undefined. The antitumor activities of decursin were investigated in B16F10 cells in vitro and in vivo. In this study, we show that treatment with decursin inhibited cell proliferation in a dose-dependent manner in B16F10 cells, but not in normal cells. Decursin also induced apoptosis in B16F10 cells, as determined by annexin V-staining assay and transferase-mediated nick-end labeling (TUNEL) staining assay. Decursin increased the phosphorylation of p38 as well as the expression of Bax while decreasing the phosphorylation of extracellular signaling-regulated kinase (ERK) and the expression of Bcl-2 in B16F10 cells. Moreover, decursin activated caspase-3 in B16F10 cells and xenograft tumor tissue. Together, these findings support further investigations into the potential use of decursin in the treatment of melanoma cells.

Label Review Training: Module 1: Label Basics, Page 21

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about types of labels.

Label Review Training: Module 1: Label Basics, Page 20

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section focuses on supplemental labeling.

Label Review Training: Module 1: Label Basics, Page 22

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about what labels require review.

Label Review Training: Module 1: Label Basics, Page 19

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section covers supplemental distributor labeling.

Label Review Training: Module 1: Label Basics, Page 18

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section discusses the types of labels.

Label Review Training: Module 1: Label Basics, Page 26

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about mandatory and advisory label statements.

Label Review Training: Module 1: Label Basics, Page 15

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the consequences of improper labeling.

Label Review Training: Module 1: Label Basics, Page 14

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about positive effects from proper labeling.

Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

PubMed Central

van Loon, A. M.; van der Logt, J. T.; Heessen, F. W.; Heeren, M. C.; Zoll, J.

1992-01-01

Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus. PMID:1312479

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

NASA Astrophysics Data System (ADS)

Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

2006-01-01

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

PubMed

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Label Review Training: Module 1: Label Basics, Page 24

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is about which labels require review.

Label Review Training: Module 1: Label Basics, Page 17

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See an overview of the importance of labels.

Label Review Training: Module 1: Label Basics, Page 27

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See examples of mandatory and advisory label statements.

Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

PubMed

Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

2016-04-01

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.

Label Review Training: Module 1: Label Basics, Page 23

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Lists types of labels that do not require review.

Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

NASA Astrophysics Data System (ADS)

Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

2016-02-01

Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

Assaying Ornithine and Arginine Decarboxylases in Some Plant Species 1

PubMed Central

Birecka, Helena; Bitonti, Alan J.; McCann, Peter P.

1985-01-01

A release of 14CO2 not related to ornithine decarboxylase activity was found in crude leaf extracts from Lycopersicon esculentum, Avena sativa, and especially from the pyrrolizidine alkaloid-bearing Heliotropium angiospermum when incubated with [1-14C]- or [U-14C]ornithine. The total 14CO2 produced was about 5- to 100-fold higher than that due to ornithine decarboxylase activities calculated from labeled putrescine (Put) found by thin-layer electrophoresis in the incubation mixtures. Partial purification with (NH4)2SO4 did not eliminate completely the interfering decarboxylation. When incubated with labeled arginine, a very significant 14CO2 release not related to arginine decarboxylase activity was observed only in extracts from H. angiospermum leaves, especially in Tris·HCl buffer. Under the assay conditions, these extracts exhibited oxidative degradation of added Put and agmatine (Agm) and also revealed a high arginase activity. Amino-guanidine at 0.1 to 0.2 millimolar prevented Put degradation and greatly decreased oxidative degradation of Agm; ornithine at 15 to 20 millimolar significantly inhibited arginase activity. A verification of the reliability of the standard 14CO2-based method by assessing labeled Put and/or Agm—formed in the presence of added aminoguanidine and/or ornithine when needed—is recommended especially when crude or semicrude plant extracts are assayed. When based on Put and/or Agm formed at 1.0 to 2.5 millimolar of substrate, the activities of ornithine decarboxylase and arginine decarboxylase in the youngest leaves of the tested species ranged between 1.1 and 3.6 and 1 and 1600 nanomoles per hour per gram fresh weight, respectively. The enzyme activities are discussed in relation to the biosynthesis of pyrrolizidine alkaloids. PMID:16664441

Label Review Training: Module 1: Label Basics, Page 16

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the importance of labels and the role in enforcement.

A Comparison of Exogenous Labels for the Histological Identification of Transplanted Neural Stem Cells

PubMed Central

Nicholls, Francesca J.; Liu, Jessie R.; Modo, Michel

2017-01-01

The interpretation of cell transplantation experiments is often dependent on the presence of an exogenous label for the identification of implanted cells. The exogenous labels Hoechst 33342, 5-bromo-2′-deoxyuridine (BrdU), PKH26, and Qtracker were compared for their labeling efficiency, cellular effects, and reliability to identify a human neural stem cell (hNSC) line implanted intracerebrally into the rat brain. Hoechst 33342 (2 mg/ml) exhibited a delayed cytotoxicity that killed all cells within 7 days. This label was hence not progressed to in vivo studies. PKH26 (5 μM), Qtracker (15 nM), and BrdU (0.2 μM) labeled 100% of the cell population at day 1, although BrdU labeling declined by day 7. BrdU and Qtracker exerted effects on proliferation and differentiation. PKH26 reduced viability and proliferation at day 1, but this normalized by day 7. In an in vitro coculture assay, all labels transferred to unlabeled cells. After transplantation, the reliability of exogenous labels was assessed against the gold standard of a human-specific nuclear antigen (HNA) antibody. BrdU, PKH26, and Qtracker resulted in a very small proportion (<2%) of false positives, but a significant amount of false negatives (~30%), with little change between 1 and 7 days. Exogenous labels can therefore be reliable to identify transplanted cells without exerting major cellular effects, but validation is required. The interpretation of cell transplantation experiments should be presented in the context of the label's limitations. PMID:27938486

A real-time high-throughput fluorescence assay for sphingosine kinases

PubMed Central

Lima, Santiago; Milstien, Sheldon; Spiegel, Sarah

2014-01-01

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format. PMID:24792926

Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

PubMed

Borrebaeck, C; Börjeson, J; Mattiasson, B

1978-06-15

Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

Early steps of supported bilayer formation probed by single vesicle fluorescence assays.

PubMed Central

Johnson, Joseph M; Ha, Taekjip; Chu, Steve; Boxer, Steven G

2002-01-01

We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles. PMID:12496104

Development of a miRNA surface-enhanced Raman scattering assay using benchtop and handheld Raman systems.

PubMed

Schechinger, Monika; Marks, Haley; Locke, Andrea; Choudhury, Mahua; Cote, Gerard

2018-01-01

DNA-functionalized nanoparticles, when paired with surface-enhanced Raman spectroscopy (SERS), can rapidly detect microRNA. However, widespread use of this approach is hindered by drawbacks associated with large and expensive benchtop Raman microscopes. MicroRNA-17 (miRNA-17) has emerged as a potential epigenetic indicator of preeclampsia, a condition that occurs during pregnancy. Biomarker detection using an SERS point-of-care device could enable prompt diagnosis and prevention as early as the first trimester. Recently, strides have been made in developing portable Raman systems for field applications. An SERS assay for miRNA-17 was assessed and translated from traditional benchtop Raman microscopes to a handheld system. Three different photoactive molecules were compared as potential Raman reporter molecules: a chromophore, malachite green isothiocyanate (MGITC), a fluorophore, tetramethylrhodamine isothiocyanate, and a polarizable small molecule 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). For the benchtop Raman microscope, the DTNB-labeled assay yielded the greatest sensitivity under 532-nm laser excitation, but the MGITC-labeled assay prevailed at 785 nm. Conversely, DTNB was preferable for the miniaturized 785-nm Raman system. This comparison showed significant SERS enhancement variation in response to 1-nM miRNA-17, implying that the sensitivity of the assay may be more heavily dependent on the excitation wavelength, instrumentation, and Raman reporter chosen than on the plasmonic coupling from DNA/miRNA-mediated nanoparticle assemblies. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Enzyme-Mediated Individual Nanoparticle Release Assay

PubMed Central

Glass, James R.; Dickerson, Janet C.; Schultz, David A.

2007-01-01

Numerous methods have been developed to measure the presence of macromolecular species in a sample, however methods that detect functional activity, or modulators of that activity are more limited. To address this limitation, an approach was developed that utilizes the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here we describe their use in a release assay format where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time is described. This approach was used to detect and discriminate between non-methylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity. PMID:16620746

A novel model for rapid induction of apoptosis in spiral ganglions of mice.

PubMed

Lee, Ji Eun; Nakagawa, Takayuki; Kim, Tae Soo; Iguchi, Fukuichiro; Endo, Tsuyoshi; Dong, Youyi; Yuki, Kazuo; Naito, Yasushi; Lee, Sang Heun; Ito, Juichi

2003-06-01

The survival of the spiral ganglion (SG) is a critical issue in preservation of hearing. Research on topics related to this issue requires a mouse experimental model because such a model has advantages including use of genetic information and knockout or "knockin" mice. Thus, the aim of the study was to establish a mouse model for induction of apoptosis of SG neurons with a definite time course. Laboratory study using experimental animals. C57BL/6 mice were used as experimental animals and were subjected to direct application of cisplatin into the inner ear. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and immunostaining for Neurofilament 200-kD (NF) and peripherin were used for analysis of SG degeneration. In addition, generation of peroxynitrite in affected spiral ganglions was examined by immunostaining for nitrotyrosine. Cellular location of activated caspase-9 and cytochrome-c in dying SG neurons were examined for analysis of cell death pathway. The TUNEL assay and immunohistochemical analysis for NF and peripherin indicated that type I neurons in spiral ganglions were deleted through the apoptotic pathway over time. Spiral ganglion neurons treated with cisplatin exhibited expression of nitrotyrosine, indicating induction of peroxynitrite by cisplatin. In dying SG neurons, expression of activated caspase-9 and translocation of cytochrome-c from mitochondria to cytoplasm were observed, indicating the mitochondrial pathway of apoptosis. The predictable fashion of induction of apoptosis in SG neurons over a well-defined time course in the model in the study will aid studies of the molecular mechanism of cell death and elucidation of a strategy for prevention of SG degeneration.

Stomatin-like protein 2 is overexpressed in cervical cancer and involved in tumor cell apoptosis

PubMed Central

Deng, Huan; Deng, Yongjian; Liu, Feiye; Chen, Jie; Li, Zheng; Zhao, Kelei; Guan, Xiaoqian; Liang, Weijiang

2017-01-01

Stomatin-like protein 2 (SLP-2) is overexpressed in numerous types of human cancer and previous studies revealed that SLP-2 may function in mitochondria. The purpose of the present study was to evaluate the expression of SLP-2 in cervical cancer and the association between SLP-2 expression and clinical features, in addition to investigating the role of SLP-2 in the apoptosis of cervical cancer cells. The expression profile of SLP-2 was determined by quantitative polymerase chain reaction, western blotting and immunohistochemical staining. The effect of SLP-2 on cell apoptosis induced by chemotherapeutics in cervical cancer cells was evaluated using Annexin V staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assays. The results indicated that SLP-2 expression in cervical cancer was significantly upregulated at the mRNA and protein levels, compared with that in normal cervical tissues. Immunohistochemical analysis revealed significant correlation between SLP-2 protein expression and clinical characteristics, including the squamous cell carcinoma antigen (P=0.003), deep stromal invasion (P=0.021), lymphovascular space involvement (P=0.044) and pelvic lymph node metastasis (P<0.001), which served as independent prognostic factors for predicting the shortening of overall survival time in patients with early-stage cervical cancer. In addition, TUNEL and Annexin V binding assays revealed that silencing SLP-2 expression significantly enhanced the sensitivity of cervical cancer cells to apoptosis induced by chemotherapeutics. Taken together, the results of the present study suggest that SLP-2 may be a progressive gene in the development of cervical cancer. Overexpression of SLP-2 serves an important role in the apoptosis of human cervical cancer cells. PMID:29181097

Urtica dioica Extract Inhibits Proliferation and Induces Apoptosis and Related Gene Expression of Breast Cancer Cells In Vitro and In Vivo.

PubMed

Mohammadi, Ali; Mansoori, Behzad; Baradaran, Pooneh Chokhachi; Khaze, Vahid; Aghapour, Mahyar; Farhadi, Mehrdad; Baradaran, Behzad

2017-10-01

Currently, because the prevalence of breast cancer and its consequent mortality has increased enormously in the female population, a number of studies have been designed to identify natural products with special antitumor properties. The main purpose of the present study was to determine the effect of Urtica dioica on triggering apoptosis and diminishing growth, size, and weight of the tumor in an allograft model of BALB/c mice. In the present study, a BALB/c mouse model of breast cancer (4T1) was used. After emergence of tumor, 2 groups of mice received the extract, 1 group at a dose of 10 mg/kg and 1 group at a dose of 20 mg/kg, by intraperitoneal injection for 28 days. During the test and after removal of the tumor mass, the size and weight of the tumor were measured. To assess the induction of apoptosis in the cancer cells, the TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling) assay was performed. The Ki-67 test was used to evaluate tumor proliferation. The results showed that the tumor size in the mice treated with the extract decreased significantly. The weight of the tumor mass in the treated mice after resection was less than that in the control group. The TUNEL assay findings revealed that apoptosis occurred in the treated group. The Ki-67 test findings also demonstrated that administration of the extract suppressed the growth of tumor cells. These results suggest that U. dioica extract can decrease the growth of breast tumors and induce apoptosis in tumor cells; thus, it might represent an ideal therapeutic tool for breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Depressed Immune Responses and Accelerated Splenic Apoptosis due to Experience of Food Deprivation and Inequality but not Unstable Social Status in Balb/c Mice.

PubMed

Aghajani, Marjan; Vaez Mahdavi, Mohammad Reza; Najafabadi, Mohsen Khalili; Ghazanfari, Tooba; Moradi, Fatemeh; Golchoobian, Ravieh; Askari, Hasan; Sanadgol, Nima; Moghaddam, Ehsan Kazemi

2017-01-01

We aimed to show that the immune system is sensitive to the detrimental effects of inequality and social injustice, and splenic vulnerability to apoptosis may also increase. In order of better determination of immune responses to chronic social stress, we implemented food deprivation, food intake inequality, and unstable social status (a change of cage-mate every 3 days) for a period of 14 days in 60 male Balb/c mice. At the end of this stress period, nitric oxide (NO) production by peritoneal adherent cells and the serum concentration of corticosterone were measured. Moreover, the viability of peritoneal adherent cells and spleen lymphocytes was evaluated by MTT assay. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was done to reveal the TUNEL-reactive apoptotic bodies in the spleen. Our results showed that food deprivation and inequality caused significant changes in the apoptosis of splenic cells in comparison with the control group (p < 0.05). Moreover, the vital activities of lymphocytes and peritoneal adherent cells, as well as NO production by the latter, increased significantly (p < 0.05). However, the experience of unstable social status did not cause a further increase in the viability of lymphocytes and peritoneal adherent cells, or NO production in animals that were food-deprived or experienced inequality. Serum concentration of corticosterone in all experimental groups, except for animals that experienced unstable social status only, significantly decreased versus the control group (p < 0.05). The results suggest that poverty and social inequality, but not unstable social status, affect immune responses and are likely involved in the induction of splenic apoptosis in mice. © 2017 S. Karger AG, Basel.

