Long-term, non-invasive FTIR detection of low-dose ionizing radiation exposure.

Non-invasive methods of detecting radiation exposure show promise to improve upon current approaches to biological dosimetry in ease, speed, and accuracy. Here we developed a pipeline that employs Fourier transform infrared (FTIR) spectroscopy in the mid-infrared spectrum to identify a signature of low dose ionizing radiation exposure in mouse ear pinnae over time. Mice exposed to 0.1 to 2 Gy total body irradiation were repeatedly measured by FTIR at the stratum corneum of the ear pinnae. We found significant discriminative power for all doses and time-points out to 90 days after exposure. Classification accuracy was maximized when testing 14 days after exposure (specificity > 0.9 with a sensitivity threshold of 0.9) and dropped by roughly 30% sensitivity at 90 days. Infrared frequencies point towards biological changes in DNA conformation, lipid oxidation and accumulation and shifts in protein secondary structure. Since only hundreds of samples were used to learn the highly discriminative signature, developing human-relevant diagnostic capabilities is likely feasible and this non-invasive procedure points toward rapid, non-invasive, and reagent-free biodosimetry applications at population scales.

Chromohalobacter salixigens Uronate Dehydrogenase: Directed Evolution for Improved Thermal Stability and Mutant CsUDH-inc X-ray Crystal Structure.

Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolution using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family library contained in total 16 mutations, only some of which when examined individually appreciably increased thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isolation, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had ΔK t 0.5 +18 °C, such that k cat was unaffected by incubation for 1 hr at ~70 °C. X-ray crystal structure of CsUDH-inc showed tight packing of the mutated residue side-chains, and comparison of rescaled B-values showed no obvious differences between wild type and mutant structures. Activity of CsUDH-inc was severely depressed on glucuronic and galacturonic acids. Combining select combinations of only three mutations resulted in good or comparable activity on these uronic acids, while maintaining some improved thermostability with ΔK t 0.5 ~+ 10 °C, indicating potential to further thermally optimize CsUDH for hyperthermophilic reaction environments.

Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens.

Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.

Vaccines train the immune system to recognize a specific virus or bacterium so that the body can be better prepared against these harmful agents. To do so, many vaccines contain viral molecules called glycoproteins, which are specific to each type of virus. Glycoproteins that sit at the surface of the virus can act as 'keys' that recognize and unlock the cells of certain organisms, leading to viral infection. To ensure a stronger immune response, glycoproteins in vaccines are often arranged on a protein scaffolding which can mimic the shape of the virus of interest and trigger a strong immune response. Many scaffoldings, however, are currently made from natural proteins which cannot always display viral glycoproteins. Here, Ueda, Antanasijevic et al. developed a method that allows for the design of artificial proteins which can serve as scaffolding for viral glycoproteins. This approach was tested using three viruses: influenza, HIV, and RSV ­ a virus responsible for bronchiolitis. The experiments showed that in each case, the relevant viral glycoproteins could attach themselves to the scaffolding. These structures could then assemble themselves into vaccine particles with predicted geometrical shapes, which mimicked the virus and maximized the response from the immune system. Designing artificial scaffolding for viral glycoproteins gives greater control over vaccine design, allowing scientists to manipulate the shape of vaccine particles and test the impact on the immune response. Ultimately, the approach developed by Ueda, Antanasijevic et al. could lead to vaccines that are more efficient and protective, including against viruses for which there is currently no suitable scaffolding.

Minimally disruptive optical control of protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Author Correction: Coherent diffractive imaging of microtubules using an X-ray laser.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Coherent diffractive imaging of microtubules using an X-ray laser.

X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2 nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4 nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.

Bone Marrow-Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration.

BACKGROUND: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. HYPOTHESIS: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. STUDY DESIGN: Controlled laboratory study. METHODS: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques-BM aspiration and BM collection-after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. RESULTS: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45-CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56- cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. CONCLUSION: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. CLINICAL RELEVANCE: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.

Evolutionarily Conserved Allosteric Communication in Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are an important class of regulatory enzymes that exhibit aberrant activities in a wide range of diseases. A detailed mapping of allosteric communication in these enzymes could, thus, reveal the structural basis of physiologically relevant-and, perhaps, therapeutically informative-perturbations (i.e., mutations, post-translational modifications, or binding events) that influence their catalytic states. This study combines detailed biophysical studies of protein tyrosine phosphatase 1B (PTP1B) with bioinformatic analyses of the PTP family to examine allosteric communication in this class of enzymes. Results of X-ray crystallography, molecular dynamics simulations, and sequence-based statistical analyses indicate that PTP1B possesses a broadly distributed allosteric network that is evolutionarily conserved across the PTP family, and findings from both kinetic studies and mutational analyses show that this network is functionally intact in sequence-diverse PTPs. The allosteric network resolved in this study reveals new sites for targeting allosteric inhibitors of PTPs and helps explain the functional influence of a diverse set of disease-associated mutations.

