Measurement of cell microrheology by magnetic twisting cytometry with frequency domain demodulation.

Magnetic twisting cytometry (MTC) (Wang N, Butler JP, and Ingber DE, Science 260: 1124-1127, 1993) is a useful technique for probing cell micromechanics. The technique is based on twisting ligand-coated magnetic microbeads bound to membrane receptors and measuring the resulting bead rotation with a magnetometer. Owing to the low signal-to-noise ratio, however, the magnetic signal must be modulated, which is accomplished by spinning the sample at approximately 10 Hz. Present demodulation approaches limit the MTC range to frequencies <0.5 Hz. We propose a novel demodulation algorithm to expand the frequency range of MTC measurements to higher frequencies. The algorithm is based on coherent demodulation in the frequency domain, and its frequency range is limited only by the dynamic response of the magnetometer. Using the new algorithm, we measured the complex modulus of elasticity (G*) of cultured human bronchial epithelial cells (BEAS-2B) from 0.03 to 16 Hz. Cells were cultured in supplemented RPMI medium, and ferromagnetic beads (approximately 5 microm) coated with an RGD peptide were bound to the cell membrane. Both the storage (G', real part of G*) and loss (G", imaginary part of G*) moduli increased with frequency as omega(alpha) (2 pi x frequency) with alpha approximately equal to 1/4. The ratio G"/G' was approximately 0.5 and varied little with frequency. Thus the cells exhibited a predominantly elastic behavior with a weak power law of frequency and a nearly constant proportion of elastic vs. frictional stresses, implying that the mechanical behavior conformed to the so-called structural damping (or constant-phase) law (Maksym GN, Fabry B, Butler JP, Navajas D, Tschumperlin DJ, LaPorte JD, and Fredberg JJ, J Appl Physiol 89: 1619-1632, 2000). We conclude that frequency domain demodulation dramatically increases the frequency range that can be probed with MTC and reveals that the mechanics of these cells conforms to constant-phase behavior over a range of frequencies approaching three decades.     

Genome engineering in actinomycetes using site-specific recombinases

The rational modification of the actinomycetes genomes has a variety of applications in research, medicine, and biotechnology. The use of site-specific recombinases allows generation of multiple mutations, large DNA deletions, integrations, and inversions and may lead to significant progress in all of these fields. Despite their huge potential, site-specific recombinase-based technologies have primarily been used for simple marker removal from a chromosome. In this review, we summarise the site-specific recombination approaches for genome engineering in various actinomycetes.     

Donor CD8 T cells and IFN-gamma are critical for sex-based differences in donor CD4 T cell engraftment and lupus-like phenotype in short-term chronic graft-versus-host disease mice

The transfer of unfractionated DBA/2J (DBA) splenocytes into B6D2F(1) (DBA → F(1)) mice results in greater donor CD4 T cell engraftment in females at day 14 that persists long-term and mediates greater female lupus-like renal disease. Although donor CD8 T cells have no demonstrated role in lupus pathogenesis in this model, we recently observed that depletion of donor CD8 T cells prior to transfer eliminates sex-based differences in renal disease long-term. In this study, we demonstrate that greater day 14 female donor CD4 engraftment is also critically dependent on donor CD8 T cells. Male DBA → F(1) mice exhibit stronger CD8-dependent day 8-10 graft-versus-host (GVH) and counter-regulatory host-versus-graft (HVG) responses, followed by stronger homeostatic contraction (days 10-12). The weaker day 10-12 GVH and HVG in females are followed by persistent donor T cell activation and increasing proliferation, expansion, and cytokine production from days 12 to 14. Lastly, greater female day 14 donor T cell engraftment, activation, and cytokine production were lost with in vivo IFN-γ neutralization from days 6 to 14. We conclude the following: 1) donor CD8 T cells enhance day 10 proliferation of donor CD4 T cells in both sexes; and 2) a weaker GVH/HVG in females allows prolonged survival of donor CD4 and CD8 T cells, allowing persistent activation. These results support the novel conclusion that sex-based differences in suboptimal donor CD8 CTL activation are critical for shaping sex-based differences in donor CD4 T cell engraftment at 2 wk and lupus-like disease long-term.     

