'Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification ... More
Reduction in muscle fibre number during the adaptive radiation of notothenioid fishes: a phylogenetic perspective.
AuthorsJohnston IA, Fernández DA, Calvo J, Vieira VL, North AW, Abercromby M, Garland T
JournalJ Exp Biol
PubMed ID12819266
'The fish fauna of the continental shelf of the Southern Ocean is dominated by a single sub-order of Perciformes, the Notothenioidei, which have unusually large diameter skeletal muscle fibres. We tested the hypothesis that in fast myotomal muscle a high maximum fibre diameter (FD(max)) was related to a reduction in ... More
Cytoskeletal-assisted dynamics of the mitochondrial reticulum in living cells.
AuthorsKnowles MK, Guenza MG, Capaldi RA, Marcus AH
JournalProc Natl Acad Sci U S A
PubMed ID12417764
'Subcellular organelle dynamics are strongly influenced by interactions with cytoskeletal filaments and their associated motor proteins, and lead to complex multiexponential relaxations that occur over a wide range of spatial and temporal scales. Here we report spatio-temporal measurements of the fluctuations of the mitochondrial reticulum in osteosarcoma cells by using ... More
High-content single-cell drug screening with phosphospecific flow cytometry.
'Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against ... More
Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.
AuthorsKrutzik PO, Nolan GP
JournalNat Methods
PubMed ID16628206
'Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, ... More
Laser scanning cytometry and tissue microarray analysis of salinity effects on killifish chloride cells.
AuthorsLima RN, Kültz D
JournalJ Exp Biol
PubMed ID15073205
'The effects of salinity on chloride cells (CC) and Na(+)/K(+)-ATPase content in gill epithelium of euryhaline killifish Fundulus heteroclitus were analyzed using laser scanning cytometry (LSC) and tissue microarrays (TMAs). Salinity acclimations consisted of acute transfer from freshwater (FW) to 1x seawater (SW) and gradual transfer from FW to 2.4x ... More
Synthesis of novel fluorinated coumarins: excellent UV-light excitable fluorescent dyes.
AuthorsSun WC, Gee KR, Haugland RP
JournalBioorg Med Chem Lett
PubMed ID9873685
'Two new fluorinated fluorescent dyes, 6,8-difluoro-7-hydroxy-4-methylcoumarin (Marina Blue) and 3-carboxy-6,8-difluoro-7-hydroxycoumarin (Pacific Blue), exhibit excellent photophysical properties among a series of novel fluorinated 7-hydroxycoumarins. Most of these fluorinated coumarins have quantum yields (0.63 to 0.89) equal to or higher than that of the parent compound (0.63), which, in combination with their ... More
Enhanced amine and amino acid analysis using Pacific Blue and the Mars Organic Analyzer microchip capillary electrophoresis system.
'The fluorescent amine reactive probe Pacific Blue succinimidyl ester (PB) is used for the detection of trace amounts of amines and amino acids by microchip capillary electrophoresis on the Mars Organic Analyzer (MOA). The spectral and chemical properties of PB provide a 200-fold increase in sensitivity and improved resolution compared ... More
Evolution and biogeography of deep-sea vent and seep invertebrates.
Deep-sea hydrothermal vents and cold seeps are submarine springs where nutrient-rich fluids emanate from the sea floor. Vent and seep ecosystems occur in a variety of geological settings throughout the global ocean and support food webs based on chemoautotrophic primary production. Most vent and seep invertebrates arrive at suitable habitats ... More
Effect of Small-Molecule Modification on Single-Cell Pharmacokinetics of PARP Inhibitors.
AuthorsThurber GM, Reiner T, Yang KS, Kohler RH, Weissleder R,
Journal
PubMed ID24552776
The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging, given the complex tumor microenvironment including intra- and intertumor heterogeneity. The difficulty in studying this distribution is even ... More
Alloreactive cytotoxic T cells provide means to decipher the immunopeptidome and reveal a plethora of tumor-associated self-epitopes.
AuthorsKumari S, Wälchli S, Fallang LE, Yang W, Lund-Johansen F, Schumacher TN, Olweus J,
Journal
PubMed ID24344295
HLA molecules presenting peptides derived from tumor-associated self-antigens (self-TAA) are attractive targets for T-cell-based immunotherapy of cancer. However, detection of such epitopes is hampered by self-tolerance and limitations in the sensitivity of mass spectrometry. Here, we used T cells from HLA-A2-negative donors as tools to detect HLA-A2-bound peptides from two ... More
T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells.
