Alveolar mimics with periodic strain and its effect on the cell layer formation

We report on the development of a new model of alveolar air–tissue interface on a chip. The model consists of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40–80 µm at the air–liquid interface by application of air pressure in the range of 200–1,000 Pa. With respect to the diameter of the monolayers, that is, 500 µm, this displacement corresponds to a linear strain of 2–10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time of 1–3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz for 1 hr to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy.     

Toward Long‐Term Stable and Highly Efficient Perovskite Solar Cells via Effective Charge Transporting Materials

Perovskite solar cells (PSCs) have advanced quickly with their power conversion efficiency approaching the record of silicon solar cells. However, there is still a big challenge to obtain both high efficiency and long‐term stability for future commercialization of PSCs. The major instability issue is associated with the decomposition or phase transition of perovskite materials that are believed to be intrinsically unstable under outdoor working conditions. Herein, the authors review the approaches that marked important progress in developing new functional electron/hole transporting materials that enabled highly efficient and stable PSCs. The findings that accelerate charge diffusion and that suppress the irrevocable loss of ions diffusing out of perovskite materials and other diffusion processes are highlighted. In addition, derivative interface engineering methods to control the diffusion process of charges/ions/molecules are also reviewed. Finally, the authors propose key research issues in charge transporting materials and interface engineering with regard to the important diffusion processes that will be one of the keys to realize highly efficient and long‐term stable PSCs.     

Parameterization and classification of the protein universe via geometric techniques

We present a scheme for the classification of 3487 non-redundant protein structures into 1207 non-hierarchical clusters by using recurring structural patterns of three to six amino acids as keys of classification. This results in several signature patterns, which seem to decide membership of a protein in a functional category. The patterns provide clues to the key residues involved in functional sites as well as in protein-protein interaction. The discovered patterns include a "glutamate double bridge" of superoxide dismutase, the functional interface of the serine protease and inhibitor, interface of homo/hetero dimers, and functional sites of several enzyme families. We use geometric invariants to decide superimposability of structural patterns. This allows the parameterization of patterns and discovery of recurring patterns via clustering. The geometric invariant-based approach eliminates the computationally explosive step of pair-wise comparison of structures. The results provide a vast resource for the biologists for experimental validation of the proposed functional sites, and for the design of synthetic enzymes, inhibitors and drugs.     

Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein,BCR, and Regulates Levels of Rac1-GTP

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.     

ConfocalVR: Immersive Visualization for Confocal Microscopy

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of two-dimensional images throughout the specimen. Current software applications reconstruct the three-dimensional (3D) image and render it as a two-dimensional projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade VR systems to fully immerse the user in the 3D cellular image. In this virtual environment, the user can (1) adjust image viewing parameters without leaving the virtual space, (2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and (3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits.     

Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1–2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.     

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter,ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.