Effect of different wavelengths of light on the antioxidant and immunity status of juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress

NASA Astrophysics Data System (ADS)

Choe, Jong Ryeol; Shin, Yoon Sub; Choi, Ji Yong; Kim, Tae Hwan; Jung, Min-Min; Choi, Cheol Young

2017-12-01

We investigated the effect of light wavelengths on antioxidant and immunity parameters in juvenile rock bream, Oplegnathus fasciatus, exposed to thermal stress (25 and 30°C). We exposed the fish to light emitting diodes (LEDs) emitting green (520 nm) and red light (630 nm) of 0.25 and 0.5 W/m2 intensity, and measured the activity, and mRNA and protein expression levels of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. We also determined the levels of plasma hydrogen peroxide (H2O2), melatonin, and lysozyme. Furthermore, the mRNA and protein levels of caspase-3 were measured and terminal transferase dUTP nick end labeling (TUNEL) assays were performed. We observed that mRNA expression and activities of antioxidant enzymes and plasma H2O2 levels were significantly higher after exposure to high temperatures. However, increases in these parameters were significantly lower after exposure to green LED light. The plasma melatonin and lysozyme levels were significantly lower in the different groups after exposure to high temperatures; however, in groups exposed to green LED light, their levels were significantly higher than those in the control group. The expression pattern of caspase-3 mRNA was similar to that of H2O2. The TUNEL assay showed that apoptosis was markedly higher at higher water temperatures than that at 20°C. These results indicate that high water temperatures induce oxidative stress and decrease the immunity in juvenile rock bream but green LED light inhibits the rise in oxidative stress and combats the decrease in immunity and should, thus, be useful in the culture of rock bream.

High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

2013-12-01

The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

A ribonuclease protection assay can distinguish spring viremia of carp virus from pike fry rhabdovirus

USGS Publications Warehouse

Ahne, W.; Kurath, G.; Winton, J.R.

1998-01-01

Thirteen rhabdovirus isolates from 10 teleost fish species as well as reference strains of spring viraemia of carp virus (SVCV) and pike fry rhabdovirus (PFRV) cross-reacted in an indirect immunofluorescence assay and were thus indistinguishable by this method. A ribonuclease protection assay (RPA) using a super(32)P-labeled RNA probe made from a cloned copy of the full length SVCV glycoprotein (G) gene was able to discriminate clearly between the type strains of SVCV and PFRV and among the 13 rhabdovirus isolates. Results for the RPA were generally in agreement with standard serum neutralisation assays; however, the RPA was also able to detect genomic differences between isolates of SVCV. These results have implications for fish disease control programs for SVCV.

Emission Behavior of Fluorescently Labeled Silver Nanoshell: Enhanced Self-Quenching by Metal Nanostructure.

PubMed

Zhang, Jian; Fu, Yi; Lakowicz, Joseph R

2007-02-08

Labeled silica beads with an average diameter of 100 nm were synthesized by incorporating with 20-600 μM Ru(bpy)(3) (2+) complexes. Silver shells were deposited on the beads layer-by-layer with the shell thickness of 5-50 nm. The emission band became narrower and the intensity was enhanced depending on the shell thickness. Self-quenching of the probe was observed at high concentration. Poisson statistics were employed to analyze self-quenching of the fluorophores. The estimated quenching distance was extended from 6 to 16 nm with shell growth from 0 to 50 nm. Moreover, the silver shells were also labeled with Rhodamine 6G. Fluorescence enhancement and reduced lifetime were also observed for silver-silica shell containing R6G. We found that by adjustment of probe concentration and silver shell thickness, a Ru(bpy)(3) (2+)-labeled particle could be 600 times brighter than an isolated Ru(bpy)(3) (2+) molecule. We expect labeled metal core-shell structures can become useful probes for high sensitivity and/or single particle assay.

Development of a novel method for quantification of autophagic protein degradation by AHA labeling.

PubMed

Zhang, Jianbin; Wang, Jigang; Ng, Shukie; Lin, Qingsong; Shen, Han-Ming

2014-05-01

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems.

Development of a novel method for quantification of autophagic protein degradation by AHA labeling

PubMed Central

Zhang, Jianbin; Wang, Jigang; Ng, Shukie; Lin, Qingsong; Shen, Han-Ming

2014-01-01

Autophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for L-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems. PMID:24675368

Measuring the labeling efficiency of pseudocontinuous arterial spin labeling.

PubMed

Chen, Zhensen; Zhang, Xingxing; Yuan, Chun; Zhao, Xihai; van Osch, Matthias J P

2017-05-01

Optimization and validation of a sequence for measuring the labeling efficiency of pseudocontinuous arterial spin labeling (pCASL) perfusion MRI. The proposed sequence consists of a labeling module and a single slice Look-Locker echo planar imaging readout. A model-based algorithm was used to calculate labeling efficiency from the signal acquired from the main brain-feeding arteries. Stability of the labeling efficiency measurement was evaluated with regard to the use of cardiac triggering, flow compensation and vein signal suppression. Accuracy of the measurement was assessed by comparing the measured labeling efficiency to mean brain pCASL signal intensity over a wide range of flip angles as applied in the pCASL labeling. Simulations show that the proposed algorithm can effectively calculate labeling efficiency when correcting for T1 relaxation of the blood spins. Use of cardiac triggering and vein signal suppression improved stability of the labeling efficiency measurement, while flow compensation resulted in little improvement. The measured labeling efficiency was found to be linearly (R = 0.973; P < 0.001) related to brain pCASL signal intensity over a wide range of pCASL flip angles. The optimized labeling efficiency sequence provides robust artery-specific labeling efficiency measurement within a short acquisition time (∼30 s), thereby enabling improved accuracy of pCASL CBF quantification. Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Recommendations for the generation, quantification, storage and handling of peptides used for mass spectrometry-based assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoofnagle, Andrew N.; Whiteaker, Jeffrey R.; Carr, Steven A.

2015-12-30

The Clinical Proteomic Tumor Analysis Consortium (1) (CPTAC) of the National Cancer Institute (NCI) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust technologies and workflows for the quantitative measurements of proteins. The Assay Development Working Group of the CPTAC Program aims to foster broad uptake of targeted mass spectrometry-based assays employing isotopically labeled peptides for confident assignment and quantification, including multiple reaction monitoring (MRM; also referred to as Selected Reaction Monitoring), parallel reaction monitoring (PRM), and other targeted methods.

Self-Assembled Superparamagnetic Iron Oxide Nanoclusters for Universal Cell Labeling and MRI

NASA Astrophysics Data System (ADS)

Chen, Shuzhen; Zhang, Jun; Jiang, Shengwei; Lin, Gan; Luo, Bing; Yao, Huan; Lin, Yuchun; He, Chengyong; Liu, Gang; Lin, Zhongning

2016-05-01

Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used in a variety of biomedical applications, especially as contrast agents for magnetic resonance imaging (MRI) and cell labeling. In this study, SPIO nanoparticles were stabilized with amphiphilic low molecular weight polyethylenimine (PEI) in an aqueous phase to form monodispersed nanocomposites with a controlled clustering structure. The iron-based nanoclusters with a size of 115.3 ± 40.23 nm showed excellent performance on cellular uptake and cell labeling in different types of cells, moreover, which could be tracked by MRI with high sensitivity. The SPIO nanoclusters presented negligible cytotoxicity in various types of cells as detected using MTS, LDH, and flow cytometry assays. Significantly, we found that ferritin protein played an essential role in protecting stress from SPIO nanoclusters. Taken together, the self-assembly of SPIO nanoclusters with good magnetic properties provides a safe and efficient method for universal cell labeling with noninvasive MRI monitoring capability.

Rapid screening test for detection of oxytetracycline residues in milk using lateral flow assay.

PubMed

Naik, Laxmana; Sharma, Rajan; Mann, Bimlesh; Lata, Kiran; Rajput, Y S; Surendra Nath, B

2017-03-15

A rapid, semi-quantitative lateral flow assay (LFA) was developed to screen the oxytetracycline (OTC) antibiotics residues in milk samples. In this study a competitive immuno-assay format was established. Colloidal gold nano-particles (GNP) were prepared and used as labelling material in LFA. Polyclonal antibodies were generated against OTC molecule (anti-OTC), purified and the quality was assessed by enzyme linked immuno sorbet assay. For the first time membrane components required for LFA in milk system was optimized. GNP and anti-OTC stable conjugate preparation method was standardized, and then these components were placed over the conjugate pad. OTC coupled with carrier protein was placed on test line; species specific secondary antibodies were placed on the control line of the membrane matrix. Assay was validated by spiking OTC to antibiotic free milk samples and results could be accomplished within 5min. without need of any equipment. The visual detection limit was 30ppb. Copyright © 2016 Elsevier Ltd. All rights reserved.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

1989-01-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin.

PubMed

Hicks, G R; Rayle, D L; Jones, A M; Lomax, T L

1989-07-01

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

The effects of Crataegus aronia var. dentata Browicz extract on biochemical indices and apoptosis in partially hepatectomized liver in rats

PubMed Central

Keskin, Nazan; Mammadov, Ramazan; Ili, Pinar

2012-01-01

Crataegus species have been widely used in herbal medicine, especially for the hearth diseases. In the present study, the effect of Crataegus aronia var. dentata Browicz extract on partially hepatectomized rats was investigated with biochemical and TUNEL apoptosis assays. The extracts of the plant at the concentrations of 0.5 and 1 ml/100 g body weight/day were administered orally to the two experimental groups including partially hepatectomized rats for 42 days. At the end of the experimental period, animals were sacrificed, blood was collected for the assessment of serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT), and the liver tissue was used for TUNEL assay. In biochemical assay, it was found a significant decrease in the levels of serum ALT and AST in the experimental groups. On the other hand, the plant extract did not cause any significant changes in the level of GGT in these groups. In apoptosis assay, TUNEL positive hepatocytes could not be detected in both experimental groups. The present findings can suggest that Crataegus aronia var. dentata Browicz extract can decrease the levels of serum ALT and AST and play a role in apoptosis of hepatocytes in the liver of partially hepatectomized rats. However, further studies are required to confirm the effects of the plant extract on hepatoprotection and apoptosis in the regenerating liver after partial hepatectomy in animal models. PMID:22938545

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PubMed

Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

Silencing of B7-H4 suppresses the tumorigenicity of the MGC-803 human gastric cancer cell line and promotes cell apoptosis via the mitochondrial signaling pathway.

PubMed

Zhou, Donghui; Zhou, Yong; Li, Chao; Yang, Lina

2018-04-01

B7-H4 is a transmembrane protein which is a member of the B7 superfamily. It is overexpressed in various types of cancer, including gastric cancer. However, the effects of B7-H4 on the tumorigenicity of gastric cancer and the underlying mechanisms have not yet been fully explored. Thus, the aim of this study was to examine the effects of B7-H4 on the tumorigenicity of gastric cancer cells and to elucidate the underlying mechanisms. For this purpose, B7-H4 expression in gastric cancer tissues was detected by immunohistochemical staining. The effects of B7-H4 on the biological behavior of the MGC-803 human gastric cancer cell line were examined by Cell Counting kit-8 (CCK-8) assay, cell cycle analysis, wound healing assay, Annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Moreover, the expression levels of apoptotic markers, such as cleaved caspase‑3, cleaved caspase‑9, Bcl-2 and Bax were examined by western blot analysis. Immunohistochemical staining revealed that a high expression of B7-H4 was found in about 41.8% of tissues obtained from patients with gastric cancer. Comparative analysis revealed that B7-H4 expression significantly correlated with lymph node metastasis and the TNM stage. The results of CCK-8 assay, cell cycle analysis, wound healing assay, Annexin V/propidium iodide staining assay and TUNEL assay all demonstrated that the silencing of B7-H4 by small interfering RNA decreased cell proliferation, suppressed cell motility, and induced cell cycle arrest and the apoptosis of MGC-803 human gastric cancer cells. Furthermore, the results of western blot analysis indicated that the downregulation of B7-H4 induced the apoptosis of the MGC-803 cells via the mitochondrial signaling pathway through the activation of caspase‑3 and caspase‑9, and by altering the Bax/Bcl-2 ratio in a manner that favored apoptosis. Based on the findings on human gastric cancer cell line MGC-803, the

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

PubMed

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

Fully automated assay of glycohemoglobin with the Abbott IMx analyzer: novel approaches for separation and detection.

PubMed

Wilson, D H; Bogacz, J P; Forsythe, C M; Turk, P J; Lane, T L; Gates, R C; Brandt, D R

1993-10-01

We describe a novel assay for measuring glycohemoglobin directly from anticoagulated whole blood with the Abbott IMx analyzer. The glycohemoglobin is labeled with a soluble polyanionic affinity reagent and the anionic complex is then captured with a cationic solid-phase matrix. Glycohemoglobin is quantified by measuring the quenching by heme of the static fluorescence from an added fluorophore. The assay is standardized to report both percent total glycohemoglobin (%GHb) and percent hemoglobin A1c (%HbA1c). Glucose, bilirubin, triglycerides, labile fraction, and hemoglobin variants do not interfere in the assay. Within- and between-run CVs are approximately 4-5%, with total CVs of approximately 6.5%. Highly significant linear correlations (r > 0.97) were obtained in comparison studies with two major assay methodologies. The time to obtain one result is approximately 10 min (including assay of a control), 56 min for 22 results. We describe the development, standardization, and validation of this new method.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

PubMed Central

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8–13% and reliably measures NK cell- or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies. PMID:17617419

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

PubMed

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Comparison and Correlation of Neisseria meningitidis Serogroup B Immunologic Assay Results and Human Antibody Responses following Three Doses of the Norwegian Meningococcal Outer Membrane Vesicle Vaccine MenBvac

PubMed Central

Findlow, Jamie; Taylor, Stephen; Aase, Audun; Horton, Rachel; Heyderman, Robert; Southern, Jo; Andrews, Nick; Barchha, Rita; Harrison, Ewan; Lowe, Ann; Boxer, Emma; Heaton, Charlotte; Balmer, Paul; Kaczmarski, Ed; Oster, Philipp; Gorringe, Andrew; Borrow, Ray; Miller, Elizabeth

2006-01-01

The prediction of efficacy of Neisseria meningitidis serogroup B (MenB) vaccines is currently hindered due to the lack of an appropriate correlate of protection. For outer membrane vesicle (OMV) vaccines, immunogenicity has primarily been determined by the serum bactericidal antibody (SBA) assay and OMV enzyme-linked immunosorbent assay (ELISA). However, the opsonophagocytic assay (OPA), surface labeling assay, whole blood assay (WBA), and salivary antibody ELISA have been developed although correlation with protection is presently undetermined. Therefore, the aim of the study was to investigate further the usefulness of, and relationships between, MenB immunologic assays. A phase II trial of the OMV vaccine, MenBvac, with proven efficacy was initiated to compare immunologic assays incorporating the vaccine and six heterologous strains. Correlations were achieved between the SBA assay, OMV ELISA, and OPA using human polymorphonuclear leukocytes and human complement but not between an OPA using HL60 phagocytic cells and baby rabbit complement. Correlations between the surface labeling assay, the SBA assay, and the OMV ELISA were promising, although target strain dependent. Correlations between the salivary antibody ELISA and other assays were poor. Correlations to the WBA were prevented since many samples had results greater than the range of the assay. The study confirmed the immunogenicity and benefit of a third dose of MenBvac against the homologous vaccine strain using a variety of immunologic assays. These results emphasize the need for standardized methodologies that would allow a more robust comparison of assays between laboratories and promote their further evaluation as correlates of protection against MenB disease. PMID:16861642