AuthorsKalland ME, Oberprieler NG, Vang T, Taskén K, Torgersen KM,
JournalJ Immunol
PubMed ID22013130
To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution ... More
B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression.
Human tumors contain populations of both cancerous and host immune cells whose malignant signaling interactions may define each patient's disease trajectory. We used multiplexed phospho-flow cytometry to profile single cells within human follicular lymphoma tumors and discovered a subpopulation of lymphoma cells with impaired B cell antigen receptor (BCR) signaling. ... More
FRET and competing processes between conjugated polymer and dye substituted DNA strands: a comparative study of probe selection in DNA detection.
AuthorsAl Attar HA, Monkman AP,
JournalBiomacromolecules
PubMed ID19334782
Fluorescence resonance energy transfer (FRET) between water-soluble conjugated polymer, poly-(9,9-bis(6'-N,N,N-trimethylammonium)-hexyl-fluorene phenylene) bromide, and ssDNA's labeled with four different types of dyes (Pacific-blue, Alexa-fluor 430, Fluorescein, and ROX) has been investigated. The effect of spectral overlap and Stokes-shift on the efficiency and properties of FRET were studied. In the DNA sequence ... More
Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.
AuthorsCox WG, Singer VL,
JournalBiotechniques
PubMed ID14740493
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized ... More
An anti-CD45RO immunotoxin kills HIV-latently infected cells from individuals on HAART with little effect on CD8 memory.
AuthorsSaavedra-Lozano J, Cao Y, Callison J, Sarode R, Sodora D, Edgar J, Hatfield J, Picker L, Peterson D, Ramilo O, Vitetta ES
JournalProc Natl Acad Sci U S A
PubMed ID14983037
CD4+ CD45RO+ T cells are the major latent viral reservoir in HIV-infected individuals and hence a major obstacle in curing the disease. An anti-CD45RO immunotoxin (IT) can decrease the number of both productively and latently infected CD4+ T cells obtained from HIV-infected individuals with detectable viremia. In this study, we ... More
Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes.
AuthorsMarras SA, Kramer FR, Tyagi S
JournalNucleic Acids Res
PubMed ID12409481
An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is the efficiency of energy transfer between the fluorophore and quencher used to label the probes. We have determined the efficiency of energy transfer for a large number of combinations of commonly used fluorophores and quenchers. We ... More
Comparative trends in shortening velocity and force production in skeletal muscles.
AuthorsMedler S
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID12121850
Skeletal muscles are diverse in their properties, with specific contractile characteristics being matched to particular functions. In this study, published values of contractile properties for >130 diverse skeletal muscles were analyzed to detect common elements that account for variability in shortening velocity and force production. Body mass was found to ... More
Role of secretory carrier membrane protein SCAMP2 in granule exocytosis.
AuthorsLiu L, Guo Z, Tieu Q, Castle A, Castle D
JournalMol Biol Cell
PubMed ID12475951
In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of (35)S-labeled secretogranin with the same ... More
Locomotor performance of closely related Tropidurus species: relationships with physiological parameters and ecological divergence.
AuthorsKohlsdorf T, James RS, Carvalho JE, Wilson RS, Dal Pai-Silva M, Navas CA
JournalJ Exp Biol
PubMed ID14978059
Tropidurid lizards have colonized a variety of Brazilian open environments without remarkable morphological variation, despite ecological and structural differences among habitats used. This study focuses on two Tropidurus sister-species that, despite systematic proximity and similar morphology, exhibit great ecological divergence and a third ecologically generalist congeneric species providing an outgroup ... More
The lipopolysaccharide and beta-1,3-glucan binding protein gene is upregulated in white spot virus-infected shrimp (Penaeus stylirostris).
AuthorsRoux MM, Pain A, Klimpel KR, Dhar AK
JournalJ Virol
PubMed ID12072514
Pattern recognition proteins such as lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) play an important role in the innate immune response of crustaceans and insects. Random sequencing of cDNA clones from a hepatopancreas cDNA library of white spot virus (WSV)-infected shrimp provided a partial cDNA (PsEST-289) that showed similarity to the ... More
Screening for aneuploidies of ten different chromosomes in two rounds of FISH: a short and reliable protocol.
AuthorsBaart EB, Martini E, Van Opstal D
JournalPrenat Diagn
PubMed ID15614916
OBJECTIVE: To develop a DNA labelling protocol for the simultaneous detection of five different fluorescent chromosomal DNA probes within one round of hybridisation. In combination with a commercial five-colour probemix for the second round of hybridisation, this results in a fast and reliable Fluorescence in situ Hybridization (FISH) protocol, enabling ... More