Abietane-Type Diterpenoids Inhibit Protein Tyrosine Phosphatases by Stabilizing an Inactive Enzyme Conformation.

Protein tyrosine phosphatases (PTPs) contribute to a striking variety of human diseases, yet they remain vexingly difficult to inhibit with uncharged, cell-permeable molecules; no inhibitors of PTPs have been approved for clinical use. This study uses a broad set of biophysical analyses to evaluate the use of abietane-type diterpenoids, a biologically active class of phytometabolites with largely nonpolar structures, for the development of pharmaceutically relevant PTP inhibitors. Results of nuclear magnetic resonance analyses, mutational studies, and molecular dynamics simulations indicate that abietic acid can inhibit protein tyrosine phosphatase 1B, a negative regulator of insulin signaling and an elusive drug target, by binding to its active site in a non-substrate-like manner that stabilizes the catalytically essential WPD loop in an inactive conformation; detailed kinetic studies, in turn, show that minor changes in the structures of abietane-type diterpenoids (e.g., the addition of hydrogens) can improve potency (i.e., lower IC50) by 7-fold. These findings elucidate a previously uncharacterized mechanism of diterpenoid-mediated inhibition and suggest, more broadly, that abietane-type diterpenoids are a promising source of structurally diverse-and, intriguingly, microbially synthesizable-molecules on which to base the design of new PTP-inhibiting therapeutics.

Resonant Soft X-Ray Scattering Provides Protein Structure with Chemical Specificity.

We introduce resonant soft X-ray scattering (RSoXS) as an approach to study the structure of proteins and other biological molecules in solution. Scattering contrast calculations suggest that RSoXS has comparable or even higher sensitivity than hard X-ray scattering because of contrast generated at the absorption edges of constituent elements, such as carbon and oxygen. Here, we demonstrate that working near the carbon edge reveals the envelope function of bovine serum albumin, using scattering volumes of 10-5 µL that are multiple orders of magnitude lower than traditional scattering experiments. Furthermore, tuning the X-ray energy within the carbon absorption edge provides different signatures of the size and shape of the protein by revealing the density of different types of bonding motifs within the protein. The combination of chemical specificity, smaller sample size, and enhanced X-ray contrast will propel RSoXS as a complementary tool to existing techniques for the study of biomolecular structure.

Immune Cell Induced Migration of Osteoprogenitor Cells Is Mediated by TGF-ß Dependent Upregulation of NOX4 and Activation of Focal Adhesion Kinase.

The cytokines secreted by immune cells have a large impact on the tissue, surrounding a fracture, e.g., by attraction of osteoprogenitor cells. However, the underlying mechanisms are not yet fully understood. Thus, this study aims at investigating molecular mechanisms of the immune cell-mediated migration of immature primary human osteoblasts (phOBs), with transforming growth factor beta (TGF-ß), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and focal adhesion kinase (FAK) as possible regulators. Monocyte- and macrophage (THP-1 cells ± phorbol 12-myristate 13-acetate (PMA) treatment)-conditioned media, other than the granulocyte-conditioned medium (HL-60 cells + dimethyl sulfoxide (DMSO) treatment), induce migration of phOBs. Monocyte- and macrophage (THP-1 cells)-conditioned media activate Smad3-dependent TGF-ß signaling in the phOBs. Stimulation with TGF-ß promotes migration of phOBs. Furthermore, TGF-ß treatment strongly induces NOX4 expression on both mRNA and protein levels. The associated reactive oxygen species (ROS) accumulation results in phosphorylation (Y397) of FAK. Blocking TGF-ß signaling, NOX4 activity and FAK signaling effectively inhibits the migration of phOBs towards TGF-ß. In summary, our data suggest that monocytic- and macrophage-like cells induce migration of phOBs in a TGF-ß-dependent manner, with TGF-ß-dependent induction of NOX4, associated production of ROS and resulting activation of FAK as key mediators.

Xi-cam: a versatile interface for data visualization and analysis.

Xi-cam is an extensible platform for data management, analysis and visualization. Xi-cam aims to provide a flexible and extensible approach to synchrotron data treatment as a solution to rising demands for high-volume/high-throughput processing pipelines. The core of Xi-cam is an extensible plugin-based graphical user interface platform which provides users with an interactive interface to processing algorithms. Plugins are available for SAXS/WAXS/GISAXS/GIWAXS, tomography and NEXAFS data. With Xi-cam's `advanced' mode, data processing steps are designed as a graph-based workflow, which can be executed live, locally or remotely. Remote execution utilizes high-performance computing or de-localized resources, allowing for the effective reduction of high-throughput data. Xi-cam's plugin-based architecture targets cross-facility and cross-technique collaborative development, in support of multi-modal analysis. Xi-cam is open-source and cross-platform, and available for download on GitHub.

[Implantation of a Modular Distal Femoral Replacement with Compressive Osseointegration as a Salvage Procedure in a Complex Femoral Posttraumatic Setting]. / Implantation einer modularen Knietotalendoprothese mit distalem Femurersatz mit kompressiver Osteointegration als Rückzugsoption bei komplexer femoraler posttraumatischer Ausgangssituation.