Isorhamnetin Attenuates Atherosclerosis by Inhibiting Macrophage Apoptosis via PI3K/AKT Activation and HO-1 Induction

PubMed Central

Luo, Yun; Sun, Guibo; Dong, Xi; Wang, Min; Qin, Meng; Yu, Yingli; Sun, Xiaobo

2015-01-01

Background and Purpose Isorhamnetin (Iso) is a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L. Previous studies have revealed its anti-cancer, anti-inflammatory, and anti-oxidant activities. This study investigated the ability of Iso to inhibit oxidized low-density lipoprotein (ox-LDL)-induced cell apoptosis in THP-1-derived macrophages. The effects of Iso on atherosclerosis in vivo were also evaluated in apolipoprotein E knockout (ApoE-/-) mice fed a high fat diet. Methods and Results Iso showed significant inhibitory effects on ox-LDL-induced THP-1-derived macrophage injuries via decreasing reactive oxygen species levels, lipid deposition, and caspase-3 activation, restoring mitochondrial membrane potential, reducing the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells, and regulating apoptosis-related proteins. We also determined the protective effects of Iso by PI3K/AKT activation and HO-1 induction. Iso reduced the atherosclerotic plaque size in vivo in ApoE-/- mice as assessed by oil red O, Sudan IV staining, and CD68-positive cells, and reduced macrophage apoptosis as assessed by caspase-3 and TUNEL assays in lesions. Conclusion In conclusion, our results show that Iso inhibited atherosclerotic plaque development in ApoE-/- mice by PI3K/AKT activation and HO-1 induction. PMID:25799286

Neuroprotective effect of nicorandil through inhibition of apoptosis by the PI3K/Akt1 pathway in a mouse model of deep hypothermic low flow.

PubMed

Yu, Di; Fan, Changfeng; Zhang, Weiyan; Wen, Zhongyuan; Hu, Liang; Yang, Lei; Feng, Yu; Yin, Ke-Jie; Mo, Xuming

2015-10-15

Nicorandil exerts a protective effect on ischemia-reperfusion (I/R) injury in the brain and kidney through anti-apoptotic mechanisms. However, the mechanism by which nicorandil protects against I/R injury induced by deep hypothermic low flow (DHLF) remains unclear. We used a cerebral I/R model induced by DHLF to determine the neuroprotective effects and possible mechanisms of nicorandil. Hematoxylin-eosin (HE) staining and in situ terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) assay were used to detect changes in cell morphology and the number of apoptotic cells in hippocampus, respectively. The apoptotic regulators including Bcl-2, Bax, Akt, and p-Akt (the active, phosphorylated form of Akt) were examined by Western blot (WB). Histopathological findings showed that nicorandil significantly alleviated morphological damage in hippocampal and reduced the number of TUNEL-positive nuclei induced by DHLF. Nicorandil also increased the expression of Bcl-2 and decreased the expression of Bax, while increasing p-Akt level. Consistent with these results, nicorandil-mediated neuroprotection was reduced in the Akt1+/- mutant mice and inhibited by LY294002, a PI3K inhibitor. These findings showed that nicorandil provides a neuroprotective role in DHLF-induced I/R injury by inhibiting apoptosis via activation of the PI3K/Akt1 signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

PubMed

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-06-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.

PubMed

Zylicz-Stachula, Agnieszka; Polska, Katarzyna; Skowron, Piotr; Rak, Janusz

2014-07-07

DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of tritium labelled endomorphin II and its stability in the radioreceptor assay

NASA Astrophysics Data System (ADS)

Tömböly, Cs.; Spetea, M.; Borsodi, A.; Tóth, G.

1999-01-01

Endomorphine I (Tyr-Pro-Trp-Phe-NH2) and endomorphin II (Tyr-Pro-Phe-Phe-NH2) are recently isolated neuropeptides. They have the highest specificity and affinity for the μ-opiate receptor among all the endogenous substances so far described, and they may be natural ligands for this receptor [1]. We prepared the [3H] endomorphin II with high specific radioactivity (53.4 Ci/mmol) by catalytic dehalotritiation. The precursor [(3,5-I2)Tyr1]-endomorphin II was synthesized by solid phase peptide synthesis using Boc chemistry. Labelled endomorphin II was used to investigate its binding properties in rat brain membrane. The stability of [3H]endomorphin II toward enzymatic degradation in membrane preparation was examined by RP-HPLC and by using a radioactivity detector.

Preparation of near-infrared-labeled targeted contrast agents for clinical translation

NASA Astrophysics Data System (ADS)

Olive, D. Michael

2011-03-01

Targeted fluorophore-labeled contrast agents are moving toward translation to human surgical use. To prepare for future clinical use, we examined the performance of potential ligands targeting the epidermal growth factor receptor, α5β3 integrins, and GLUT transporters for their suitability as directed contrast agents. Each agent was labeled with IRDye 800CW, and near-infrared dye with excitation/emission wavelengths of 789/805 nm, which we determined had favorable toxicity characteristics. The probe molecules examined consisted of Affibodies, nanobodies, peptides, and the sugar 2-deoxy-D-glucose. Each probe was tested for specific and non-specific binding in cell based assays. All probe types showed good performance in mouse models for detecting either spontaneous tumors or tumor xenografts in vivo. Each of the probes tested show promise for future human clinical studies.

A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

PubMed

Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

2014-11-11

Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

Enzyme-linked immunosorbent assays for determination of plasma aldosterone using highly specific polyclonal antibodies.

PubMed

Schwartz, F; Hadas, E; Harnik, M; Solomon, B

1990-01-01

Two enzyme-linked immunosorbent assays were established and compared for the estimation of plasma aldosterone. In the first method immobilized aldosterone-protein complexes on the ELISA plates compete with aldosterone to be determined for the binding of certain amount of anti-aldosterone antibodies. The sensitivity of this method depends on the protein carrier used to conjugate with aldosterone. In the second method, anti-aldosterone antibodies adsorbed on ELISA plates compete for binding of known amount of the enzyme-labeled aldosterone and aldosterone to be determined. The highly specific rabbit anti-aldosterone antibodies were obtained by injection of aldosterone-oxime thyroglobulin. The detection limit of aldosterone in both methods ranged between 2-20 pg. The proposed assays are suitable for the determination of aldosterone in biological fluids compared with other reported ELISA assays, as well as with RIA.

ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.

PubMed

Bushnell, S; Budde, J; Catino, T; Cole, J; Derti, A; Kelso, R; Collins, M L; Molino, G; Sheridan, P; Monahan, J; Urdea, M

1999-05-01

The sensitivity and specificity of branched DNA (bDNA) assays are derived in part through the judicious design of the capture and label extender probes. To minimize non-specific hybridization (NSH) events, which elevate assay background, candidate probes must be computer screened for complementarity with generic sequences present in the assay. We present a software application which allows for rapid and flexible design of bDNA probesets for novel targets. It includes an algorithm for estimating the magnitude of NSH contribution to background, a mechanism for removing probes with elevated contributions, a methodology for the simultaneous design of probesets for multiple targets, and a graphical user interface which guides the user through the design steps. The program is available as a commercial package through the Pharmaceutical Drug Discovery program at Chiron Diagnostics.

Novel Fitc-Labeled Igy Antibody: Fluorescence Imaging Toxoplasma Gondii In Vitro.

PubMed

Sert, Mehtap; Cakir Koc, Rabia; Budama Kilinc, Yasemin

2017-04-12

Toxoplasmosis is caused by T. gondii and can create serious health problems in humans and also worldwide economic harm. Because of the clinical and veterinary importance of toxoplasmosis, its timely and accurate diagnosis has a major impact on disease-fighting strategies. T. gondii surface antigen 1 (SAG1), an immunodominant-specific antigen, is often used as a diagnostic tool. Therefore, the aim of this study was the optimization of novel fluorescein isothiocyanate (FITC) labeling of the SAG1-specific IgY antibody to show the potential for immunofluorescence imaging of T. gondii in vitro. The specificity of IgY antibodies was controlled by an enzyme-linked immunosorbent assay (ELISA), and the concentration of the IgY antibody was detected using a spectrophotometer. The optimum incubation time and FITC concentration were determined with a fluorescence spectrometer. The obtained FITC-labeled IgY was used for marking T. gondii tachyzoites, which were cultured in vitro and viewed using light microscopy. The interaction of the fluorescence-labeled antibody and the T. gondii tachyzoites was examined with a fluorescence microscope. In this study, for the first time, a FITC-labeled anti-SAG1 IgY antibody was developed according to ELISA, fluorescence spectrometer, and fluorescence imaging of cell culture. In the future, the obtained FITC-labeled T. gondii tachyzoites' specific IgY antibodies may be used as diagnostic tools for the detection of T. gondii infections in different samples.

N-Sulfonyl-β-lactam hapten as an effective labeling reagent for aldolase mAb.

PubMed

Inokuma, Tsubasa; Fuller, Roberta P; Barbas, Carlos F

2015-04-15

Utilization of chemically programmed antibodies (cpAbs) is regarded to be one of the most efficient methods for the development of therapeutic systems. cpAbs can extend the half-life of programming reagents, activate immune systems via the Fc region of antibodies and achieve universal vaccination by attaching varieties of small, programmed molecules. In the current study, we aimed to develop a novel labeling reagent for the preparation of cpAbs and found that N-sulfonyl-β-lactams (NSBLs) were optimal. NSBL can be synthesized from readily available 4-(bromomethyl)benzenesulfonyl chloride via few simple manipulations and can label the aldolase monoclonal antibody (mAb) 84G3, which could not be labeled effectively by the conventional labeling reagent, N-acyl-β-lactam (NABL). We also demonstrated that the conjugate, which consists of mAb 84G3 and an NSBL bearing a biotin moiety, maintained strong binding activity to streptavidin. In addition, the stability assay of NSBL revealed that NSBLs can tolerate aqueous media without significant decomposition over 24h. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

PubMed Central

D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

2013-01-01

Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

Label-free nano-biosensing on the road to tuberculosis detection.

PubMed

Golichenari, Behrouz; Velonia, Kelly; Nosrati, Rahim; Nezami, Alireza; Farokhi-Fard, Aref; Abnous, Khalil; Behravan, Javad; Tsatsakis, Aristidis M

2018-08-15

Tuberculosis, an ailment caused by the bacterium Mycobacterium tuberculosis (Mtb) complex, is one of the catastrophic transmittable diseases that affect human. Reports published by WHO indicate that in 2017 about 6.3 million people progressed to TB and 53 million TB patients died from 2000 to 2016. Therefore, early diagnosis of the disease is of great importance for global health care programs. Common diagnostics like the traditional PPD test and antibody-assisted assays suffer the lack of sensitivity, long processing time and cumbersome post-test proceedings. These shortcomings restrict their use and encourage innovations in TB diagnostics. In recent years, the biosensor concept opened up new horizons in sensitive and fast detection of the disease, reducing the interval time between sampling and diagnostic result. Among new diagnostics, label-free nano-biosensors are highly promising for sensitive and accessible detection of tuberculosis. Various specific label-free nano-biosensors have been recently reported detecting the whole cell of M. tuberculosis, mycobacterial proteins and IFN-γ as crucial markers in early diagnosis of TB. This article provides a focused overview on nanomaterial-based label-free biosensors for tuberculosis detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

PubMed

Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

2016-04-01

A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology. Copyright © 2015 Elsevier Inc. All rights reserved.

Micromethod for phosphonoformate inhibition assay of hepatitis B viral DNA polymerase.

PubMed

Lin, H J; Wu, P C; Lai, C L; Chak, W

1984-04-01

A micromethod for the specific measurement of hepatitis B viral DNA polymerase in serum is presented, based on the phosphonoformate inhibition assay (J Med Virol 12: 61-70, 1983). In the micromethod, sample volume is reduced to 120 microL and the ultracentrifugation step is eliminated. The method allows good discrimination between serum infected with hepatitis B virus and uninfected serum. The cutoff value for rate of nucleotide incorporation, based on assays of 41 serum specimens negative for hepatitis B serological markers, was about 15 nU/L (90th percentile). Serum containing hepatitis B surface and antigens exhibited rates of phosphonoformate-inhibitive nucleotide incorporation of 150 (SD 150) nU/L, with an upper 90th percentile range of 17 to 667 nU/L (n = 41). The micromethod makes use of commercially available [32P]dCTP (specific activity about 7000 kCi/mol). 125I-labeled dCTP was found to be unsuitable for this assay. Human DNA polymerases in serum are detected by this method but are excluded from the phosphonoformate-inhibitive fraction.

Food Labels

MedlinePlus

... Staying Safe Videos for Educators Search English Español Food Labels KidsHealth / For Teens / Food Labels What's in ... to have at least 95% organic ingredients. Making Food Labels Work for You The first step in ...

Potential protection of green tea polyphenols against 1800 MHz electromagnetic radiation-induced injury on rat cortical neurons.

PubMed

Liu, Mei-Li; Wen, Jian-Qiang; Fan, Yu-Bo

2011-10-01

Radiofrequency electromagnetic fields (EMF) are harmful to public health, but the certain anti-irradiation mechanism is not clear yet. The present study was performed to investigate the possible protective effects of green tea polyphenols against electromagnetic radiation-induced injury in the cultured rat cortical neurons. In this study, green tea polyphenols were used in the cultured cortical neurons exposed to 1800 MHz EMFs by the mobile phone. We found that the mobile phone irradiation for 24 h induced marked neuronal cell death in the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and protective effects of green tea polyphenols on the injured cortical neurons were demonstrated by testing the content of Bcl-2 Assaciated X protein (Bax) in the immunoprecipitation assay and Western blot assay. In our study results, the mobile phone irradiation-induced increases in the content of active Bax were inhibited significantly by green tea polyphenols, while the contents of total Bax had no marked changes after the treatment of green tea polyphenols. Our results suggested a neuroprotective effect of green tea polyphenols against the mobile phone irradiation-induced injury on the cultured rat cortical neurons.

Anticancer Activity of Indian Stingless Bee Propolis: An In Vitro Study

PubMed Central

Choudhari, Milind K.; Haghniaz, Reihaneh; Rajwade, Jyutika M.; Paknikar, Kishore M.

