Broadband infrared absorber based on a sputter deposited hydrogenated carbon multilayer enhancing MEMS-based CMOS thermopile performance.

Based on pulsed DC sputter deposition of hydrogenated carbon, an absorber optical coating with maximized broadband infrared absorptance is reported. Enhanced broadband (2.5-20 µm) infrared absorptance (>90%) with reduced infrared reflection is achieved by combining a low-absorptance antireflective (hydrogenated carbon) overcoat with a broadband-absorptance carbon underlayer (nonhydrogenated). The infrared optical absorptance of sputter deposited carbon with incorporated hydrogen is reduced. As such, hydrogen flow optimization to minimize reflection loss, maximize broadband absorptance, and achieve stress balance is described. Application to complementary metal-oxide-semiconductor (CMOS) produced microelectromechanical systems (MEMS) thermopile device wafers is described. A 220% increase in thermopile output voltage is demonstrated, in agreement with modeled prediction.

Cisplatin uptake and release in pH sensitive zeolitic imidazole frameworks.

Cancer remains hard to treat, partially due to the non-specificity of chemotherapeutics. Metal-organic frameworks (MOFs) are promising carriers for targeted chemotherapy, yet, to date, there have been few detailed studies to systematically enhance drug loading while maintaining controlled release. In this work, we investigate which molecular simulation methods best capture the experimental uptake and release of cisplatin from UiO-66 and UiO-66(NH2). We then screen a series of biocompatible, pH-sensitive zeolitic imidazolate frameworks (ZIFs) for their ability to retain cisplatin in healthy parts of the patient and release it in the vicinity of a tumor. Pure-component GCMC simulations show that the maximum cisplatin loading depends on the pore volume. To achieve this maximum loading in the presence of water, either the pore size needs to be large enough to occupy both cisplatin and its solvation shell or the MOF-cisplatin interaction must be more favorable than the cisplatin-shell interaction. Both solvated and non-solvated simulations show that cisplatin release rates can be controlled by either decreasing the pore limiting diameters or by manipulating framework-cisplatin interaction energies to create strong, dispersed adsorption sites. The latter method is preferable if cisplatin loading is performed from solution into a pre-synthesized framework as weak interaction energies and small pore window diameters will hinder cisplatin uptake. Here, ZIF-82 is most promising. If it is possible to load cisplatin during crystallization, ZIF-11 would outcompete the other MOFs screened as cisplatin cannot pass through its pore windows; therefore, release rates would be purely driven by the pH triggered framework degradation.

Monoclonal antibody stability can be usefully monitored using the excitation-energy-dependent fluorescence edge-shift.

Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.

TCR-induced alteration of primary MHC peptide anchor residue.

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.

Exposing the Interplay Between Enzyme Turnover, Protein Dynamics, and the Membrane Environment in Monoamine Oxidase B.

There is an increasing realization that structure-based drug design may show improved success by understanding the ensemble of conformations accessible to an enzyme and how the environment affects this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinson's disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear, and routes to drugging this target would be valuable. Evidence of a radical in either the transition state or the resting state of MAO-B is present throughout the literature and is suggested to be a flavin semiquinone, a tyrosyl radical, or both. Here we see evidence of a resting-state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. On the basis of enzyme kinetic studies, enzyme variants, and molecular dynamics simulations, we find evidence for the importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity and protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these targets.

Intrinsic Flexibility of the EMT Zeolite Framework under Pressure.

The roles of organic additives in the assembly and crystallisation of zeolites are still not fully understood. This is important when attempting to prepare novel frameworks to produce new zeolites. We consider 18-crown-6 ether (18C6) as an additive, which has previously been shown to differentiate between the zeolite EMC-2 (EMT) and faujasite (FAU) frameworks. However, it is unclear whether this distinction is dictated by influences on the metastable free-energy landscape or geometric templating. Using high-pressure synchrotron X-ray diffraction, we have observed that the presence of 18C6 does not impact the EMT framework flexibility-agreeing with our previous geometric simulations and suggesting that 18C6 does not behave as a geometric template. This was further studied by computational modelling using solid-state density-functional theory and lattice dynamics calculations. It is shown that the lattice energy of FAU is lower than EMT, but is strongly impacted by the presence of solvent/guest molecules in the framework. Furthermore, the EMT topology possesses a greater vibrational entropy and is stabilised by free energy at a finite temperature. Overall, these findings demonstrate that the role of the 18C6 additive is to influence the free energy of crystallisation to assemble the EMT framework as opposed to FAU.

'Something in the way she moves': The functional significance of flexibility in the multiple roles of protein disulfide isomerase (PDI).

