Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

Dynamic secretome of bone marrow-derived stromal cells reveals a cardioprotective biochemical cocktail.

Transplanted stromal cells have demonstrated considerable promise as therapeutic agents in diverse disease settings. Paracrine signaling can be an important mediator of these therapeutic effects at the sites of acute or persistent injury and inflammation. As many stromal cell types, including bone marrow-derived stromal cells (BMSCs), display tissue-specific responses, there is a need to explore their secretory dynamics in the context of tissue and injury type. Paracrine signals are not static, and could encode contextual dynamics in the kinetic changes of the concentrations of the secreted ligands. However, precise measurement of dynamic and context-specific cellular secretory signatures, particularly in adherent cells, remains challenging. Here, by creating an experimental and computational analysis platform, we reconstructed dynamic secretory signatures of cells based on a very limited number of time points. By using this approach, we demonstrate that the secretory signatures of CD133-positive BMSCs are uniquely defined by distinct biological contexts, including signals from injured cardiac cells undergoing oxidative stress, characteristic of cardiac infarction. Furthermore, we show that the mixture of recombinant factors reproducing the dynamics of BMSC-generated secretion can mediate a highly effective rescue of cells injured by oxidative stress and an improved cardiac output. These results support the importance of the dynamic multifactorial paracrine signals in mediating remedial effects of stromal stem cells, and pave the way for stem cell-inspired cell-free treatments of cardiac and other injuries.

A microphysiological model of the bronchial airways reveals the interplay of mechanical and biochemical signals in bronchospasm.

In asthma, the contraction of the airway smooth muscle and the subsequent decrease in airflow involve a poorly understood set of mechanical and biochemical events. Organ-level and molecular-scale models of the airway are frequently based on purely mechanical or biochemical considerations and do not account for physiological mechanochemical couplings. Here, we present a microphysiological model of the airway that allows for the quantitative analysis of the interactions between mechanical and biochemical signals triggered by compressive stress on epithelial cells. We show that a mechanical stimulus mimicking a bronchospastic challenge triggers the marked contraction and delayed relaxation of airway smooth muscle, and that this is mediated by the discordant expression of cyclooxygenase genes in epithelial cells and regulated by the mechanosensor and transcriptional co-activator Yes-associated protein. A mathematical model of the intercellular feedback interactions recapitulates aspects of obstructive disease of the airways, which include pathognomonic features of severe difficult-to-treat asthma. The microphysiological model could be used to investigate the mechanisms of asthma pathogenesis and to develop therapeutic strategies that disrupt the positive feedback loop that leads to persistent airway constriction.

Self-induced mechanical stress can trigger biofilm formation in uropathogenic Escherichia coli.

Bacterial biofilms represent an important medical problem; however, the mechanisms of the onset of biofilm formation are poorly understood. Here, using new controlled methods allowing high-throughput and reproducible biofilm growth, we show that biofilm formation is linked to self-imposed mechanical stress. In growing uropathogenic Escherichia coli colonies, we report that mechanical stress can initially emerge from the physical stress accompanying colony confinement within micro-cavities or hydrogel environments reminiscent of the cytosol of host cells. Biofilm formation can then be enhanced by a nutrient access-modulated feedback loop, in which biofilm matrix deposition can be particularly high in areas of increased mechanical and biological stress, with the deposited matrix further enhancing the stress levels. This feedback regulation can lead to adaptive and diverse biofilm formation guided by the environmental stresses. Our results suggest previously unappreciated mechanisms of the onset and progression of biofilm growth.

Brain-on-a-chip model enables analysis of human neuronal differentiation and chemotaxis.

Migration of neural progenitors in the complex tissue environment of the central nervous system is not well understood. Progress in this area has the potential to drive breakthroughs in neuroregenerative therapies, brain cancer treatments, and neurodevelopmental studies. To a large extent, advances have been limited due to a lack of controlled environments recapitulating characteristics of the central nervous system milieu. Reductionist cell culture models are frequently too simplistic, and physiologically more relevant approaches such as ex vivo brain slices or in situ experiments provide little control and make information extraction difficult. Here, we present a brain-on-chip model that bridges the gap between cell culture and ex vivo/in vivo conditions through recapitulation of self-organized neural differentiation. We use a new multi-layer silicone elastomer device, over the course of four weeks to differentiate pluripotent human (NTERA2) cells into neuronal clusters interconnected with thick axonal bundles and interspersed with astrocytes, resembling the brain parenchyma. Neurons within the device express the neurofilament heavy (NF200) mature axonal marker and the microtubule-associated protein (MAP2ab) mature dendritic marker, demonstrating that the devices are sufficiently biocompatible to allow neuronal maturation. This neuronal-glial environment is interfaced with a layer of human brain microvascular endothelial cells showing characteristics of the blood-brain barrier including the expression of zonula occludens (ZO1) tight junctions and increased trans-endothelial electrical resistance. We used this device to model migration of human neural progenitors in response to chemotactic cues within a brain-tissue setting. We show that in the presence of an environment mimicking brain conditions, neural progenitor cells show a significantly enhanced chemotactic response towards shallow gradients of CXCL12, a key chemokine expressed during embryonic brain development and in pathological tissue regions of the central nervous system. Our brain-on-chip model thus provides a convenient and scalable model of neural differentiation and maturation extensible to analysis of complex cell and tissue behaviors.