2013-01-01

Indian stingless bee propolis has a complex chemical nature and is reported to possess various medicinal properties. In the present study, anticancer activity of the ethanolic extract of propolis (EEP) was explored by testing the cytotoxic and apoptotic effect in four different cancer cell lines, namely, MCF-7 (human breast cancer), HT-29 (human colon adenocarcinoma), Caco-2 (human epithelial colorectal adenocarcinoma), and B16F1 (murine melanoma), at different concentrations. Cytotoxicity was evaluated by MTT assay and Trypan blue dye exclusion assay. EEP at a concentration of 250 μg/mL exhibited ≥50% mortality in all cell lines tested (i.e., IC50 value). EEP revealed a concentration and time dependent cytotoxic effect. Apoptosis was estimated by differential staining (ethidium bromide/acridine orange) and TUNEL (deoxynucleotidyl transferase-dUTP nick end labeling) assay. Light microscopy and atomic force microscopy demonstrated morphological features of apoptosis in all the cell lines after treatment with 250 μg/mL EEP for 24 h. Thus, early onset of apoptosis is the reason for anticancer activity of Indian stingless bee propolis. Further, the antioxidant potential of Indian stingless bee propolis was demonstrated to substantiate its anticancer activity. PMID:23762169

Anticancer activity of Indian stingless bee propolis: an in vitro study.

PubMed

Choudhari, Milind K; Haghniaz, Reihaneh; Rajwade, Jyutika M; Paknikar, Kishore M

2013-01-01

Indian stingless bee propolis has a complex chemical nature and is reported to possess various medicinal properties. In the present study, anticancer activity of the ethanolic extract of propolis (EEP) was explored by testing the cytotoxic and apoptotic effect in four different cancer cell lines, namely, MCF-7 (human breast cancer), HT-29 (human colon adenocarcinoma), Caco-2 (human epithelial colorectal adenocarcinoma), and B16F1 (murine melanoma), at different concentrations. Cytotoxicity was evaluated by MTT assay and Trypan blue dye exclusion assay. EEP at a concentration of 250 μg/mL exhibited ≥50% mortality in all cell lines tested (i.e., IC50 value). EEP revealed a concentration and time dependent cytotoxic effect. Apoptosis was estimated by differential staining (ethidium bromide/acridine orange) and TUNEL (deoxynucleotidyl transferase-dUTP nick end labeling) assay. Light microscopy and atomic force microscopy demonstrated morphological features of apoptosis in all the cell lines after treatment with 250 μg/mL EEP for 24 h. Thus, early onset of apoptosis is the reason for anticancer activity of Indian stingless bee propolis. Further, the antioxidant potential of Indian stingless bee propolis was demonstrated to substantiate its anticancer activity.

Real-time PCR-based infectivity assay for the titration of turkey hemorrhagic enteritis virus, an adenovirus, in live vaccines.

PubMed

Mahsoub, Hassan M; Evans, Nicholas P; Beach, Nathan M; Yuan, Lijuan; Zimmerman, Kurt; Pierson, Frank W

2017-01-01

The current in vitro titration method for turkey hemorrhagic enteritis virus (THEV) is the end-point dilution assay (EPD) in suspension cell culture (CC). This assay is subjective and results in high variability among vaccine lots. In this study, a new in vitro infectivity method combining a SYBR Green I-based qPCR assay and CC was developed for titration of live hemorrhagic enteritis (HE) CC vaccines. The qPCR was used to determine the virus genome copy number (vGCN) of the internalized virus particles following inoculation of susceptible RP19 cells with 1 vaccine label dose. The measured vGCN represents the number of infectious viral particles (IVP) per 1 dose. This method was used to compare 9 vaccine lots from 3 companies in the United States. Significant lot-to-lot variations within the same company and among the various companies were found in genomic and qPCR-based infectious titer per label dose. A positive linear relationship was found between qPCR infectious titer and genomic titer. Further, considerable variations in CCID 50 titers were found among tested vaccine lots, indicating the high variability of the current titration methods. The new method provides an alternative to classical titration assays and can help reduce variation among HE vaccine products. Copyright © 2016 Elsevier B.V. All rights reserved.

Label Review Training: Module 1: Label Basics, Page 25

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review: clarity, accuracy, consistency with EPA policy, and enforceability.

Label Review Training: Module 1: Label Basics, Page 29

EPA Pesticide Factsheets

This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is a quiz on Module 1.

An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range.

PubMed

Wei, Yanli; Chen, Yanxia; Li, Huanhuan; Shuang, Shaomin; Dong, Chuan; Wang, Gufeng

2015-01-15

A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.

Enhanced UV-B radiation during pupal stage reduce body mass and fat content, while increasing deformities, mortality and cell death in female adults of solitary bee Osmia bicornis.

PubMed

Wasielewski, Oskar; Wojciechowicz, Tatiana; Giejdasz, Karol; Krishnan, Natraj

2015-08-01

The effects of enhanced UV-B radiation on the oogenesis and morpho-anatomical characteristics of the European solitary red mason bee Osmia bicornis L. (Hymenoptera: Megachilidae) were tested under laboratory conditions. Cocooned females in the pupal stage were exposed directly to different doses (0, 9.24, 12.32, and 24.64 kJ/m(2) /d) of artificial UV-B. Our experiments revealed that enhanced UV-B radiation can reduce body mass and fat body content, cause deformities and increase mortality. Following UV exposure at all 3 different doses, the body mass of bees was all significantly reduced compared to the control, with the highest UV dose causing the largest reduction. Similarly, following UV-B radiation, in treated groups the fat body index decreased and the fat body index was the lowest in the group receiving the highest dose of UV radiation. Mortality and morphological deformities, between untreated and exposed females varied considerably and increased with the dose of UV-B radiation. Morphological deformities were mainly manifested in the wings and mouthparts, and occurred more frequently with an increased dose of UV. Cell death was quantified by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (DNA fragmentation) during early stages of oogenesis of O. bicornis females. The bees, after UV-B exposure exhibited more germarium cells with fragmented DNA. The TUNEL test indicated that in germarium, low doses of UV-B poorly induced the cell death during early development. However, exposure to moderate UV-B dose increased programmed cell death. In females treated with the highest dose of UV-B the vast majority of germarium cells were TUNEL-positive. © 2014 Institute of Zoology, Chinese Academy of Sciences.

Effect of Topically Applied Azithromycin on Corneal Epithelial and Endothelial Apoptosis in a Rat Model of Corneal Alkali Burn.

PubMed

Arikan, Sedat; Karaca, Turan; Ertekin, Yusuf Haydar; Comez, Arzu Taskiran; Ersan, Ismail; Demirtas, Selim; Elmas, Sait; Tufan, Hasan Ali; Turkon, Hakan

2016-04-01

To investigate the antiapoptotic effect of topically administered azithromycin (AZM) on corneal epithelial and endothelial cells in a rat model of corneal alkali burn. Twenty-four Wistar albino rats were divided into 4 equal groups as pseudovehicle (group 1), control (group 2), alkali burned (group 3), and treatment (group 4) groups. Alkali injury was induced only in the right corneas of rats belonging to groups 3 and 4 using 1N NaOH. The rats in group 3 and the rats in group 4 were respectively treated either with an artificial tear gel or with 1.5% AZM eye drops for 5 days. At the fifth day of the experiment, the apoptosis in the corneal epithelium and endothelium of all rats was assessed using a terminal dUTP nick-end labeling (TUNEL) assay. In addition, tumor necrosis factor-alpha (TNF-α) density in the corneal epithelium was measured in all rats. The mean numbers of TUNEL+ cells in the corneal epithelium and endothelium of rats in group 3 were 117.1 ± 23.8 and 34.6.± 11.3, respectively, whereas in group 4, they were 75.8 ± 15.7 and 14.7 ± 3.5, respectively. Also the mean TNF-α densities in the corneal epithelium in group 3 and group 4 were 2.65 ± 1.3 and 1.65 ± 1.1, respectively. There was a significant decrease in the mean number of TUNEL+ cells in the corneal epithelium and endothelium and in the mean TNF-α density in the corneal epithelium of rats in group 4, when compared with group 3. Topically applied AZM can decrease TNF-α-induced apoptosis in corneal alkali burn.

Photoaffinity Labeling Studies on a Promoter of Dendritic Spine Formation

NASA Astrophysics Data System (ADS)

Sibucao, Kevin Carlo Abril

The small molecule BTA-EG4 has been shown to be a promoter of dendritic spine formation. The mechanism behind this phenomenon, however, is not well understood. The work in this dissertation is motivated by this gap in knowledge. The first part of this dissertation focuses on photoaffinity labeling studies to identify the cellular targets of BTA-EG4. Chapter 1 provides a summary of Alzheimer's disease, the rational design of BTA-EG 4, and methods to determine targets of small molecules. In Chapter 2, the synthesis of a BTA-EG4-based photoaffinity labeling probe and photodegradation studies are presented. Kinetic studies demonstrate that the probe photolyzes rapidly under UV light. In Chapter 3, photoaffinity labeling studies and subsequent protein identification experiments are reported. Competition experiments with the photoaffinity labeling probe and BTA-EG4 demonstrate that the probe labels a 55-kDa protein specifically. Tandem mass spectrometry revealed that the 55-kDa protein is the actin binding protein fascin 1. The second part of this dissertation focuses on the major protein identified from photoaffinity labeling studies, fascin 1. Chapter 4 provides a brief survey of the structure and function of fascin 1. In Chapter 5, characterizations of the interaction between BTA-EG4 and fascin 1 are reported. Isothermal titration calorimetry confirms the physical binding between fascin 1 and BTA-EG6, a BTA-EG4 analog. Slow speed sedimentation assays reveal that BTA-EG4 does not affect the actin-bundling activity of fascin 1. However, GST pull-down experiments show that BTA-EG4 inhibits the binding of fascin 1 with the GTPase Rab35. In addition, this work demonstrates that BTA-EG4 may be mechanistically distinct from the known fascin inhibitor G2.

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

PubMed

Garver, W S; Kemp, J D; Kuehn, G D

1992-12-01

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

Multiplexed chemiluminescent assays in ArrayPlates for high-throughput measurement of gene expression

NASA Astrophysics Data System (ADS)

Martel, Ralph R.; Rounseville, Matthew P.; Botros, Ihab W.; Seligmann, Bruce E.

2002-06-01

Multiplexed Molecular Profiling (MMP) assays for drug discovery are performed in ArrayPlates. ArrayPlates are 96- well microtiter plates that contain a 16-element array at the bottom of each well. Each element within an array measures one analyte in a sample. A CCD imager records the quantitative chemiluminescent readout of all 1,536 elements in a 96-well plate simultaneously. Since array elements are reagent modifiable by the end-user, ArrayPlates can be adapted to a broad range of nucleic acid- and protein-based assays. Such multiplexed assays are rapidly established, flexible, robust, automation-friendly and cost-effective. Nucleic acid assays in ArrayPlates can detect DNA and RNA, including SNPs and ESTs. A multiplexed mRNA assay to measure the expression of 16 genes is described. The assay combines a homogeneous nuclease protection assay with subsequent probe immobilization to the array by means of a sandwich hybridization followed with chemiluminescent detection. This assay was used to examine cells grown and treated in microplates and avoided cloning, transfection, RNA insolation, reverse transcription, amplification and fluorochrome labeling. Standard deviations for the measurement of 16 genes ranged from 3 percent to 13 percent in samples of 30,000 cells. Such ArrayPlates transcription assays are useful in drug discovery and development for target validation, screening, lead optimization, metabolism and toxicity profiling. Chemiluminescent detection provides ArrayPlates assays with high signal-to-noise readout and simplifies imager requirements. Imaging a 2D surface that contains arrays simplifies lens requirements relative to imaging columns of liquid in microtiter plate wells. The Omix imager for ArrayPlates is described.

A simple heterogeneous one-step assay for screening estrogenic compounds.

PubMed

Huovinen, Tuomas; Rytkönen, Kalle; Lamminmäki, Urpo; Pellinen, Teijo

2013-01-01

Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ERα. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ERα, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E(2)) and wide dynamic range of more than four orders of magnitude.

Development of a Targeted Urine Proteome Assay for kidney diseases.

PubMed

Cantley, Lloyd G; Colangelo, Christopher M; Stone, Kathryn L; Chung, Lisa; Belcher, Justin; Abbott, Thomas; Cantley, Jennifer L; Williams, Kenneth R; Parikh, Chirag R

2016-01-01

Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM. The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry assays and its application in supporting microdose absolute bioavailability studies.

PubMed

Gu, Huidong; Wang, Jian; Aubry, Anne-Françoise; Jiang, Hao; Zeng, Jianing; Easter, John; Wang, Jun-sheng; Dockens, Randy; Bifano, Marc; Burrell, Richard; Arnold, Mark E

2012-06-05

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

A label distance maximum-based classifier for multi-label learning.

PubMed

Liu, Xiaoli; Bao, Hang; Zhao, Dazhe; Cao, Peng

2015-01-01

Multi-label classification is useful in many bioinformatics tasks such as gene function prediction and protein site localization. This paper presents an improved neural network algorithm, Max Label Distance Back Propagation Algorithm for Multi-Label Classification. The method was formulated by modifying the total error function of the standard BP by adding a penalty term, which was realized by maximizing the distance between the positive and negative labels. Extensive experiments were conducted to compare this method against state-of-the-art multi-label methods on three popular bioinformatic benchmark datasets. The results illustrated that this proposed method is more effective for bioinformatic multi-label classification compared to commonly used techniques.

Less label, more free: approaches in label-free quantitative mass spectrometry.

PubMed

Neilson, Karlie A; Ali, Naveid A; Muralidharan, Sridevi; Mirzaei, Mehdi; Mariani, Michael; Assadourian, Gariné; Lee, Albert; van Sluyter, Steven C; Haynes, Paul A

2011-02-01

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

PubMed Central

Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

2010-01-01

We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

CPTAC Assay Portal: a repository of targeted proteomic assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2014-06-27

To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigatorsmore » to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.« less

Chlorella vulgaris triggers apoptosis in hepatocarcinogenesis-induced rats*

PubMed Central

Mohd Azamai, Emey Suhana; Sulaiman, Suhaniza; Mohd Habib, Shafina Hanim; Looi, Mee Lee; Das, Srijit; Abdul Hamid, Nor Aini; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

2009-01-01

Chlorella vulgaris (CV) has been reported to have antioxidant and anticancer properties. We evaluated the effect of CV on apoptotic regulator protein expression in liver cancer-induced rats. Male Wistar rats (200~250 g) were divided into eight groups: control group (normal diet), CDE group (choline deficient diet supplemented with ethionine in drinking water to induce hepatocarcinogenesis), CV groups with three different doses of CV (50, 150, and 300 mg/kg body weight), and CDE groups treated with different doses of CV (50, 150, and 300 mg/kg body weight). Rats were sacrificed at various weeks and liver tissues were embedded in paraffin blocks for immunohistochemistry studies. CV, at increasing doses, decreased the expression of anti-apoptotic protein, Bcl-2, but increased the expression of pro-apoptotic protein, caspase 8, in CDE rats, which was correlated with decreased hepatoctyes proliferation and increased apoptosis as determined by bromodeoxy-uridine (BrdU) labeling and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay, respectively. Our study shows that CV has definite chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats. PMID:19198018

Label-free and non-contact optical biosensing of glucose with quantum dots.