Protein disulfide isomerase (PDI) has diverse functions in the endoplasmic reticulum as catalyst of redox transfer, disulfide isomerization and oxidative protein folding, as molecular chaperone and in multi-subunit complexes. It interacts with an extraordinarily wide range of substrate and partner proteins, but there is only limited structural information on these interactions. Extensive evidence on the flexibility of PDI in solution is not matched by any detailed picture of the scope of its motion. A new rapid method for simulating the motion of large proteins provides detailed molecular trajectories for PDI demonstrating extensive changes in the relative orientation of its four domains, great variation in the distances between key sites and internal motion within the core ligand-binding domain. The review shows that these simulations are consistent with experimental evidence and provide insight into the functional capabilities conferred by the extensive flexible motion of PDI.

The flexibility and dynamics of protein disulfide isomerase.

We have studied the mobility of the multidomain folding catalyst, protein disulfide isomerase (PDI), by a coarse-graining approach based on flexibility. We analyze our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We find that there is interdomain flexibility at every interdomain junction but these show very different characteristics. The extent of interdomain flexibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures-and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The flexibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain flexibility and clear differences between the domains in their propensity for internal motion. Our results suggest that PDI flexibility enables it to interact with many different partner molecules of widely different sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI. Proteins 2016; 84:1776-1785. © 2016 Wiley Periodicals, Inc.

Characterization of folding cores in the cyclophilin A-cyclosporin A complex.

Determining the folding core of a protein yields information about its folding process and dynamics. The experimental procedures for identifying the amino acids that make up the folding core include hydrogen-deuterium exchange and Φ-value analysis and can be expensive and time consuming. Because of this, there is a desire to improve upon existing methods for determining protein folding cores theoretically. We have obtained HDX data for the complex of cyclophilin A with the immunosuppressant cyclosporin A. We compare these data, as well as literature values for uncomplexed cyclophilin A, to theoretical predictions using a combination of rigidity analysis and coarse-grained simulations of protein motion. We find that in this case, the most specific prediction of folding cores comes from a combined approach that models the rigidity of the protein using the first software suite and the dynamics of the protein using the froda tool.

Structures of mesophilic and extremophilic citrate synthases reveal rigidity and flexibility for function.

Citrate synthase (CS) catalyses the entry of carbon into the citric acid cycle and is highly-conserved structurally across the tree of life. Crystal structures of dimeric CSs are known in both "open" and "closed" forms, which differ by a substantial domain motion that closes the substrate-binding clefts. We explore both the static rigidity and the dynamic flexibility of CS structures from mesophilic and extremophilic organisms from all three evolutionary domains. The computational expense of this wide-ranging exploration is kept to a minimum by the use of rigidity analysis and rapid all-atom simulations of flexible motion, combining geometric simulation and elastic network modeling. CS structures from thermophiles display increased structural rigidity compared with the mesophilic enzyme. A CS structure from a psychrophile, stabilized by strong ionic interactions, appears to display likewise increased rigidity in conventional rigidity analysis; however, a novel modified analysis, taking into account the weakening of the hydrophobic effect at low temperatures, shows a more appropriate decreased rigidity. These rigidity variations do not, however, affect the character of the flexible dynamics, which are well conserved across all the structures studied. Simulation trajectories not only duplicate the crystallographically observed symmetric open-to-closed transitions, but also identify motions describing a previously unidentified antisymmetric functional motion. This antisymmetric motion would not be directly observed in crystallography but is revealed as an intrinsic property of the CS structure by modeling of flexible motion. This suggests that the functional motion closing the binding clefts in CS may be independent rather than symmetric and cooperative.

High-resolution NMR studies of structure and dynamics of human ERp27 indicate extensive interdomain flexibility.

ERp27 (endoplasmic reticulum protein 27.7 kDa) is a homologue of PDI (protein disulfide-isomerase) localized to the endoplasmic reticulum. ERp27 is predicted to consist of two thioredoxin-fold domains homologous with the non-catalytic b and b' domains of PDI. The structure in solution of the N-terminal b-like domain of ERp27 was solved using high-resolution NMR data. The structure confirms that it has the thioredoxin fold and that ERp27 is a member of the PDI family. (15)N-NMR relaxation data were obtained and ModelFree analysis highlighted limited exchange contributions and slow internal motions, and indicated that the domain has an average order parameter S(2) of 0.79. Comparison of the single-domain structure determined in the present study with the equivalent domain within full-length ERp27, determined independently by X-ray diffraction, indicated very close agreement. The domain interface inferred from NMR data in solution was much more extensive than that observed in the X-ray structure, suggesting that the domains flex independently and that crystallization selects one specific interdomain orientation. This led us to apply a new rapid method to simulate the flexibility of the full-length protein, establishing that the domains show considerable freedom to flex (tilt and twist) about the interdomain linker, consistent with the NMR data.

Aseptic loosening of the option stemmed tibial tray in the Zimmer NexGen LPS total knee arthroplasty system.

