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1.
EMBO Rep ; 17(6): 901-13, 2016 06.
Article in English | MEDLINE | ID: mdl-27170622

ABSTRACT

During meiosis, cohesin complexes mediate sister chromatid cohesion (SCC), synaptonemal complex (SC) assembly and synapsis. Here, using super-resolution microscopy, we imaged sister chromatid axes in mouse meiocytes that have normal or reduced levels of cohesin complexes, assessing the relationship between localization of cohesin complexes, SCC and SC formation. We show that REC8 foci are separated from each other by a distance smaller than 15% of the total chromosome axis length in wild-type meiocytes. Reduced levels of cohesin complexes result in a local separation of sister chromatid axial elements (LSAEs), as well as illegitimate SC formation at these sites. REC8 but not RAD21 or RAD21L cohesin complexes flank sites of LSAEs, whereas RAD21 and RAD21L appear predominantly along the separated sister-chromatid axes. Based on these observations and a quantitative distribution analysis of REC8 along sister chromatid axes, we propose that the high density of randomly distributed REC8 cohesin complexes promotes SCC and prevents illegitimate SC formation.


Subject(s)
Chromatids/genetics , Chromatids/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Synaptonemal Complex , Animals , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Male , Meiosis/genetics , Mice , Mice, Knockout , Nuclear Proteins/genetics , Phosphoproteins/genetics , Protein Subunits/metabolism , Sister Chromatid Exchange , Spermatocytes/metabolism , Cohesins
2.
Front Immunol ; 7: 119, 2016.
Article in English | MEDLINE | ID: mdl-27092139

ABSTRACT

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

3.
J Phys Chem B ; 117(45): 14151-6, 2013 Nov 14.
Article in English | MEDLINE | ID: mdl-24134567

ABSTRACT

The fluorescence spectrum of CdSe core-CdS/ZnS shell colloidal quantum dots (QDs) embedded in porous alumina membrane was studied. Small peaks, superimposed on the principal QD fluorescence spectrum, were observed. Finite-difference time-domain simulation indicates that the QD point radiation emitting from within the membrane is strongly modulated by the photonic band structure introduced by the membrane pores, leading to the observed fine spectral features. Moreover, the principal QD fluorescence peak red-shifted when the optical excitation power was increased, which is attributed to QD material heating due to emitted phonons when the photoexcited electron and hole relax nonradiatively from high-energy states to the ground exciton state before fluorescence.


Subject(s)
Aluminum Oxide/chemistry , Quantum Dots/chemistry , Cadmium Compounds/chemistry , Colloids/chemistry , Electrons , Photons , Porosity , Selenium Compounds/chemistry , Spectrometry, Fluorescence , Sulfides/chemistry , Zinc Compounds/chemistry
4.
Hear Res ; 298: 93-103, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23296212

ABSTRACT

In animals, hearing loss resulting from cochlear mechanosensory cell damage can be mitigated by antioxidants such as d-methionine (d-met) and acetyl-l-carnitine (ALCAR). The systemic routes of administration of these compounds, that must of necessity transit trough the cochlear fluids, may affect the antioxidant levels in the cochlea and the resulting oto-protective effect. In this study, we analyzed the pharmacokinetics of [(14)C]d-met in the cochlea and four other tissues after intratracheal (IT), intranasal (IN), and oral by gavage (OG) administration and compared it to intravenous administration (IV). We then analyzed the effect of these four routes on the antioxidant content of the cochlear fluids after d-met or ALCAR administration, by liquid chromatography/mass spectrometry. Our results showed that the concentration of methionine and ALCAR in cochlear fluids significantly increased after their respective systemic administration. Interestingly, d-met administration also contributed to an increase of ALCAR. Our results also showed that the delivery routes differently affected the bioavailability of administered [(14)C]d-met as well as the concentrations of methionine, ALCAR and the ratio of oxidized to reduced glutathione. Overall, pulmonary delivery via IT administration achieved high concentrations of methionine, ALCAR, and oxidative-related metabolites in cochlear fluids, in some cases surpassing IV administration, while IN route appeared to be the least efficacious. To our knowledge, this is the first report of the direct measurements of antioxidant levels in cochlear fluids after their systemic administration. This report also demonstrates the validity of the pulmonary administration of antioxidants and highlights the different contributions of d-met and ALCAR allowing to further investigate their impact on oxidative stress in the cochlear microenvironment.