PubMed

Khan, Saara A; Smith, Gennifer T; Seo, Felix; Ellerbee, Audrey K

2015-02-15

We present a label-free, optical sensor for biomedical applications based on changes in the visible photoluminescence (PL) of quantum dots in a thin polymer film. Using glucose as the target molecule, the screening of UV excitation due to pre-absorption by the product of an enzymatic assay leads to quenching of the PL of quantum dots (QDs) in a non-contact scheme. The irradiance changes in QD PL indicate quantitatively the level of glucose present. The non-contact nature of the assay prevents surface degradation of the QDs, which yields an efficient, waste-free, cost-effective, portable, and sustainable biosensor with attractive market features. The limit of detection of the demonstrated biosensor is ~3.5 µm, which is competitive with existing contact-based bioassays. In addition, the biosensor operates over the entire clinically relevant range of glucose concentrations of biological fluids including urine and whole blood. The comparable results achieved across a range of cost-affordable detectors, including a spectrophotometer, portable spectrometer, and iPhone camera, suggest that label-free and visible quantification of glucose with QD films can be applied to low-cost, point-of-care biomedical sensing as well as scientific applications in the laboratory for characterizing glucose or other analytes. Copyright © 2014 Elsevier B.V. All rights reserved.

The Anticancer Effects of Radachlorin-mediated Photodynamic Therapy in the Human Endometrial Adenocarcinoma Cell Line HEC-1-A.

PubMed

Kim, Su-Mi; Rhee, Yun-Hee; Kim, Jong-Soo

2017-11-01

We investigated the effect of photodynamic therapy (PDT) using radachlorin on invasion, vascular formation and apoptosis by targeting epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in the HEC-1-A endometrial adenocarcinoma cell line. To investigate the apoptotic pathway, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and western blot analysis. We also evaluated the effects of PDT on tubular capillary formation in and invasion by HEC-1-A cells with a tube formation assay, invasion assay, prostaglandin E2 (PGE2) assay, and western blot analysis. PDT had anticancer effects on HEC-1-A through activation of the intrinsic pathway of apoptosis via caspase-9 and poly-(ADP-ribose) polymerase (PARP). PDT also inhibited tubular capillary formation in and invasion by HEC-1-A under VEGF pretreatment, that resulted from down-regulation of VEGFR2, EGFR, Ras homolog gene family/ member A (RhoA) and PGE2. These results are indicative of the specificity of radachlorin-mediated PDT to VEGF. The major advantage of radachlorin-mediated PDT is its selectivity for cancer tissue while maintaining adjacent normal endometrial tissue. Therefore, radachlorin-mediated PDT might offer high anticancer efficacy for endometrial adenocarcinoma and an especially useful modality for preserving fertility. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

PubMed

Li, R; Kast, J

2017-01-01

Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

In vitro adherence of radioactively labeled Escherichia coli in normal and cystitis-prone females

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parsons, C.L.; Anwar, H.; Stauffer, C.

Numerous investigators report data obtained using an in vitro quantitative assay for measuring bacterial adherence to epithelial cells. In the modified assay described here, we eliminated the need for visual counting of bacteria by incorporating the use of radioactively labeled Escherichia coli. This allowed quantitation of bacterial adherence to as many as 50,000 vaginal cells, whereas the visual counting system limits the determination to perhaps 50 cells. Using the modified method, we found no statistically significant differences among values for adherence of E. coli type 04 to the vaginal cells of control and cystitis-prone women at either pH 6.4 ormore » 4.0.« less

Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

USDA-ARS?s Scientific Manuscript database

The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

High-throughput, dual probe biological assays based on single molecule detection

DOEpatents

Hollars, Christopher W [Brentwood, CA; Huser, Thomas R [Livermore, CA; Lane, Stephen M [Oakland, CA; Balhorn, Rodney L [Livermore, CA; Bakajin, Olgica [San Leandro, CA; Darrow, Christopher [Pleasanton, CA; Satcher, Jr., Joe H.

2006-07-11

A method and apparatus with the sensitivity to detect and identify single target molecules through the localization of dual, fluorescently labeled probe molecules. This can be accomplished through specific attachment of the taget to a surface or in a two-dimensional (2D) flowing fluid sheet having approximate dimensions of 0.5 .mu.m.times.100 .mu.m.times.100 .mu.m. A device using these methods would have 10.sup.3 10.sup.4 greater throughput than previous one-dimensional (1D) micro-stream devices having 1 .mu.m.sup.3 interrogation volumes and would for the first time allow immuno- and DNA assays at ultra-low (femtomolar) concentrations to be performed in short time periods (.about.10 minutes). The use of novel labels (such as metal or semiconductor nanoparticles) may be incorporated to further extend the sensitivity possibly into the attomolar range.

Dual-primer self-generation SERS signal amplification assay for PDGF-BB using label-free aptamer.

PubMed

Ye, SuJuan; Zhai, XiaoMo; Wu, YanYing; Kuang, ShaoPing

2016-05-15

Highly sensitive detection of proteins, especially those associated with cancers, is essential to biomedical research as well as clinical diagnosis. In this work, a simple and novel one-two-three signal amplification surface-enhanced Raman scattering (SERS) method for the detection of protein is fabricated by using label-free aptamer and dual-primer self-generation. Platelet-derived growth factor B-chain (PDGF-BB) is selected as the model protein. The one-two-three cascade DNA amplification means one target-aptamer binding event, two hairpin DNA switches and three DNA amplification reactions. This strategy possesses some remarkable features compared to conventional signal amplification methods: (i) A smart probe including a label-free aptamer is fabricated, for suitable hybridization without hindering the affinity of the aptamer toward its target. (ii) Using the unique structure switch of the aptamer and cooperator, a one-two-three working mode is developed to amplify the SERS signal. The amplification efficiency is enhanced. Given the unique and attractive characteristics, a simple and universal strategy is designed to accomplish ultrasensitive detection of proteins. The detection limit of PDGF-BB via SERS detection is 0.42 pM, with the linear range from 1.0×10(-12)M to 10(-8)M. It is potentially universal because the aptamer can be easily designed for biomolecules whose aptamers undergo similar conformational changes. Copyright © 2015 Elsevier B.V. All rights reserved.

Investigating rhodamine B-labeled peptoids: scopes and limitations of its applications.

PubMed

Birtalan, Esther; Rudat, Birgit; Kölmel, Dominik K; Fritz, Daniel; Vollrath, Sidonie B L; Schepers, Ute; Bräse, Stefan

2011-01-01

The fluorophore rhodamine B is often used in biological assays. It is inexpensive, robust under a variety of reaction conditions, can be covalently linked to bioactive molecules, and has suitable spectral properties in terms of absorption and fluorescence wavelength. Nonetheless, there are some drawbacks: it can readily form a spirolactam compound, which is nonfluorescent, and therefore may not be the dye of choice for all fluorescence microscopy applications. Herein this spirolactam formation was observed by purifying such a labeled peptoid with high performance liquid chromatography (HPLC) and monitored in detail by making a series of analytical HPLC runs over time. Additionally, a small library of eight peptoids with rhodamine B as label was synthesized. Analysis of the absorption properties of these molecules demonstrated that the problem of fluorescence loss can be overcome by coupling secondary amines with rhodamine B.

SiO2@antisense molecules covered by nepetalactone, extracted from Nepeta gloeocephala, inhibits ILK phosphorylation and downstream PKB/AKT signaling in HeLa cells.

PubMed

Dehghany Ashkezary, M; Aboee-Mehrizi, F; Moradi, P

2017-01-01

In this study, the anticancer property of SiO 2 @antisense molecules (SiO 2 @AMs) and SiO 2 @AM covered by nepetalactone (SiO 2 @AM/CN), extracted from Nepeta gloeocephala, was investigated. Here integrin-linked kinase (ILK) phosphorylation and protein kinase B/AKT (PKB/AKT) signaling was studied when HeLa cells were exposed to SiO 2 @AM and SiO 2 @AM/CN. First, N. gloeocephala was identified at the Iranian National Herbarium. Then, its essential oil (EO) was obtained by the hydrodistillation method. In the next step, 4aα,7α,7aα-nepetalactone was extracted from the EO, based on the spectroscopic data. To obtain SiO 2 @AM/CN, 1 ml of SiO 2 @AM was mixed with extracted nepetalactone and then strongly shaken for 30 min. Finally, serial concentrations (100, 50, 25 and 12.5 μg ml -1 ) of SiO 2 @AM and SiO 2 @AM/CN were prepared and then exposed to HeLa cells (2 × 10 5 cells per ml) for 24 h at 37 °C. After incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell-cycle analysis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and western blots were carried out. To find ILK phosphorylation and PKB/AKT signaling, the expression of threonine-173 (Thr-173), serine-246 (Ser-246), total ILK, AKT-Ser473, AKT-Thr308 and total AKT was investigated. HeLa cells that were treated with SiO 2 @AM/CN had G2/M arrest. Based on the TUNEL assay, many apoptotic cells have been shown when they were exposed to SiO 2 @AM/CN. Importantly, SiO 2 @AM/CN decreased ILK phosphorylation at Thr-173 and Ser-246 without affecting total ILK levels. Moreover, SiO 2 @AM/CN decreased AKT-Ser473 and AKT-Thr308 phosphorylation without affecting total PKB/AKT protein.

The α7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to taxanes.

PubMed

Tu, Chao-Chiang; Huang, Chien-Yu; Cheng, Wan-Li; Hung, Chin-Sheng; Uyanga, Batzorig; Wei, Po-Li; Chang, Yu-Jia

2016-04-01

Gastric cancer is difficult to cure because most patients are diagnosed at an advanced disease stage. Systemic chemotherapy remains an important therapy for gastric cancer, but both progression-free survival and disease-free survival associated with various combination regimens are limited because of refractoriness and chemoresistance. Accumulating evidence has revealed that the homomeric α7-nicotinic acetylcholine receptor (A7-nAChR) promotes human gastric cancer by driving cancer cell proliferation, migration, and metastasis. Therefore, A7-nAChR may serve as a potential therapeutic target for gastric cancer. However, the role of A7-nAChR in taxane therapy for gastric cancer was unclear. Cells were subjected to A7-nAChR knockdown (A7-nAChR KD) using short interfering RNA (siRNA). The anti-proliferative effects of taxane were assessed via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and cell cycle distribution assays. A7-nAChR-KD cells exhibited low resistance to docetaxel and paclitaxel treatment, as measured by the MTT assay. Following paclitaxel treatment, the proportion of apoptotic cells was higher among A7-nAChR-KD cells than among scrambled control cells, as measured by cell cycle distribution and TUNEL assays. Further molecular analyses showed a reduction in the pAKT levels and a dramatic increase in the Bad levels in paclitaxel-treated A7-nAChR-KD cells but not in scrambled control cells. Following paclitaxel treatment, the level of Bax was slightly increased in both cell populations, whereas Poly (ADP-ribose) polymerase (PARP) cleavage was increased only in A7-nAChR-KD cells. These findings indicate that A7-nAChR-KD cells are more sensitive to paclitaxel treatment. We conclude that A7-nAChR may be a key biomarker for assessing the chemosensitivity of gastric cancer cells to taxane.

Systematic Comparison of Label-Free, Metabolic Labeling, and Isobaric Chemical Labeling for Quantitative Proteomics on LTQ Orbitrap Velos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhou; Adams, Rachel M; Chourey, Karuna

2012-01-01

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification. Isobaricmore » chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. Based on the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.« less

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry

PubMed Central

Ornatsky, Olga I.; Kinach, Robert; Bandura, Dmitry R.; Lou, Xudong; Tanner, Scott D.; Baranov, Vladimir I.; Nitz, Mark; Winnik, Mitchell A.

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping. PMID:19122859

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

PubMed

Ornatsky, Olga I; Kinach, Robert; Bandura, Dmitry R; Lou, Xudong; Tanner, Scott D; Baranov, Vladimir I; Nitz, Mark; Winnik, Mitchell A

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

Development of an event-specific hydrolysis probe quantitative real-time polymerase chain reaction assay for Embrapa 5.1 genetically modified common bean (Phaseolus vulgaris).

PubMed

Treml, Diana; Venturelli, Gustavo L; Brod, Fábio C A; Faria, Josias C; Arisi, Ana C M

2014-12-10

A genetically modified (GM) common bean event, namely Embrapa 5.1, resistant to the bean golden mosaic virus (BGMV), was approved for commercialization in Brazil. Brazilian regulation for genetically modified organism (GMO) labeling requires that any food containing more than 1% GMO be labeled. The event-specific polymerase chain reaction (PCR) method has been the primary trend for GMO identification and quantitation because of its high specificity based on the flanking sequence. This work reports the development of an event-specific assay, named FGM, for Embrapa 5.1 detection and quantitation by use of SYBR Green or hydrolysis probe. The FGM assay specificity was tested for Embrapa 2.3 event (a noncommercial GM common bean also resistant to BGMV), 46 non-GM common bean varieties, and other crop species including maize, GM maize, soybean, and GM soybean. The FGM assay showed high specificity to detect the Embrapa 5.1 event. Standard curves for the FGM assay presented a mean efficiency of 95% and a limit of detection (LOD) of 100 genome copies in the presence of background DNA. The primers and probe developed are suitable for the detection and quantitation of Embrapa 5.1.

Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

NASA Astrophysics Data System (ADS)

Chirvi, Sajal

Biomolecular interaction analysis (BIA) plays vital role in wide variety of fields, which include biomedical research, pharmaceutical industry, medical diagnostics, and biotechnology industry. Study and quantification of interactions between natural biomolecules (proteins, enzymes, DNA) and artificially synthesized molecules (drugs) is routinely done using various labeled and label-free BIA techniques. Labeled BIA (Chemiluminescence, Fluorescence, Radioactive) techniques suffer from steric hindrance of labels on interaction site, difficulty of attaching labels to molecules, higher cost and time of assay development. Label free techniques with real time detection capabilities have demonstrated advantages over traditional labeled techniques. The gold standard for label free BIA is surface Plasmon resonance (SPR) that detects and quantifies the changes in refractive index of the ligand-analyte complex molecule with high sensitivity. Although SPR is a highly sensitive BIA technique, it requires custom-made sensor chips and is not well suited for highly multiplexed BIA required in high throughput applications. Moreover implementation of SPR on various biosensing platforms is limited. In this research work spectral domain phase sensitive interferometry (SD-PSI) has been developed for label-free BIA and biosensing applications to address limitations of SPR and other label free techniques. One distinct advantage of SD-PSI compared to other label-free techniques is that it does not require use of custom fabricated biosensor substrates. Laboratory grade, off-the-shelf glass or plastic substrates of suitable thickness with proper surface functionalization are used as biosensor chips. SD-PSI is tested on four separate BIA and biosensing platforms, which include multi-well plate, flow cell, fiber probe with integrated optics and fiber tip biosensor. Sensitivity of 33 ng/ml for anti-IgG is achieved using multi-well platform. Principle of coherence multiplexing for multi

101 Labeled Brain Images and a Consistent Human Cortical Labeling Protocol

PubMed Central

Klein, Arno; Tourville, Jason

2012-01-01

We introduce the Mindboggle-101 dataset, the largest and most complete set of free, publicly accessible, manually labeled human brain images. To manually label the macroscopic anatomy in magnetic resonance images of 101 healthy participants, we created a new cortical labeling protocol that relies on robust anatomical landmarks and minimal manual edits after initialization with automated labels. The “Desikan–Killiany–Tourville” (DKT) protocol is intended to improve the ease, consistency, and accuracy of labeling human cortical areas. Given how difficult it is to label brains, the Mindboggle-101 dataset is intended to serve as brain atlases for use in labeling other brains, as a normative dataset to establish morphometric variation in a healthy population for comparison against clinical populations, and contribute to the development, training, testing, and evaluation of automated registration and labeling algorithms. To this end, we also introduce benchmarks for the evaluation of such algorithms by comparing our manual labels with labels automatically generated by probabilistic and multi-atlas registration-based approaches. All data and related software and updated information are available on the http://mindboggle.info/data website. PMID:23227001

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.

