Tyrosine/Cysteine Cluster Sensitizing Human γD-Crystallin to Ultraviolet Radiation-Induced Photoaggregation in Vitro

Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of HγD-Crys by replacing pairs with alanines and monitoring photoaggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light-scattering aggregates. Further mutant studies implicated Y16 as important for photoaggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The data suggest an initial photoaggregation pathway for HγD-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.National Eye Institute (EY015834

UV-radiation Induced Disruption of Dry-Cavities in Human γD-crystallin Results in Decreased Stability and Faster Unfolding

Age-onset cataracts are believed to be expedited by the accumulation of UV-damaged human γD-crystallins in the eye lens. Here we show with molecular dynamics simulations that the stability of γD-crystallin is greatly reduced by the conversion of tryptophan to kynurenine due to UV-radiation, consistent with previous experimental evidences. Furthermore, our atomic-detailed results reveal that kynurenine attracts more waters and other polar sidechains due to its additional amino and carbonyl groups on the damaged tryptophan sidechain, thus breaching the integrity of nearby dry center regions formed by the two Greek key motifs in each domain. The damaged tryptophan residues cause large fluctuations in the Tyr-Trp-Tyr sandwich-like hydrophobic clusters, which in turn break crucial hydrogen-bonds bridging two β-strands in the Greek key motifs at the “tyrosine corner”. Our findings may provide new insights for understanding of the molecular mechanism of the initial stages of UV-induced cataractogenesis.International Business Machines Corporation (IBM Blue Gene Program

Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions

Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of ~3, compared with conventional cryoEM. The reconstruction was carried out to 13 Å resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.National Institutes of Health (U.S.) (Grant P41RR002250)National Institutes of Health (U.S.) (Grant R01AI0175208)National Institutes of Health (U.S.) (Grant PN1EY016525)Robert Welch Foundation (Q1242

Assessment of Knowledge-Based Planning for Prostate Intensity Modulated Proton Therapy

Purpose: To assess the performance of a proton-specific knowledge based planning (KBPP) model in creation of robustly optimized intensity-modulated proton therapy (IMPT) plans for treatment of patients with prostate cancer. Materials and Methods: Forty-five patients with localized prostate cancer, who had previously been treated with volumetric modulated arc therapy, were selected and replanned with robustly optimized IMPT. A KBPP model was generated from the results of 30 of the patients, and the remaining 15 patient results were used for validation. The KBPP model quality and accuracy were evaluated with the model-provided organ-at-risk regression plots and metrics. The KBPP quality was also assessed through comparison of expert and KBPP-generated IMPT plans for target coverage and organ-at-risk sparing. Results: The resulting R (2) (mean ± SD, 0.87 ± 0.07) between dosimetric and geometric features, as well as the χ(2) test (1.17 ± 0.07) between the original and estimated data, showed the model had good quality. All the KBPP plans were clinically acceptable. Compared with the expert plans, the KBPP plans had marginally higher dose-volume indices for the rectum V65Gy (0.8% ± 2.94%), but delivered a lower dose to the bladder (-1.06% ± 2.9% for bladder V65Gy). In addition, KBPP plans achieved lower hotspot (-0.67Gy ± 2.17Gy) and lower integral dose (-0.09Gy ± 0.3Gy) than the expert plans did. Moreover, the KBPP generated better plans that demonstrated slightly greater clinical target volume V95 (0.1% ± 0.68%) and lower homogeneity index (-1.13 ± 2.34). Conclusions: The results demonstrated that robustly optimized IMPT plans created by the KBPP model are of high quality and are comparable to expert plans. Furthermore, the KBPP model can generate more-robust and more-homogenous plans compared with those of expert plans. More studies need to be done for the validation of the proton KBPP model at more-complicated treatment sites

Functional liquid structures by emulsification of graphene and other two-dimensional nanomaterials

Pickering emulsions stabilised with nanomaterials provide routes to a range of functional macroscopic assemblies. We demonstrate the formation and properties of water-in-oil emulsions prepared through liquid-phase exfoliation of graphene. Due to the functional nature of the stabiliser, the emulsions exhibit conductivity due to inter-particle tunnelling. We demonstrate a strain sensing application with a large gauge factor of ~40; the highest reported in a liquid. Our methodology can be applied to other two-dimensional layered materials opening up applications such as energy storage materials, and flexible and printable electronics

Ultrasensitive strain gauges enabled by graphene-stabilized silicone emulsions

Here, an approach is presented to incorporate graphene nanosheets into a silicone rubber matrix via solid stabilization of oil‐in‐water emulsions. These emulsions can be cured into discrete, graphene‐coated silicone balls or continuous, elastomeric films by controlling the degree of coalescence. The electromechanical properties of the resulting composites as a function of interdiffusion time and graphene loading level are characterized. With conductivities approaching 1 S m−1, elongation to break up to 160%, and a gauge factor of ≈20 in the low‐strain linear regime, small strains such as pulse can be accurately measured. At higher strains, the electromechanical response exhibits a robust exponential dependence, allowing accurate readout for higher strain movements such as chest motion and joint bending. The exponential gauge factor is found to be ≈20, independent of loading level and valid up to 80% strain; this consistent performance is due to the emulsion‐templated microstructure of the composites. The robust behavior may facilitate high‐strain sensing in the nonlinear regime using nanocomposites, where relative resistance change values in excess of 107 enable highly accurate bodily motion monitoring

Accurate model annotation of a near-atomic resolution cryo-EM map

Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo- EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly.With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.National Institutes of Health (U.S.) (Grant P41GM103832)National Institutes of Health (U.S.) (Grant R01GM079429)National Institutes of Health (U.S.) (Grant PN2EY016525)National Institutes of Health (U.S.) (Grant P01GM063210)Robert A. Welch Foundation (Grant Q1242

Size selection and thin-film assembly of MoS2 elucidates thousandfold conductivity enhancement in few-layer nanosheet networks

Printed electronics based on liquid-exfoliated nanosheet networks are limited by inter-nanosheet junctions and thick films which hinder field-effect gating. Here, few-layer molybdenum disulfide nanosheets are assembled by Langmuir deposition into thin films, and size selection is shown to lead to a thousandfold conductivity enhancement with potential applicability to all nanosheet networks

Mechanochromic and thermochromic sensors based on graphene infused polymer opals

High quality opal‐like photonic crystals containing graphene are fabricated using evaporation‐driven self‐assembly of soft polymer colloids. A miniscule amount of pristine graphene within a colloidal crystal lattice results in the formation of colloidal crystals with a strong angle‐dependent structural color and a stop band that can be reversibly shifted across the visible spectrum. The crystals can be mechanically deformed or can reversibly change color as a function of their temperature, hence their sensitive mechanochromic and thermochromic response make them attractive candidates for a wide range of visual sensing applications. In particular, it is shown that the crystals are excellent candidates for visual strain sensors or integrated time‐temperature indicators which act over large temperature windows. Given the versatility of these crystals, this method represents a simple, inexpensive, and scalable approach to produce multifunctional graphene infused synthetic opals and opens up exciting applications for novel solution‐processable nanomaterial based photonics