BACKGROUND: Large bone defects and losses play a crucial role in both tumour surgery and in complex primary and revision total knee replacement. The established options of cemented or uncemented long intramedullary stems are limited by large bone defects and are at risk from relatively high exposure to aseptic loosening. There is no general valid agreement on implant fixation of the distal femur. A further option is the cementless fixation method with compressive osseointegration, based on the so-called Wolff law of bone remodelling. This method was developed in order to reduce the high loosening rates in revision arthroplasty due to intense stress shielding and is intended to be applied in patients with huge bone losses. The so-called Compress® system (or CPS) allows such a distal femur reconstruction. It has mainly been applied and evaluated in tumor endoprosthetics. There are currently few data on the application of this system in complex distal femoral posttraumatic deformity or revision arthroplasty. PATIENTS: A case report of two male patients aged 59/56 years with a 1-year follow-up is presented. Both patients had a complex post-traumatic femoral deformity with bone loss, prior surgery and an ipsilateral hip replacement. Implantation was performed of a modular total knee replacement, consisting of a cemented modular tibia base plate and distal femoral replacement with cementless implant fixation by compressive osseointegration. Both patients were clinically and radiologically evaluated prospectively. RESULTS: Good clinical and radiological results were demonstrated in both patients after distal femoral replacement by compressive osseointegration. There was no need for further or revision surgery. Both patients were rapidly able to resume their jobs. The survival rates for CPS were comparable to published values with conventional procedures. There are yet no long-term results or extensive data for revision arthroplasty or posttraumatic cases. CONCLUSIONS: Besides distal femoral replacement with compressive osseointegration in oncological arthroplasty, the indication of complex distal femoral settings with large bone defects can be evaluated for daily clinical routine. Especially if there is ipsilateral total hip replacement, this option might be used to avoid interprosthetic stress risers.

Correlations in Scattered X-Ray Laser Pulses Reveal Nanoscale Structural Features of Viruses.

We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.

Reconstruction from limited single-particle diffraction data via simultaneous determination of state, orientation, intensity, and phase.

Free-electron lasers now have the ability to collect X-ray diffraction patterns from individual molecules; however, each sample is delivered at unknown orientation and may be in one of several conformational states, each with a different molecular structure. Hit rates are often low, typically around 0.1%, limiting the number of useful images that can be collected. Determining accurate structural information requires classifying and orienting each image, accurately assembling them into a 3D diffraction intensity function, and determining missing phase information. Additionally, single particles typically scatter very few photons, leading to high image noise levels. We develop a multitiered iterative phasing algorithm to reconstruct structural information from single-particle diffraction data by simultaneously determining the states, orientations, intensities, phases, and underlying structure in a single iterative procedure. We leverage real-space constraints on the structure to help guide optimization and reconstruct underlying structure from very few images with excellent global convergence properties. We show that this approach can determine structural resolution beyond what is suggested by standard Shannon sampling arguments for ideal images and is also robust to noise.

Water-Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase.

This study uses mutants of human carbonic anhydrase (HCAII) to examine how changes in the organization of water within a binding pocket can alter the thermodynamics of protein-ligand association. Results from calorimetric, crystallographic, and theoretical analyses suggest that most mutations strengthen networks of water-mediated hydrogen bonds and reduce binding affinity by increasing the enthalpic cost and, to a lesser extent, the entropic benefit of rearranging those networks during binding. The organization of water within a binding pocket can thus determine whether the hydrophobic interactions in which it engages are enthalpy-driven or entropy-driven. Our findings highlight a possible asymmetry in protein-ligand association by suggesting that, within the confines of the binding pocket of HCAII, binding events associated with enthalpically favorable rearrangements of water are stronger than those associated with entropically favorable ones.

Computational design of self-assembling cyclic protein homo-oligomers.

Self-assembling cyclic protein homo-oligomers play important roles in biology, and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue-pair-transform method to assess the designability of a protein-protein interface. This method is sufficiently rapid to enable the systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were characterized experimentally, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (four homodimers, six homotrimers, six homotetramers and one homopentamer) had solution small-angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each is very close to their corresponding computational model.

Principles for designing proteins with cavities formed by curved ß sheets.

Active sites and ligand-binding cavities in native proteins are often formed by curved ß sheets, and the ability to control ß-sheet curvature would allow design of binding proteins with cavities customized to specific ligands. Toward this end, we investigated the mechanisms controlling ß-sheet curvature by studying the geometry of ß sheets in naturally occurring protein structures and folding simulations. The principles emerging from this analysis were used to design, de novo, a series of proteins with curved ß sheets topped with α helices. Nuclear magnetic resonance and crystal structures of the designs closely match the computational models, showing that ß-sheet curvature can be controlled with atomic-level accuracy. Our approach enables the design of proteins with cavities and provides a route to custom design ligand-binding and catalytic sites.