PubMed

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-10-15

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. © 2017 Schvartz et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

PubMed Central

Wilson, Peter M.; LaBonte, Melissa J.; Russell, Jared; Louie, Stan; Ghobrial, Andrew A.; Ladner, Robert D.

2011-01-01

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation. PMID:21576234

In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images.

PubMed

Christiansen, Eric M; Yang, Samuel J; Ando, D Michael; Javaherian, Ashkan; Skibinski, Gaia; Lipnick, Scott; Mount, Elliot; O'Neil, Alison; Shah, Kevan; Lee, Alicia K; Goyal, Piyush; Fedus, William; Poplin, Ryan; Esteva, Andre; Berndl, Marc; Rubin, Lee L; Nelson, Philip; Finkbeiner, Steven

2018-04-19

Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Sensitive Identification of Ostrich Meat

PubMed Central

Abdulmawjood, Amir; Grabowski, Nils; Fohler, Svenja; Kittler, Sophie; Nagengast, Helga; Klein, Guenter

2014-01-01

Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP) assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes. PMID:24963709

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

PubMed Central

Fujishiro, Mitsuhiro; Okamura, Tomohisa; Fujio, Keishi; Okazaki, Hiroaki; Nomura, Seitaro; Takeda, Norifumi; Harada, Mutsuo; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Yamamoto, Kazuhiko; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays. PMID:26020647

PROGRAMMED CELL DEATH IN EXTRAOCULAR MUSCLE TENDON/SCLERA PRECURSORS

EPA Science Inventory

Abstract

Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.

Methods: Vital staining with LysoTracker Red and Nile blue sulphate as well as terminal nick end labeling (TUNEL) were utiliz...

Nanobeads-based assays. The case of gluten detection

NASA Astrophysics Data System (ADS)

Venditti, Iole; Fratoddi, Ilaria; Vittoria Russo, Maria; Bellucci, Stefano; Crescenzo, Roberta; Iozzino, Luisa; Staiano, Maria; Aurilia, Vincenzo; Varriale, Antonio; Rossi, Mosè; D'Auria, Sabato

2008-11-01

In order to verify if the use of nanobeads of poly[phenylacetylene-(co-acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200 nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media.

Do nutrition labels influence healthier food choices? Analysis of label viewing behaviour and subsequent food purchases in a labelling intervention trial.

PubMed

Ni Mhurchu, Cliona; Eyles, Helen; Jiang, Yannan; Blakely, Tony

2018-02-01

There are few objective data on how nutrition labels are used in real-world shopping situations, or how they affect dietary choices and patterns. The Starlight study was a four-week randomised, controlled trial of the effects of three different types of nutrition labels on consumer food purchases: Traffic Light Labels, Health Star Rating labels, or Nutrition Information Panels (control). Smartphone technology allowed participants to scan barcodes of packaged foods and receive randomly allocated labels on their phone screen, and to record their food purchases. The study app therefore provided objectively recorded data on label viewing behaviour and food purchases over a four-week period. A post-hoc analysis of trial data was undertaken to assess frequency of label use, label use by food group, and association between label use and the healthiness of packaged food products purchased. Over the four-week intervention, study participants (n = 1255) viewed nutrition labels for and/or purchased 66,915 barcoded packaged products. Labels were viewed for 23% of all purchased products, with decreasing frequency over time. Shoppers were most likely to view labels for convenience foods, cereals, snack foods, bread and bakery products, and oils. They were least likely to view labels for sugar and honey products, eggs, fish, fruit and vegetables, and meat. Products for which participants viewed the label and subsequently purchased the product during the same shopping episode were significantly healthier than products where labels were viewed but the product was not subsequently purchased: mean difference in nutrient profile score -0.90 (95% CI -1.54 to -0.26). In a secondary analysis of a nutrition labelling intervention trial, there was a significant association between label use and the healthiness of products purchased. Nutrition label use may therefore lead to healthier food purchases. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.

PubMed

Oakley, Robert H; Hudson, Christine C; Cruickshank, Rachael D; Meyers, Diane M; Payne, Richard E; Rhem, Shay M; Loomis, Carson R

2002-11-01

G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V(2) receptor (V(2)R), which binds arrestin with high affinity, and the beta(2)-adrenergic receptor (beta(2)AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V(2)R or the beta(2)AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V(2)R and beta(2)AR, respectively. EC(50) values agreed closely with K(d) values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC(50) doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z' values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.

An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

PubMed

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

PubMed

Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

1999-01-01

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Chemotaxis of cancer cells in three-dimensional environment monitored label-free by quantitative phase digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Schnekenburger, Jürgen; Ketelhut, Steffi

2017-02-01

We investigated the capabilities of digital holographic microscopy (DHM) for label-free quantification of the response of living single cells to chemical stimuli in 3D assays. Fibro sarcoma cells were observed in a collagen matrix inside 3D chemotaxis chambers with a Mach-Zehnder interferometer-based DHM setup. From the obtained series of quantitative phase images, the migration trajectories of single cells were retrieved by automated cell tracking and subsequently analyzed for maximum migration distance and motility. Our results demonstrate DHM as a highly reliable and efficient tool for label-free quantification of chemotaxis in 2D and 3D environments.

Sperm DNA oxidative damage and DNA adducts

PubMed Central

Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

2015-01-01

The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on

Treatment of Near-Infrared Photodynamic Therapy Using a Liposomally Formulated Indocyanine Green Derivative for Squamous Cell Carcinoma

PubMed Central

Maruyama, Tetsuro; Akutsu, Yasunori; Suganami, Akiko; Tamura, Yutaka; Fujito, Hiromichi; Ouchi, Tomoki; Akanuma, Naoki; Isozaki, Yuka; Takeshita, Nobuyoshi; Hoshino, Isamu; Uesato, Masaya; Toyota, Taro; Hayashi, Hideki; Matsubara, Hisahiro

2015-01-01

Introduction Photodynamic therapy (PDT) is a less invasive option for cancer treatment that has evolved through recent developments in nanotechnology. We have designed and synthesized a novel liposome system that includes an indocyanine green (ICG) derivative, ICG-C18, in its bilayer. In addition to its use as an optical imager to visualize blood, lymphatic, and bile flow, ICG has also been used as an optical sensitizer. In the present report, we evaluate the use of our novel liposome system, LP-ICG-C18, in PDT for squamous cell carcinoma in an autologous murine model. Materials and Methods An excitation pulse beam (300 μJ/pulse) of a single band (800 nm) was used for sensitization. The cytotoxicity of the photodynamic therapy was evaluated in terms of cellular morphology changes, methyl thiazolyl tetrazolium (MTT) assay results, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. We tested the enhanced permeability and retention effect of LP-ICG-C18 in tumor-bearing C3H/He mice using a near-infrared fluorescence imaging system and fluorescence microscopy. We also examined the antitumor effect of PDT by measuring tumor volume in tumor-bearing mice. Results Cell death and apoptosis were only observed in the PDT group receiving LP-ICG-C18. LP-ICG-C18 itself had no cytotoxic activity and showed good biocompatibility. LP-ICG-C18 accumulated on the tumor 24 hours after injection and was retained for approximately 3 weeks. Tumor cell apoptosis following PDT with LP-ICG-C18 was also observed under optical microscopy, MTT assay, and TUNEL staining. Conclusion These findings suggest that LP-ICG-C18 may be an effective intervening material in PDT for malignant disease. PMID:25850029

In vitro targeted photodynamic therapy with a pyropheophorbide--a conjugated inhibitor of prostate-specific membrane antigen.

PubMed

Liu, Tiancheng; Wu, Lisa Y; Choi, Joseph K; Berkman, Clifford E

2009-05-01

The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on PS-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 hr by HOE33342/PI double staining, becoming more intense by 4 hr. Evidence for the apoptotic caspase cascade being activated was based on the appearance of poly-ADP-ribose polymerase (PARP) p85 fragment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detected DNA fragmentation 16 hr post-PDT, confirming apoptotic events. Cell permeability by HOE33342/PI double staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. (c) 2009 Wiley-Liss, Inc.

Treatment of near-infrared photodynamic therapy using a liposomally formulated indocyanine green derivative for squamous cell carcinoma.

PubMed

Maruyama, Tetsuro; Akutsu, Yasunori; Suganami, Akiko; Tamura, Yutaka; Fujito, Hiromichi; Ouchi, Tomoki; Akanuma, Naoki; Isozaki, Yuka; Takeshita, Nobuyoshi; Hoshino, Isamu; Uesato, Masaya; Toyota, Taro; Hayashi, Hideki; Matsubara, Hisahiro

2015-01-01

Photodynamic therapy (PDT) is a less invasive option for cancer treatment that has evolved through recent developments in nanotechnology. We have designed and synthesized a novel liposome system that includes an indocyanine green (ICG) derivative, ICG-C18, in its bilayer. In addition to its use as an optical imager to visualize blood, lymphatic, and bile flow, ICG has also been used as an optical sensitizer. In the present report, we evaluate the use of our novel liposome system, LP-ICG-C18, in PDT for squamous cell carcinoma in an autologous murine model. An excitation pulse beam (300 μJ/pulse) of a single band (800 nm) was used for sensitization. The cytotoxicity of the photodynamic therapy was evaluated in terms of cellular morphology changes, methyl thiazolyl tetrazolium (MTT) assay results, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. We tested the enhanced permeability and retention effect of LP-ICG-C18 in tumor-bearing C3H/He mice using a near-infrared fluorescence imaging system and fluorescence microscopy. We also examined the antitumor effect of PDT by measuring tumor volume in tumor-bearing mice. Cell death and apoptosis were only observed in the PDT group receiving LP-ICG-C18. LP-ICG-C18 itself had no cytotoxic activity and showed good biocompatibility. LP-ICG-C18 accumulated on the tumor 24 hours after injection and was retained for approximately 3 weeks. Tumor cell apoptosis following PDT with LP-ICG-C18 was also observed under optical microscopy, MTT assay, and TUNEL staining. These findings suggest that LP-ICG-C18 may be an effective intervening material in PDT for malignant disease.

Peroxynitrite induces apoptosis of mouse cochlear hair cells via a Caspase-independent pathway in vitro.

PubMed

Cao, Zhixin; Yang, Qianqian; Yin, Haiyan; Qi, Qi; Li, Hongrui; Sun, Gaoying; Wang, Hongliang; Liu, Wenwen; Li, Jianfeng

2017-11-01

Peroxynitrite (ONOO - ) is a potent and versatile oxidant implicated in a number of pathophysiological processes. The present study was designed to investigate the effect of ONOO - on the cultured cochlear hair cells (HCs) of C57BL/6 mice in vitro as well as the possible mechanism underlying the action of such an oxidative stress. The in vitro primary cultured cochlear HCs were subjected to different concentrations of ONOO - , then, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy (TEM), the apoptosis was determined by Terminal deoxynucleotidyl transferase dUNT nick end labeling (TUNEL) assay, the mRNA expressions of Caspase-3, Caspase-8, Caspase-9, Apaf1, Bcl-2, and Bax were analyzed by RT-PCR, and the protein expressions of Caspase-3 and AIF were assessed by immunofluorescence. This work demonstrated that direct exposure of primary cultured cochlear HCs to ONOO - could result in a base-to-apex gradient injury of HCs in a concentration-dependent manner. Furthermore, ONOO - led to much more losses of outer hair cells than inner hair cells mainly through the induction of apoptosis of HCs as evidenced by TEM and TUNEL assays. The mRNA expressions of Caspase-8, Caspase-9, Apaf1, and Bax were increased and, meanwhile, the mRNA expression of Bcl-2 was decreased in response to ONOO - treatment. Of interesting, the expression of Caspase-3 had no significant change, whereas, the expression alteration of AIF was observed. These results suggested that ONOO - can effectively damage the survival of cochlear HCs via triggering the apoptotic pathway. The findings from this work suggest that ONOO - -induced apoptosis is mediated, at least in part, via a Caspase-independent pathway in cochlear HCs.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

PubMed

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Development of a nanoparticle-based surface-modified fluorescence assay for the detection of prion proteins.

PubMed

Henry, James; Anand, Ashish; Chowdhury, Mustafa; Coté, Gerard; Moreira, Rosana; Good, Theresa

2004-11-01

A nanoparticle-based immunoassay for the detection of recombinant bovine prion protein (PrP) was developed as a step in the development of screening tools for the prevention of the spread of transmissible spongiform encephalopathies. The assay is based on the competitive binding between PrP and a peptide-fluorophore to a nanoparticle-labeled antibody which is specific for a conserved prion sequence. The fluorophore, when bound to the antibody, is subject to surfaced-modified fluorescence, enabling detection of changes in the concentration of bound fluorophore in the presence of prion protein. Important factors considered during the development of the assay were ease of use, robustness, and detection level. The effects of pH and nanoparticle conjugation chemistry on surface-modified fluorescence observed in the assay were explored. Effects of concentrations of antibody and fluorophore on reproducibility and detection limits were examined. At present, the detection limits of the system are approximately equal to the antibody-peptide fluorophore equilibrium dissociation constant, which is near one nanomolar concentration. Improved assay performance could be obtained by optimization of the nanoparticle surface resonance effects. The simplicity of the assay and ease of use may make the type of assay described in this report attractive for screening purposes in the food industry.

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes.

PubMed

Sadhu, Kalyan K; Mizukami, Shin; Watanabe, Shuji; Kikuchi, Kazuya

2011-05-01

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-09-19

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

System and method for detecting components of a mixture including a valving scheme for competition assays

DOEpatents

Koh, Chung-Yan; Piccini, Matthew E.; Singh, Anup K.

2017-07-11

Examples are described including measurement systems for conducting competition assays. A first chamber of an assay device may be loaded with a sample containing a target antigen. The target antigen in the sample may be allowed to bind to antibody-coated beads in the first chamber. A control layer separating the first chamber from a second chamber may then be opened to allow a labeling agent loaded in a first portion of the second chamber to bind to any unoccupied sites on the antibodies. A centrifugal force may then be applied to transport the beads through a density media to a detection region for measurement by a detection unit.

