an-organotypic-high-throughput-system-for-characterization-drug

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

H. Lee Moffitt Cancer Center and Research Institute

To analyze muscle activation patterns during various footplate perturbations, used as proprioceptive challenges in patients with low back pain (LBP) and in controls.

Muscle activation patterns in subjects with and without low back pain.

Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 degrees C. The pH-independent equilibrium association constants for formation of the CcP.HN3 and Mb.HN3 complexes are (1.05 +/- 0.06)x10(5) and (1.6 +/- 0.8)x10(5) M(-1), respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The DeltaH0 values for formation of CcP.HN3 and Mb.HN3 are -16.4 +/- 0.7 and -9.0 +/- 0.5 kcal/mol, respectively, and the Delta S0 values are -32 +/- 2 and -16 +/- 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 +/- 0.2 and > or =8.2 for the Mb.HN3 and CcP.HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3-. CcP binds N3- very weakly, if at all, and only an upper-limit of 18 +/-5 M(-1) for the pH-independent equilibrium association constant for the CcP.N3- complex can be determined. Mb binds N3- with an association constant of (1.8 +/- 0.1)x10(4) M(-1). The ratio of the equilibrium association constants for HN3 and N3- binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.

Azide binding to yeast cytochrome c peroxidase and horse metmyoglobin: comparative thermodynamic investigation using isothermal titration calorimetry.

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site.

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain.

Increased atrial volumes predict adverse cardiovascular events. Accordingly, accurate measurement of atrial size has become increasingly important in clinical practice. The area-length method is commonly used to estimate the volume. Disagreements between atrial volumes using echocardiography and other imaging modalities have been found. It is unclear whether this has resulted from differences in the measurement method or discrepancies among imaging modalities. We compared the right atrial (RA) and left atrial (LA) volume estimates using the area-length method for transthoracic echocardiography and cardiovascular magnetic resonance (CMR) imaging. Patients undergoing echocardiography and CMR imaging within 1 month were identified retrospectively. For both modalities, the RA and LA long-axis dimension and area were measured using standard 2- and 4-chamber views, and the volume was calculated using the area-length method for both atria. The echocardiographic and CMR values were compared using the Bland-Altman method. A total of 85 patients and 18 controls were included in the present study. The atrial volumes estimated using the area-length method were significantly smaller when measured using echocardiography than when measured using CMR imaging (LA volume 35 ± 20 vs 49 ± 30 ml/m², p <0.001, and RA volume 32 ± 23 vs 43 ± 29 ml/m², p = 0.012). The mean difference (CMR imaging minus echocardiography) was 14 ± 14 ml/m² for the LA and 10 ± 16 ml/m² for the RA volume. Similar results were found in the healthy controls. No significant intra- or interobserver variability was found within each modality. In conclusion, echocardiography consistently underestimated the atrial volumes compared to CMR imaging using the area-length method.

Comparison of left and right atrial volume by echocardiography versus cardiac magnetic resonance imaging using the area-length method.

Accurate preclinical predictions of the clinical efficacy of experimental cancer drugs are highly desired but often haphazard. Such predictions might be improved by incorporating elements of the tumor microenvironment in preclinical models by providing a more physiological setting. In generating improved xenograft models, it is generally accepted that the use of primary tumors from patients are preferable to clonal tumor cell lines. Here we describe an interdisciplinary platform to study drug response in multiple myeloma, an incurable cancer of the bone marrow. This platform uses microfluidic technology to minimize the number of cells per experiment, while incorporating three-dimensional extracellular matrix and mesenchymal cells derived from the tumor microenvironment. We used sequential imaging and a novel digital imaging analysis algorithm to quantify changes in cell viability. Computational models were used to convert experimental data into dose-exposure-response "surfaces," which offered predictive utility. Using this platform, we predicted chemosensitivity to bortezomib and melphalan, two clinical multiple myeloma treatments, in three multiple myeloma cell lines and seven patient-derived primary multiple myeloma cell populations. We also demonstrated how this system could be used to investigate environment-mediated drug resistance and drug combinations that target it. This interdisciplinary preclinical assay is capable of generating quantitative data that can be used in computational models of clinical response, demonstrating its utility as a tool to contribute to personalized oncology.

A preclinical assay for chemosensitivity in multiple myeloma.

The purpose of this study was to test the hypothesis that handheld ultrasound (HHU) provides a more accurate diagnosis than physical examination in patients with suspected cardiovascular abnormalities and that its use thus reduces additional testing and overall costs.

Timothy Jacobson

Department of Cancer Imaging and Metabolism

H. Lee Moffitt Cancer Center and Research Institute

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Timothy Jacobson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cancer Imaging and Metabolism

H. Lee Moffitt Cancer Center and Research Institute

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Department of Cancer Imaging and Metabolism