Subject(s)
Acetylcarnitine/administration & dosage , Acetylcarnitine/pharmacokinetics , Antioxidants/administration & dosage , Antioxidants/pharmacokinetics , Glutathione/metabolism , Labyrinthine Fluids/metabolism , Methionine/administration & dosage , Methionine/pharmacokinetics , Administration, Inhalation , Administration, Intranasal , Administration, Oral , Animals , Biological Availability , Biotransformation , Chromatography, High Pressure Liquid , Endolymph/metabolism , Injections, Intravenous , Male , Mass Spectrometry , Oxidation-Reduction , Oxidative Stress/drug effects , Perilymph/metabolism , Rats , Rats, Sprague-Dawley
5.
J Histochem Cytochem ; 59(12): 1060-75, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22147606

ABSTRACT

Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti-MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1-, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31-, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.


Subject(s)
Antigens, Ly/metabolism , MART-1 Antigen/metabolism , Melanoma, Experimental/pathology , Membrane Proteins/metabolism , Pericytes/pathology , Animals , CD146 Antigen/metabolism , Cell Hypoxia , Cell Line, Tumor , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Flow Cytometry , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunohistochemistry , Melanoma, Experimental/blood supply , Melanoma, Experimental/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred ICR , Mice, SCID , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Neoplasm Transplantation , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Pericytes/metabolism , Skin/metabolism , Skin/pathology , Stromal Cells/metabolism , Time Factors , Tumor Microenvironment
6.
PLoS One ; 5(11): e15453, 2010 Nov 12.
Article in English | MEDLINE | ID: mdl-21103395

ABSTRACT

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.


Subject(s)
Cell Culture Techniques/methods , Cytotoxicity, Immunologic/immunology , Immunologic Surveillance/immunology , Killer Cells, Natural/immunology , Cell Culture Techniques/instrumentation , Cell Proliferation , Cell Survival/immunology , Cells, Cultured , HEK293 Cells , Humans , Killer Cells, Natural/cytology , Killer Cells, Natural/ultrastructure , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Time-Lapse Imaging/methods
7.
Lab Chip ; 10(20): 2727-32, 2010 Oct 21.
Article in English | MEDLINE | ID: mdl-20820481

ABSTRACT

We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability. We use time-lapse imaging to show that cells continuously exposed to ultrasound are able to divide and remain viable for at least 12 hours inside the device. Thus, the device can be used to induce and maintain aggregation in a parallelized fashion, facilitating long-term microscopy studies of, e.g., cell-cell interactions.


Subject(s)
Cell Aggregation/physiology , Flow Cytometry/instrumentation , Microfluidic Analytical Techniques/instrumentation , Micromanipulation/instrumentation , Ultrasonics/instrumentation , Animals , Equipment Design , Equipment Failure Analysis , Humans
8.
Ultrasonics ; 49(1): 112-9, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18701122

ABSTRACT

We demonstrate and investigate multiple localized ultrasonic manipulation functions in series in microfluidic chips. The manipulation functions are based on spatially separated and confined ultrasonic primary radiation force fields, obtained by local matching of the resonance condition of the microfluidic channel. The channel segments are remotely actuated by the use of frequency-specific external transducers with refracting wedges placed on top of the chips. The force field in each channel segment is characterized by the use of micrometer-resolution particle image velocimetry (micro-PIV). The confinement of the ultrasonic fields during single- or dual-segment actuation, as well as the cross-talk between two adjacent fields, is characterized and quantified. Our results show that the field confinement typically scales with the acoustic wavelength, and that the cross-talk is insignificant between adjacent fields. The goal is to define design strategies for implementing several spatially separated ultrasonic manipulation functions in series for use in advanced particle or cell handling and processing applications. One such proof-of-concept application is demonstrated, where flow-through-mode operation of a chip with flow splitting elements is used for two-dimensional pre-alignment and addressable merging of particle tracks.


Subject(s)
Cell Separation/methods , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Micromanipulation/methods , Sonication , Stress, Mechanical
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