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases

PubMed Central

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2014-01-01

N-hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and mycobacteria. NMOs catalyze the hydroxylation of lysine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of l-kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington’s and Alzheimer’s diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin monooxygenases. Fluorescently-labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a Kd value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with Kd values of 2.1 ± 0.2 μM and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we showed that this assay can be used to identify inhibitors of NMOs. A Z’-factor of 0.77 was calculated and we show that the assay exhibits good tolerance to temperature, incubation time, and DMSO concentration. PMID:22410281

A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

PubMed

Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

2012-06-01

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

2013-07-25

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

PubMed

Košir, Alexandra Bogožalec; Spilsberg, Bjørn; Holst-Jensen, Arne; Žel, Jana; Dobnik, David

2017-08-17

Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

NASA Astrophysics Data System (ADS)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

A simple and fast detection method for bovine milk residues in foods: a 2-site monoclonal antibody immunochromatography assay.

PubMed

Xuli, Wu; Weiyi, He; Ji, Kunmei; Wenpu, Wan; Dongsheng, Hu; Hui, Wu; Xinpin, Luo; Zhigang, Liu

2013-03-01

The ingredient declaration on food labels assumes paramount importance in the protection of food-allergic consumers. China has not implemented Food allergen labeling. A gold immunochromatography assay (GICA) was developed using 2 monoclonal antibodies (mAb) against the milk allergen β-lactoglobulin in this study. The GICA was specific for pure milk samples with a sensitivity of 0.2 ng/mL. Milk protein traces extracted from 110 food products were detected by this method. The labels of 106 were confirmed by our GICA method: 57 food samples originally labeled as containing milk were positive for β-lactoglobulin and 49 food samples labeled as not containing milk were negative for β-lactoglobulin. However, 3 food samples falsely labeled as containing milk were found to contain no β-lactoglobulin whereas 1 food sample labeled as not containing milk actually contained β-lactoglobulin. First, these negatives could be because of the addition of a casein fraction. Second, some countries demand that food manufacturers label all ingredients derived from milk as "containing milk" even though the ingredients contain no detectable milk protein by any method. Our GICA method could thus provide a fast and simple method for semiquantitatation of β-lactoglobulin in foods. The present method provides a fast, simple, semiquantitative method for the determination of milk allergens in foods. © 2013 Institute of Food Technologists®

Differential DNA damage in response to the neonatal and adult excitotoxic hippocampal lesion in rats.

PubMed

Khaing, Z Z; Weickert, C S; Weinberger, D R; Lipska, B K

2000-12-01

We examined the developmental profile of excitotoxin-induced nuclear DNA fragmentation using the transferase dUTP nick-end labelling (TUNEL) technique, as a marker of DNA damage and cell death in rats with neonatal and adult excitotoxic lesions of the ventral hippocampus. We hypothesized that infusion of neurotoxin may result in a differential pattern of cell death in neonatally and adult lesioned rats, both in the infusion site and in remote brain regions presumably involved in mediating behavioural changes observed in these animals. Brains of rats lesioned at 7 days of age and in adulthood were collected at several survival times 1-21 days after the lesion. In the lesioned neonates 1-3 days postlesion, marked increases in TUNEL-positive cells occurred in the ventral hippocampus, the site of neurotoxin infusion, and in a wide surrounding area. Adult lesioned brains showed more positive cells than controls only at the infusion site. In the lesioned neonates, TUNEL-labelled cells were also present in the striatum and nucleus accumbens 1 day postlesion but not at later survival times. Our findings indicate that cell death in remote regions is more prominent in immature than adult brains, that it may lead to distinct alterations in development of these brain regions, and thus may be responsible for functional differences between neonatally and adult lesioned rats.

Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions.

PubMed

Ponchel, Frederique; Toomes, Carmel; Bransfield, Kieran; Leong, Fong T; Douglas, Susan H; Field, Sarah L; Bell, Sandra M; Combaret, Valerie; Puisieux, Alain; Mighell, Alan J; Robinson, Philip A; Inglehearn, Chris F; Isaacs, John D; Markham, Alex F

2003-10-13

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Species and interspecies radioimmunoassays for rat type C virus p30: interviral comparisons and assay of human tumor extracts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Charman, H.P.; White, M.H.; Rahman, R.

1976-01-01

The major internal protein, p30, of rat type C virus (RaLV) was purified and utilized to establish intra- and interspecies radioimmunoassays. Three rat viruses were compared in homologous and heterologous intraspecies assays with no evidence of type specificity. The only heterologous viruses to give inhibition in these species assays were the feline (FeLV) and hamster (HaLV) type C viruses; these reactions were incomplete and required high virus concentrations. An interspecies assay using a goat antiserum prepared after sequentially immunizing with FeLV, RD 114, and woolly monkey virus p30's and labeled RaLV p30 was inhibited by all mammalian type C viruses,more » although preferentially by RaLV, FeLV, and HaLV. Thus, as in a previously reported assay developed with HaLV p30, rat, hamster, and cat p30's seem more closely related to each other than to mouse type C virus p30. High levels of specific antigen were found in all cell lines producing rat virus, whereas embryonic tissues from several rat strains and cell lines considered virus-free based on other tests were negative for p30. Rats bearing tumors containing Moloney murine sarcoma virus (RaLV) did not contain free circulating antibody to RaLV p30. Fifty-one human tumor extracts (including two tumor cell lines) were tested for activity in the RaLV species and 47 in the interspecies assays after Sephadex gel filtration and pooling of material in the 15,000- to 40,000-molecular-weight range. At a sensitivity level of 7 ng/ml (0.7 ng/assay) in the interspecies assay, all human tissues, with one exception, were negative. The one positive result is considered nonspecific based on proteolysis of the labeled antigen. Input tissue protein of the purified tumor extracts averaged 1.9 mg/ml with a range of less than 0.025 to 22 mg/ml. Tissues from NIH Swiss mice processed in the same manner were positive in the interspecies assay but negative in the intraspecies RaLV assay. (auth)« less

GEO Label: User and Producer Perspectives on a Label for Geospatial Data

NASA Astrophysics Data System (ADS)

Lush, V.; Lumsden, J.; Masó, J.; Díaz, P.; McCallum, I.

2012-04-01

One of the aims of the Science and Technology Committee (STC) of the Group on Earth Observations (GEO) was to establish a GEO Label- a label to certify geospatial datasets and their quality. As proposed, the GEO Label will be used as a value indicator for geospatial data and datasets accessible through the Global Earth Observation System of Systems (GEOSS). It is suggested that the development of such a label will significantly improve user recognition of the quality of geospatial datasets and that its use will help promote trust in datasets that carry the established GEO Label. Furthermore, the GEO Label is seen as an incentive to data providers. At the moment GEOSS contains a large amount of data and is constantly growing. Taking this into account, a GEO Label could assist in searching by providing users with visual cues of dataset quality and possibly relevance; a GEO Label could effectively stand as a decision support mechanism for dataset selection. Currently our project - GeoViQua, - together with EGIDA and ID-03 is undertaking research to define and evaluate the concept of a GEO Label. The development and evaluation process will be carried out in three phases. In phase I we have conducted an online survey (GEO Label Questionnaire) to identify the initial user and producer views on a GEO Label or its potential role. In phase II we will conduct a further study presenting some GEO Label examples that will be based on Phase I. We will elicit feedback on these examples under controlled conditions. In phase III we will create physical prototypes which will be used in a human subject study. The most successful prototypes will then be put forward as potential GEO Label options. At the moment we are in phase I, where we developed an online questionnaire to collect the initial GEO Label requirements and to identify the role that a GEO Label should serve from the user and producer standpoint. The GEO Label Questionnaire consists of generic questions to identify whether

Stereoselective quantification of methadone and a d(6)-labeled isotopomer using high performance liquid chromatography-atmospheric pressure chemical ionization mass-spectrometry: application to a pharmacokinetic study in a methadone maintained subject.

PubMed

Foster, David J R; Morton, Erin B; Heinkele, Georg; Mürdter, Thomas E; Somogyi, Andrew A

2006-08-01

There is evidence that the apparent oral clearance of rac-methadone is induced during the early phase of methadone maintenance treatment. However, it is not known if this is due to changes in bioavailability or if this phenomenon is stereoselective. This knowledge can be obtained by administering a dose of stable-labeled methadone at selected times during ongoing treatment. Therefore, the authors developed a stereoselective high performance liquid chromatography-atmospheric pressure chemical ionization mass-spectrometry assay for the quantification of the enantiomers of methadone and a d(6)-labeled isotopomer. The compounds were quantified in a single assay after liquid-liquid extraction and stereoselective high performance liquid chromatograph with atmospheric pressure chemical ionization-mass spectrometry detection. The following ions were monitored: m/z 310.15 for unlabeled methadone; m/z 316.15 for methadone-d(6); and m/z 313.15 for the methadone-d(3) (internal standard). Calibration curves ranged from 0.5 to 75 ng/mL for each compound. Extraction recovery was approximately 80% for all analytes, without evidence of differences between the unlabeled and stable-labeled compounds or concentration dependency. Minor ion promotion was observed (<15%) but this was identical for all analytes including the d(3)-labeled internal standard, with peak area ratios in extracted samples identical to control injections. The isotopomers did not alter each others' ionisation, even at 10:1 concentration ratios, and 10-fold diluted samples were within 10% of the nominal concentration. Assay performance was acceptable, with interassay and intra-assay bias and precision <10% for all compounds, including the upper and lower limits of quantitation. In conclusion, the assay was successfully applied to quantify the concentration of the methadone enantiomers of both orally administered unlabeled methadone and an intravenous 5 mg dose of methadone-d(6) in a patient receiving chronic oral

Dynamic map labeling.

PubMed

Been, Ken; Daiches, Eli; Yap, Chee

2006-01-01

We address the problem of filtering, selecting and placing labels on a dynamic map, which is characterized by continuous zooming and panning capabilities. This consists of two interrelated issues. The first is to avoid label popping and other artifacts that cause confusion and interrupt navigation, and the second is to label at interactive speed. In most formulations the static map labeling problem is NP-hard, and a fast approximation might have O(nlogn) complexity. Even this is too slow during interaction, when the number of labels shown can be several orders of magnitude less than the number in the map. In this paper we introduce a set of desiderata for "consistent" dynamic map labeling, which has qualities desirable for navigation. We develop a new framework for dynamic labeling that achieves the desiderata and allows for fast interactive display by moving all of the selection and placement decisions into the preprocessing phase. This framework is general enough to accommodate a variety of selection and placement algorithms. It does not appear possible to achieve our desiderata using previous frameworks. Prior to this paper, there were no formal models of dynamic maps or of dynamic labels; our paper introduces both. We formulate a general optimization problem for dynamic map labeling and give a solution to a simple version of the problem. The simple version is based on label priorities and a versatile and intuitive class of dynamic label placements we call "invariant point placements". Despite these restrictions, our approach gives a useful and practical solution. Our implementation is incorporated into the G-Vis system which is a full-detail dynamic map of the continental USA. This demo is available through any browser.

Development of a Kinetic Assay for Late Endosome Movement.

PubMed

Esner, Milan; Meyenhofer, Felix; Kuhn, Michael; Thomas, Melissa; Kalaidzidis, Yannis; Bickle, Marc

2014-08-01

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering. © 2014 Society for Laboratory Automation and Screening.

Association of Ki-67 Labelling Index and IL-17A with Pituitary Adenoma.

PubMed

Glebauskiene, Brigita; Liutkeviciene, Rasa; Vilkeviciute, Alvita; Gudinaviciene, Inga; Rocyte, Aurelija; Simonaviciute, Dovile; Mazetyte, Ruta; Kriauciuniene, Loresa; Zaliuniene, Dalia

2018-01-01

The aim of the present study was to determine if the Ki-67 labelling index reflects invasiveness of pituitary adenoma and to evaluate IL-17A concentration in blood serum of pituitary adenoma patients. The study was conducted in the Hospital of Lithuanian University of Health Sciences. All pituitary adenomas were analysed based on magnetic resonance imaging findings. The suprasellar extension and sphenoid sinus invasion by pituitary adenoma were classified according to Hardy classification modified by Wilson. Knosp classification system was used to quantify the invasion of the cavernous sinus. The Ki-67 labelling index was obtained by immunohistochemical analysis with the monoclonal antibody, and serum levels of IL-17A were determined by enzyme-linked immunosorbent assay (ELISA). Sixty-nine PA tissue samples were investigated. Serum levels of IL-17A were determined in 60 patients with PA and 64 control subjects. Analysis revealed statistically significantly higher Ki-67 labelling index in invasive compared to noninvasive pituitary adenomas. Median serum IL-17A level was higher in the pituitary adenoma patients than in the control group. Conclusion . IL-17A might be a significant marker for patients with pituitary adenoma and Ki-67 labelling index in case of invasive pituitary adenomas.

Label-free fluorescent detection of protein kinase activity based on the aggregation behavior of unmodified quantum dots.

PubMed

Xu, Xiahong; Liu, Xin; Nie, Zhou; Pan, Yuliang; Guo, Manli; Yao, Shouzhuo

2011-01-01

Herein, we present a novel label-free fluorescent assay for monitoring the activity and inhibition of protein kinases based on the aggregation behavior of unmodified CdTe quantum dots (QDs). In this assay, cationic substrate peptides induce the selective aggregation of unmodified QDs with anionic surface charge, whereas phosphorylated peptides do not. Phosphorylation by kinase alters the net charge of peptides and subsequently inhibits the aggregation of unmodified QDs, causing an enhanced fluorescence with a 45 nm blue-shift in emission and a yellow-to-green emission color change. Hence the fluorescence response allows this QD-based method to easily probe kinase activity by a spectrometer or even by the naked eye. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.47 mU μL(-1)). On the basis of the fluorescence response of QDs on the concentration of PKA inhibitor H-89, the IC(50) value, the half maximal inhibitory concentration, was estimated, which was in agreement with the literature value. Moreover, the system can be applicable to detect the Forskolin/3-isobutyl-1-methylxantine (IBMX)-stimulated activation of PKA in cell lysate. Unlike the existing QD-based enzyme activity assays in which the modification process of QDs is essential, this method relies on unmodified QDs without the requirement of peptide labeling and QDs' modification, presenting a promising candidate for cost-effective kinase activity and inhibitor screening assays.

A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

PubMed

Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

2015-07-07

Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

NASA Astrophysics Data System (ADS)

Gober, Isaiah Nathaniel

This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the

Understanding Food Labels

MedlinePlus

... Healthy eating for girls Understanding food labels Understanding food labels There is lots of info on food ... need to avoid because of food allergies. Other food label terms top In addition to the Nutrition ...

78 FR 66826 - Prior Label Approval System: Generic Label Approval

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-07

... container of a misleading form or size.\\1\\ FSIS has interpreted these provisions as requiring that the...-evaluating-labeling . Labels submitted as an extraordinary circumstance are given the highest priority for... submissions to FSIS headquarters, thus increasing the availability of FSIS labeling staff. Upon publication of...

Use of a capillary electrophoresis instrument with laser-induced fluorescence detection for DNA quantitation. Comparison of YO-PRO-1 and PicoGreen assays.

PubMed

Guillo, Christelle; Ferrance, Jerome P; Landers, James P

2006-04-28

Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R2 > 0.999) and reproducible (RSD < 9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20 pg and 60 pg, respectively) than the YO-PRO-1 assay (60 pg and 260 pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80 min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required).