Photophysics of PbS Quantum Dot Films Capped with Arsenic Sulfide Ligands

PbS quantum dots (QDs) of different sizes capped with short (NH₄)₃AsS₃ inorganic ligands are produced via ligand exchange processes from oleate‐capped PbS QDs. The solid‐state photophysical properties of the control organic‐capped and the inorganic‐ligand‐capped QDs are investigated to determine their potential for optoelectronic applications. Ultrafast transient transmission shows that in the oleate‐capped QDs, carrier recombination at sub‐nanosecond scales occurs via Auger recombination, traps, and surface states. At longer times, intense signals associated with radiative recombination are obtained. After ligand exchange, the QDs become decorated with (NH₄)₃AsS₃ complexes and relaxation is dominated by efficient carrier transfer to the ligand states on timescales as fast as ≈2 ps, which competes with carrier thermalization to the QD band edge states. Recombination channels present in the oleate‐capped QDs, such as radiative and Auger recombination, appear quenched in the inorganic‐capped QDs. Evidence of efficient carrier trapping at shallow ligand states, which appears more intense under excitation above the (NH₄)₃AsS₃ gap, is provided. A detailed band diagram of the various relaxation and recombination processes is proposed that comprehensively describes the photophysics of the QD systems studied.     

Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry

Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.     

The functional response of U937 macrophage-like cells is modulated by extracellular matrix proteins and mechanical strain.

Extracellular matrix proteins (ECMs) play a significant role in the transfer of mechanical strain to monocyte-derived macrophages (MDMs) affecting morphological changes in a foreign body reaction. This study investigated how the functional responses of U937 macrophage-like cells differed when subjected to 2 dynamic strain types (nonuniform biaxial or uniform uniaxial strain) while cultured on siloxane membranes coated with either collagen type I or RGD peptide repeats (ProNectin). Biaxial strain caused an increase in intracellular esterase and acid phosphatase (AP) activities, as well as monocyte-specific esterase (MSE) protein levels in cells that were seeded on either uncoated surfaces (shown previously) or collagen, but not ProNectin. Released AP activity, but not released esterase activity, was increased on all surfaces. Biaxial strain increased IL-6, but not IL-8 on all surfaces. When cells were subjected to uniaxial strain, intracellular esterase increased on coated surfaces only, whereas intracellular AP activity was unaffected. Both esterase and AP released activities increased on all surfaces. Uniaxial strain increased the release of IL-6 on all surfaces, but IL-8 on coated surfaces only. This study demonstrated for the first time that ECM proteins could specifically modulate cellular responses to different types of strain. Using this approach with an in vitro cell system may help to unravel the complex function of MDMs in the foreign-body reaction.     

Chronic oscillatory strain induces MLCK associated rapid recovery from acute stretch in airway smooth muscle cells

A deep inspiration (DI) temporarily relaxes agonist-constricted airways in normal subjects, but in asthma airways are refractory and may rapidly renarrow, possibly due to changes in the structure and function of airway smooth muscle (ASM). Chronic largely uniaxial cyclic strain of ASM cells in culture causes several structural and functional changes in ASM similar to that in asthma, including increases in contractility, MLCK content, shortening velocity, and shortening capacity. However, changes in recovery from acute stretch similar to a DI have not been measured. We have therefore measured the response and recovery to large stretches of cells modified by chronic stretching and investigated the role of MLCK. Chronic, 10% uniaxial cyclic stretch, with or without a strain gradient, was administered for up to 11 days to cultured cells grown on Silastic membranes. Single cells were then removed from the membrane and subjected to 1 Hz oscillatory stretches up to 10% of the in situ cell length. These oscillations reduced stiffness by 66% in all groups (P < 0.05). Chronically strained cells recovered stiffness three times more rapidly than unstrained cells, while the strain gradient had no effect. The stiffness recovery in unstrained cells was completely inhibited by the MLCK inhibitor ML-7, but recovery in strained cells exhibiting increased MLCK was slightly inhibited. These data suggest that chronic strain leads to enhanced recovery from acute stretch, which may be attributable to the strain-induced increases in MLCK. This may also explain in part the more rapid renarrowing of activated airways following DI in asthma.     

Mechanical strain increases cell stiffness through cytoskeletal filament reorganization

We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G'). G' was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10-12 days. G' was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G' Para was 24 +/- 5% (+/- SE) greater compared with Perp (P < 0.05), and 35% +/- 6 greater compared with control (Cont, P < 0.01). The difference between strained and control cells was enhanced by KCl, increasing G' 171 +/- 7% Para compared with 125 +/- 6% Perp and 129 +/- 8% Cont (P < 10-5 both cases). The decrease in G' from baseline due to relaxant agonists isoproterenol and dibutyryl cAMP was similar in all groups. Long-term oscillatory loading of airway smooth muscle (ASM) cells caused stiffness to increase and become anisotropic. These findings are consistent with the hypothesis that cytoskeletal reorganization can enhance ASM stiffness and contractility. They imply, furthermore, that oscillatory loading of ASM may contribute to airway narrowing and failure of airway dilation in asthma.