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In JoVE (1)

Other Publications (10)

Articles by Jason Roszik in JoVE

 JoVE Immunology and Infection

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center


JoVE 50058

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Other articles by Jason Roszik on PubMed

[Free Radial Forearm Flap in the Reconstruction of the Mouth Floor After Removal of a Tumor by Pull-through Method]

Free radial forearm flap was used to replace the tissue defect caused by pull-through operation for cancer of the mouth in 13 cases. There was no total or partial flap failure. The greatest advantage of the method is that the restriction of the tongue movement is less when compared to the results of other methods since the thin forearm flaps are well adaptable and are of a pliable nature. The newly developed lower alveolar crest makes the prosthetic rehabilitation possible.

Radial Forearm Flap in Maxillo-facial Surgery

Thirty consecutive patients treated with a free radial forearm flap were reviewed. This flap was used in the reconstruction of intraoral defect in 24 patients and of extraoral defect in 6 patients. There were no total or partial flap failures. Donor site complication included a partial loss of skin graft in 4 and radial fracture occurred in 1 patient. The authors considered the application of the radial forearm flap a reliable method for resurfacing large skin defect of the face. However, according to their conviction the most important field of the forearm flap is its use in intraoral reconstruction after pull-through operation. They drew attention to the limitation of the use of osteocutaneous flap in the replacement of segmental mandibular defect.

Radial Forearm Flap in Maxillo-facial Surgery

Thirty consecutive patients treated with a free radial forearm flap are reviewed. The flap was used in the reconstruction of intraoral defect in 24 patients and of extraoral defect in 6 patients. There were no total or partial flap failures. Donor site complication included a partial loss of skin graft in 4 and radial fracture occurred in 1 patient. The authors considered the application of the radial forearm flap a reliable method for resurfacing large skin defects of the face. However, according to their conviction the most important field of the forearm flap is its use in intraoral reconstruction after pull-through operation. Attention is drawn to the limitation of the use of osteocutaneous flap in the replacement of segmental mandibular defect.

AccPbFRET: an ImageJ Plugin for Semi-automatic, Fully Corrected Analysis of Acceptor Photobleaching FRET Images

The acceptor photobleaching fluorescence resonance energy transfer (FRET) method is widely used for monitoring molecular interactions in cells. This method of FRET, while among those with the simplest mathematics, is robust, self-controlled and independent of fluorophore amounts and ratios.

Cylindrospermopsin Induces Alterations of Root Histology and Microtubule Organization in Common Reed (Phragmites Australis) Plantlets Cultured in Vitro

We aimed to study the histological and cytological alterations induced by cylindrospermopsin (CYN), a protein synthesis inhibitory cyanotoxin in roots of common reed (Phragmites australis). Reed is an ecologically important emergent aquatic macrophyte, a model for studying cyanotoxin effects. We analyzed the histology and cytology of reed roots originated from tissue cultures and treated with 0.5-40 microg ml(-1) (1.2-96.4 microM) CYN. The cyanotoxin decreased root elongation at significantly lower concentrations than the elongation of shoots. As general stress responses of plants to phytotoxins, CYN increased root number and induced the formation of a callus-like tissue and necrosis in root cortex. Callus-like root cortex consisted of radially swollen cells that correlated with the reorientation of microtubules (MTs) and the decrease of MT density in the elongation zone. Concomitantly, the cyanotoxin did not decrease, rather it increased the amount of beta-tubulin in reed plantlets. CYN caused the formation of double preprophase bands; the disruption of mitotic spindles led to incomplete sister chromatid separation and disrupted phragmoplasts in root tip meristems. This work shows that CYN alters reed growth and anatomy through the alteration of MT organization.

Evaluation of Intensity-based Ratiometric FRET in Image Cytometry--approaches and a Software Solution

The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results.

T-cell Synapse Formation Depends on Antigen Recognition but Not CD3 Interaction: Studies with TCR:ζ, a Candidate Transgene for TCR Gene Therapy

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.

ErbB Protein Modifications Are Secondary to Severe Cell Membrane Alterations Induced by Elisidepsin Treatment

Elisidepsin is a marine-derived anti-tumor agent with unique mechanism of action. It has been suggested to induce necrosis associated with severe membrane damage. Since indirect evidence points to the involvement of ErbB receptor tyrosine kinases and lipid rafts in the mechanism of action of elisidepsin, we investigated the effect of the drug on the distribution of ErbB proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing ErbB receptors. Stable expression of a single member of the ErbB family (ErbB1-3) or co-transfection of ErbB2 and ErbB3 did not modify the elisidepsin sensitivity of CHO and A431 cells. However, elisidepsin induced the redistribution of ErbB3 and two GPI-anchored proteins (transfected GPI-anchored eGFP and placental alkaline phosphatase) from the plasma membrane to intracellular vesicles without comparable effects on ErbB1 and ErbB2. Elisidepsin increased the binding of a conformational sensitive anti-ErbB3 antibody without modifying the binding of other ErbB2 or ErbB3 antibodies, and it decreased the homoassociation of both ErbB2 and ErbB3. We also found that elisidepsin decreased the fluorescence anisotropy of a membrane specific fluorescent probe and induced a blue shift in the emission spectrum of Laurdan pointing to significant changes in the order of the plasma membrane possibly associated with the formation of liquid ordered domains. Although the distribution of ErbB proteins is preferentially altered by elisidepsin, our data question their role in determining sensitivity to the drug. We assume that induction of liquid ordered domains is the primary action of elisidepsin leading to all the other observed changes.

Imaging of T Cells Expressing Chimeric Antigen Receptors

Microcystin-LR, a Protein Phosphatase Inhibitor, Induces Alterations in Mitotic Chromatin and Microtubule Organization Leading to the Formation of Micronuclei in Vicia Faba

Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies.

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