Detection Techniques for Biomolecules using Semi-Conductor Nanocrystals and Magnetic Beads as Labels

NASA Astrophysics Data System (ADS)

Chatterjee, Esha

Continued interest in the development of miniaturized and portable analytical platforms necessitates the exploration of sensitive methods for the detection of trace analytes. Nanomaterials, on account of their unique physical and chemical properties, are not only able to overcome many limitations of traditional detection reagents but also enable the exploration of many new signal transduction technologies. This dissertation presents a series of investigations of alternative detection techniques for biomolecules, involving the use of semi-conductor nanocrystals and magnetic beads as labels. Initial research focused on the development of quantum dot-encapsulating liposomes as a novel fluorescent label for immunoassays. This hybrid nanomaterial was anticipated to overcome the drawbacks presented by traditional fluorophores as well as provide significant signal amplification. Quantum dot-encapsulating liposomes were synthesized by the method of thin film hydration and characterized. The utility of these composite nanostructures for bioanalysis was demonstrated. However, the longterm instability of the liposomes hampered quantitative development. A second approach for assay development exploited the ability of gold nanoparticles to quench the optical signals obtained from quantum dots. The goal of this study was to demonstrate the feasibility of using aptamer-linked nanostructures in FRET-based quenching for the detection of proteins. Thrombin was used as the model analyte in this study. Experimental parameters for the assay were optimized. The assay simply required the mixing of the sample with the reagents and could be completed in less than an hour. The limit of detection for thrombin by this method was 5 nM. This homogeneous assay can be easily adapted for the detection of a wide variety of biochemicals. The novel technique of ferromagnetic resonance generated in magnetic bead labels was explored for signal transduction. This inductive detection technique lends

Abandoning a label doesn’t make it disappear: The perseverance of labeling effects

PubMed Central

Foroni, Francesco; Rothbart, Myron

2012-01-01

Labels exert strong influence on perception and judgment. The present experiment examines the possibility that such effects may persist even when labels are abandoned. Participants judged the similarity of pairs of silhouette drawings of female body types, ordered on a continuum from very thin to very heavy, under conditions where category labels were, and were not, superimposed on the ordered stimuli. Consistent with earlier research, labels had strong effects on perceived similarity, with silhouettes sharing the same label judged as more similar than those having different labels. Moreover, when the labels were removed and no longer present, the effect of the labels, although diminished, persisted. It did not make any difference whether the labels were simply abandoned or, in addition, had their validity challenged. The results are important for our understanding of categorization and labeling processes. The potential theoretical and practical implications of these results for social processes are discussed. PMID:23105148

Garlic (Allium sativum) Fresh Juice Induces Apoptosis in Human Oral Squamous Cell Carcinoma: The Involvement of Caspase-3, Bax and Bcl-2

PubMed Central

Farhadi, Farrokh; Jahanpour, Salar; Hazem, Kameliya; Aghbali, Amirala; Baradran, Behzad; Vahid Pakdel, Seyyed Mahdi

2015-01-01

Background and aims. There is no report on the apoptotic impact of Allium sativum L.(Garlic) on the oral squamous cell carcinoma (KB); hence, this study was designed to survey the apoptotic effects of garlic fresh juice (GFJ) on the KB cells. Materials and methods. MTTassay (MicrocultureTetrazolium Assay) was carried out to evaluate the cytotoxicity of GFJ on KB cells. Furthermore, TUNEL(Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling)and DNA fragmentation tests were performed to determine if GFJ is able to induce apoptosis in KB cells. Also a standard kit was used to assess caspase-3 activity in KB cells. Also western blotting was employed to evaluate the effect of GFJ on Bax:Bcl-2 ratio. Results. Significant cytotoxic effects were observed for the minimum used concentration (1μg/mL) as calculated to be 77.97±2.3% for 24 h and 818±3.1% for 36h of incubation (P < 0.001). Furthermore, TUNEL and DNA fragmentation tests corroborated the apoptosis inducing activity of GFJ. Consistently, after treating KB cells with GFJ(1μg/mL), caspase-3 activity and Bax:Bcl-2 ratio were raised by 7.3±0.6 and (P <0.001) folds, respectively. Conclusion. The results of this study advanced that GFJ induces apoptosis in the KB cells through increasing caspase-3 activity and Bax:Bcl2 ratio which could be attributed to its organo-sulfurcomponents. PMID:26889365

Influence of mild traumatic brain injury during pediatric stage on short-term memory and hippocampal apoptosis in adult rats.

PubMed

Park, Mi-Sook; Oh, Hyean-Ae; Ko, Il-Gyu; Kim, Sung-Eun; Kim, Sang-Hoon; Kim, Chang-Ju; Kim, Hyun-Bae; Kim, Hong

2014-06-01

Traumatic brain injury (TBI) is a leading cause of neurological deficit in the brain, which induces short- and long-term brain damage, cognitive impairment with/without structural alteration, motor deficits, emotional problems, and death both in children and adults. In the present study, we evaluated whether mild TBI in childhood causes persisting memory impairment until adulthood. Moreover, we investigated the influence of mild TBI on memory impairment in relation with hippocampal apoptosis. For this, step-down avoidance task, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and immunohistochemistry for caspase-3 were performed. Male Sprague-Dawley rats were used in the experiments. The animals were randomly divided into two groups: sham-operation group and TBI-induction group. The mild TBI model was created with an electromagnetic contusion device activated at a velocity of 3.0 m/sec. The results showed that mild TBI during the pediatric stage significantly decreased memory retention. The numbers of TUNEL-positive and caspase-3-positive cells were increased in the TBI-induction group compared to those in the sham-operation group. Defective memory retention and apoptosis sustained up to the adult stage. The present results shows that mild TBI induces long-lasting cognitive impairment from pediatric to adult stages in rats through the high level of apoptosis. The finding of this study suggests that children with mild TBI may need intensive treatments for the reduction of long-lasting cognitive impairment by secondary neuronal damage.

Time-specific blockade of PDGFR with Imatinib (Glivec®) causes cataract and disruption of lens fiber cells in neonatal mice.

PubMed

Zhou, Yin-Pin; He, Yang-Tao; Chen, Cheng-Li; Ji, Jun; Niu, Jian-Qin; Wang, Han-Zhi; Li, Shi-Feng; Huang, Lan; Mei, Feng

2011-03-01

This study aimed at investigating the response of lens epithelial cells in postnatal mice to Imatinib (Glivec®, a potent inhibitor of platelet-derived growth factor receptor (PDGFR)) treatment. Mouse eyes were sampled 10 days after administration of Imatinib (0.5 mg·g(-1)·day(-1)) for 3 days, at either 7, 14, or 21 days postpartum. Structural changes of lens were revealed by routine H.E. staining. Levels of proliferation and apoptosis were revealed by BrdU incorporation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively, and immunofluorescent staining with anti-PDGFRα antibody was carried out on the sections of eyeball. PDGFRα and p-PDGFRαprotein levels were evaluated by Western blot. Our results indicated that administration of Imatinib led to blockade of PDGFR signaling. Formation of cataracts was found only in those mice where treatment started from 7 days postpartum (P7), but was not observed in those samples from P14 nor P21. Fiber cells were disorganized in cataract lens core as observed histologically, and migration of epithelial cells was also inhibited. No apoptosis was detected with the TUNEL method. Our results indicated blockade of PDGFR at the neonatal stage (P7) would lead to cataracts and lens fiber cells disorganization, suggesting that PDGFR signaling plays a time-specific and crucial role in the postnatal development of lens in the mouse, and also may provide a new approach to produce a congenital cataract animal model.

Novel double-layer Silastic testicular prosthesis with controlled release of testosterone in vitro, and its effects on castrated rats

PubMed Central

Chen, Hui-Xing; Yang, Shi; Ning, Ye; Shao, Hai-Hao; Ma, Meng; Tian, Ru-Hui; Liu, Yu-Fei; Gao, Wei-Qiang; Li, Zheng; Xia, Wei-Liang

2017-01-01

Testicular prostheses have been used to deal with anorchia for nearly 80 years. Here, we evaluated a novel testicular prosthesis that can controllably release hormones to maintain physiological levels of testosterone in vivo for a long time. Silastic testicular prostheses with controlled release of testosterone (STPT) with different dosages of testosterone undecanoate (TU) were prepared and implanted into castrated Sprague-Dawley rats. TU oil was applied by oral administration to a separate group of castrated rats. Castrated untreated and sham-operated groups were used as controls. Serum samples from every group were collected to measure the levels of testosterone (T), follicle-stimulating hormone and luteinizing hormone (LH). Maximum intracavernous penile pressure (ICPmax) was recorded. The prostates and seminal vesicles were weighed and subjected to histology, and a terminal dexynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) assay was used to evaluate apoptosis. Our results revealed that the weights of these tissues and the levels of T and LH showed significant statistical differences in the oral administration and TU replacement groups compared with the castrated group (P < 0.05). Compared with the sham-operated group, the ICPmax, histology and TUNEL staining for apoptosis, showed no significant differences in the hormone replacement groups implanted with medium and high doses of STPT. Our results suggested that this new STPT could release TU stably through its double semi-permeable membranes with excellent biocompatibility. The study provides a new approach for testosterone replacement therapy. PMID:27174160

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

PubMed Central

Iqbal, Asif J.; Regan-Komito, Daniel; Christou, Ivy; White, Gemma E.; McNeill, Eileen; Kenyon, Amy; Taylor, Lewis; Kapellos, Theodore S.; Fisher, Edward A.; Channon, Keith M.; Greaves, David R.

2013-01-01

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators. PMID:23516549

Functionalized magnetic-fluorescent hybrid nanoparticles for cell labelling.

PubMed

Lou, Lei; Yu, Ke; Zhang, Zhengli; Li, Bo; Zhu, Jianzhong; Wang, Yiting; Huang, Rong; Zhu, Ziqiang

2011-05-01

A facile method of synthesizing 60 nm magnetic-fluorescent core-shell bifunctional nanocomposites with the ability to label cells is presented. Hydrophobic trioctylphosphine oxide (TOPO)-capped CdSe@ZnS quantum dots (QDs) were assembled on polyethyleneimine (PEI)-coated Fe(3)O(4) nanoparticles (MNP). Polyethyleneimine was utilized for the realization of multifunction, including attaching 4 nm TOPO capped CdSe@ZnS quantum dots onto magnetite particles, altering the surface properties of quantum dots from hydrophobic to hydrophilic as well as preventing the formation of large aggregates. Results show that these water-soluble hybrid nanocomposites exhibit good colloidal stability and retain good magnetic and fluorescent properties. Because TOPO-capped QDs are assembled instead of their water-soluble equivalents, the nanocomposites are still highly luminescent with no shift in the PL peak position and present long-term fluorescence stability. Moreover, TAT peptide (GRKKRRQRRRPQ) functionalized hybrid nanoparticles were also studied due to their combined magnetic enrichment and optical detection for cell separation and rapid cell labelling. A cell viability assay revealed good biocompatibility of these hybrid nanoparticles. The potential application of the new magnetic-fluorescent nanocomposites in biological and medicine is demonstrated. © The Royal Society of Chemistry 2011

Gold Nanoparticle Labeling Based ICP-MS Detection/Measurement of Bacteria, and Their Quantitative Photothermal Destruction

PubMed Central

Lin, Yunfeng

2015-01-01

Bacteria such as Salmonella and E. coli present a great challenge in public health care in today’s society. Protection of public safety against bacterial contamination and rapid diagnosis of infection require simple and fast assays for the detection and elimination of bacterial pathogens. After utilizing Salmonella DT104 as an example bacterial strain for our investigation, we report a rapid and sensitive assay for the qualitative and quantitative detection of bacteria by using antibody affinity binding, popcorn shaped gold nanoparticle (GNPOPs) labeling, surfance enchanced Raman spectroscopy (SERS), and inductively coupled plasma mass spectrometry (ICP-MS) detection. For qualitative analysis, our assay can detect Salmonella within 10 min by Raman spectroscopy; for quantitative analysis, our assay has the ability to measure as few as 100 Salmonella DT104 in a 1 mL sample (100 CFU/mL) within 40 min. Based on the quantitative detection, we investigated the quantitative destruction of Salmonella DT104, and the assay’s photothermal efficiency in order to reduce the amount of GNPOPs in the assay to ultimately to eliminate any potential side effects/toxicity to the surrounding cells in vivo. Results suggest that our assay may serve as a promising candidate for qualitative and quantitative detection and elimination of a variety of bacterial pathogens. PMID:26417447

Simple, rapid /sup 125/I-labeled cyclosporine double antibody/polyethylene glycol radioimmunoassay used in a pediatric cardiac transplant program

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berk, L.S.; Webb, G.; Imperio, N.C.

1986-01-01

We modified the Sandoz cyclosporine radioimmunoassay because of our need for frequent clinical monitoring of cyclosporine drug levels in allo- and xenograft pediatric cardiac transplant patients. With application of a commercially available (/sup 125/I)cyclosporine label in place of (/sup 3/H)cyclosporine and a second antibody/polyethylene glycol (PEG) method of separation in place of charcoal separation, we simplified and enhanced the speed and precision of assay performance. Studies of 140 whole blood samples comparing this new method to the (/sup 3/H)cyclosporine radioimmunoassay (RIA) method of Berk and colleagues yielded a coefficient of correlation of 0.96 (p less than 0.00001) with means ofmore » 626 and 667 ng/ml for (/sup 3/H)RIA and (/sup 125/I)RIA, respectively, and a regression equation of y = 28 + 1.02x. The major advantages are that total assay time is reduced to approximately 1 h; (/sup 125/I)cyclosporine label is used, avoiding the problems associated with liquid scintillation counting; and precision is enhanced by separating bound and free fractions with second antibody/PEG. These modifications should provide for greater ease of assay performance and improved clinical utility of cyclosporine monitoring not only in the pediatric but also in the adult transplant patient.« less

Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides

NASA Astrophysics Data System (ADS)

Cui, Wei; Parker, Laurie L.

2016-07-01

Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

PubMed

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

Applying the erythrocyte Pig-a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation.

PubMed

Ji, Zhiying; LeBaron, Matthew J

2017-08-01

The Pig-a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)-anchored cell surface markers. We hypothesized that the erythrocyte Pig-a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI-anchored CD59 as the Pig-a mutation marker and examined the frequency of CD59-negative sperm using flow cytometry. A reconstruction experiment that spiked un-labeled sperm (mutant-mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant-mimic sperm, demonstrating the analytical ability for CD59-negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N-ethyl-N-nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid-lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose-dependent increase in the frequency of CD59-negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59-negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment-related changes were detected in the frequency of CD59-negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59-negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig-a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485-493, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

PubMed

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive

Introduction to Pesticide Labels

EPA Pesticide Factsheets

Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV).

PubMed

Magalhães, Luana; de Oliveira, Arthur Henrique Cavalcante; de Souza Vasconcellos, Raphael; Mariotini-Moura, Christiane; de Cássia Firmino, Rafaela; Fietto, Juliana Lopes Rangel; Cardoso, Carmen Lúcia

2016-01-01