Migration Phenotype of Brain-Cancer Cells Predicts Patient Outcomes.

Glioblastoma multiforme is a heterogeneous and infiltrative cancer with dismal prognosis. Studying the migratory behavior of tumor-derived cell populations can be informative, but it places a high premium on the precision of in vitro methods and the relevance of in vivo conditions. In particular, the analysis of 2D cell migration may not reflect invasion into 3D extracellular matrices in vivo. Here, we describe a method that allows time-resolved studies of primary cell migration with single-cell resolution on a fibrillar surface that closely mimics in vivo 3D migration. We used this platform to screen 14 patient-derived glioblastoma samples. We observed that the migratory phenotype of a subset of cells in response to platelet-derived growth factor was highly predictive of tumor location and recurrence in the clinic. Therefore, migratory phenotypic classifiers analyzed at the single-cell level in a patient-specific way can provide high diagnostic and prognostic value for invasive cancers.

COX-2 dependent regulation of mechanotransduction in human breast cancer cells.

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7

Miniature photonic-crystal hydrophone optimized for ocean acoustics.

This work reports on an optical hydrophone that is insensitive to hydrostatic pressure, yet capable of measuring acoustic pressures as low as the background noise in the ocean in a frequency range of 1 Hz to 100 kHz. The miniature hydrophone consists of a Fabry-Perot interferometer made of a photonic-crystal reflector interrogated with a single-mode fiber and is compatible with existing fiber-optic technologies. Three sensors with different acoustic power ranges placed within a sub-wavelength sized hydrophone head allow a high dynamic range in the excess of 160 dB with a low harmonic distortion of better than -30 dB. A method for suppressing cross-coupling between sensors in the same hydrophone head is also proposed. A prototype was fabricated, assembled, and tested. The sensitivity was measured from 100 Hz to 100 kHz, demonstrating a sound-pressure-equivalent noise spectral density down to 12 µPa/Hz(1/2), a flatband wider than 10 kHz, and very low distortion.

Analysis of guided-resonance-based polarization beam splitting in photonic crystal slabs.

We present an analysis of the phase and amplitude responses of guided resonances in a photonic crystal slab. Through this analysis, we obtain the general rules and conditions under which a photonic crystal slab can be employed as a general elliptical polarization beam splitter, separating an incoming beam equally into its two orthogonal constituents, so that half the power is reflected in one polarization state, and half the power is transmitted in the other state. We show that at normal incidence a photonic crystal slab acts as a dual quarter-wave retarder in which the fast and slow axes are switched for reflection and transmission. We also analyze the case where such a structure operates at oblique incidences. As a result we show that the effective dielectric constant of the photonic crystal slab imposes the Brewster angle as a boundary, separating two ranges of angles with different mechanisms of polarization beam splitting. We show that the diattenuation can be tuned from zero to one to make the structure a circular or linear polarization beam splitter. We verify our analytical analysis through finite-difference time-domain simulations and experimental measurements at infrared wavelengths.

Controlling uncoupled resonances in photonic crystals through breaking the mirror symmetry.

We show that modes in a photonic crystal slab that are uncoupled to outside radiation in a symmetric structure can be excited by breaking the mirror symmetry through introducing a protrusion on the side of the photonic crystal holes. We show that coupling to these resonances can be controlled by the strength of this asymmetry, and that it is also possible to choose among modes to couple to, through the shape of the asymmetry introduced. We provide simple theoretical arguments that explain the effect, and present eigenmode simulations and time-domain simulations. We confirm this predicted behavior with measurements on a photonic crystal with a broken mirror symmetry that exhibits an additional sharp resonant feature with a linewidth of 0.5 nm, in agreement with both calculated and simulated predictions.

Photonic crystal slabs demonstrating strong broadband suppression of transmission in the presence of disorders.

We characterize the transmission spectra of out-of-plane, normal-incidence light of two-dimensional silicon photonic crystal slabs and observe excellent agreement between the measured data and finite-difference time-domain simulations over the 1050-1600-nm wavelength range. Crystals that are 340 nm thick and have holes of 330-nm radius on a square lattice of 998-nm pitch show 20-dB extinction in transmission from 1220 to 1255 nm. Increasing the hole radius to 450 nm broadens the extinction band further, and we obtain >85% extinction from 1310 to 1550 nm. Discrepancies between simulation and measurement are ascribed to disorder in the photonic lattice, which is measured through image processing on high-resolution scanning electron micrographs. Analysis of crystal imperfections indicates that they tend to average out narrowband spectral features, while having relatively small effects on broadband features.

Angular and polarization properties of a photonic crystal slab mirror.

It was recently demonstrated that a photonic crystal slab can function as a mirror for externally incident light along a normal direction with near-complete reflectivity over a broad wavelength range. We analyze the angular and polarization properties of such photonic crystal slab mirror, and show such reflectivity occurs over a sizable angular range for both polarizations. We also show that such mirror can be designed to reflect one polarization completely, while allowing 100% transmission for the other polarization, thus behaving as a polarization splitter with a complete contrast. The theoretical analysis is validated by comparing with experimental measurements.