BACKGROUND: We investigated the relationship between the backside deformation of polyethylene (PE) tibial inserts and aseptic loosening of the Option stemmed tibial tray used with Zimmer NexGen posterior-stabilised (PS) devices. We hypothesized that explanted inserts used in PS designs would exhibit greater extents of PE backside deformation than those used in equivalent cruciate retaining (CR) designs and that PE inserts retrieved from total knee arthroplasties (TKAs) revised for aseptic tibial tray loosening would exhibit greater extents of backside deformation than TKAs revised for other reasons. METHODS: A total of 73 explanted fixed-bearing TKAs (42 CR and 31 PS) were examined. PE components underwent geometric examination with a coordinate measuring machine using validated techniques. Multiple regression modelling was used to identify variables associated with revision secondary to aseptic loosing and to determine factors associated with increased PE backside deformation. RESULTS: PE inserts retrieved from TKAs with aseptic loosening had significantly greater backside deformation than those retrieved from TKAs revised for other reasons (p < 0.001). Greater PE backside deformation was significantly associated with larger tray/insert clearance heights (p < 0.001), thinner inserts (p < 0.001) and PS TKAs (p = 0.001). CONCLUSION: PE backside deformation was significantly greater in the PS TKAs. This may provide one explanation for the increased rate of aseptic loosening reported with the Option tibial tray used with the Legacy Posterior Stabilised (LPS) system.

Metal artefact reduction in digital tomosynthesis using component decomposition.

We propose a technique for metal artefact reduction in digital tomosynthesis reconstruction. Although the problem was addressed earlier in the literature, we suggest another approach, which is, in our opinion, simpler, and easier to implement. It is a two-stage algorithm. At the first stage, attenuation images are segmented by decomposing their intensity distributions into gaussian-like components. Statistical information contained in each component is used for pixel classification. Components corresponding to metallic objects are identified, and a pixel threshold value separating regions occupied by metal objects from the rest of the image is found. Based on this value, at the second stage, a smooth mapping of image intensity is applied. This makes dense regions transparent, resulting in the artefact reduction in reconstruction. The methodology is demonstrated by several examples.

Glycosylation increases active site rigidity leading to improved enzyme stability and turnover.

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its turnover and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRP's thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its nonglycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data show that glycosylation does not just have a passive effect on HRP stability but can exert long-range effects that mediate the 'native' enzyme's activity and stability through changes in inherent dynamics.

Exploring the energy landscapes of protein folding simulations with Bayesian computation.

Nested sampling is a Bayesian sampling technique developed to explore probability distributions localized in an exponentially small area of the parameter space. The algorithm provides both posterior samples and an estimate of the evidence (marginal likelihood) of the model. The nested sampling algorithm also provides an efficient way to calculate free energies and the expectation value of thermodynamic observables at any temperature, through a simple post processing of the output. Previous applications of the algorithm have yielded large efficiency gains over other sampling techniques, including parallel tempering. In this article, we describe a parallel implementation of the nested sampling algorithm and its application to the problem of protein folding in a Go-like force field of empirical potentials that were designed to stabilize secondary structure elements in room-temperature simulations. We demonstrate the method by conducting folding simulations on a number of small proteins that are commonly used for testing protein-folding procedures. A topological analysis of the posterior samples is performed to produce energy landscape charts, which give a high-level description of the potential energy surface for the protein folding simulations. These charts provide qualitative insights into both the folding process and the nature of the model and force field used.

Stereoselective metabolism of chloramphenicol by bacteria isolated from wastewater, and the importance of stereochemistry in environmental risk assessments for antibiotics.

Wastewater treatment plants have been highlighted as a potential hotspot for the development and spread of antibiotic resistance. Although antibiotic resistant bacteria in wastewater present a public health threat, it is also possible that these bacteria play an important role in the bioremediation through the metabolism of antibiotics before they reach the wider environment. Here we address this possibility with a particular emphasis on stereochemistry using a combination of microbiology and analytical chemistry tools including the use of supercritical-fluid chromatography coupled with mass spectrometry for chiral analysis and high-resolution mass spectrometry to investigate metabolites. Due to the complexities around chiral analysis the antibiotic chloramphenicol was used as a proof of concept to demonstrate stereoselective metabolism due to its relatively simple chemical structure and availability over the counter in the U.K. The results presented here demonstrate the chloramphenicol can be stereoselectively transformed by the chloramphenicol acetyltransferase enzyme with the orientation around the first stereocentre being key for this process, meaning that accumulation of two isomers may occur within the environment with potential impacts on ecotoxicity and emergence of bacterial antibiotic resistance within the environment.

The ALS Nutrition/NIPPV Study: design, feasibility, and initial results.

Our objective was to investigate the nutritional requirements in ALS and to determine the feasibility of early intervention with NIPPV. Subjects were enrolled into one of two arms. In the nutrition arm, total daily energy expenditure (TDEE) was determined longitudinally over 48 weeks using the Doubly Labeled Water method. In the NIPPV arm, NIPPV was offered at 80% vs. 50% FVC. Additional measurements were obtained in both arms to ultimately formulate equations to predict TDEE and to estimate sample size for a phase III study of early NIPPV. Eighty subjects were enrolled in the nutrition arm and 73 in the NIPPV arm. Baseline characteristics of the participants are described. TDEE was available for 80 subjects in 249 independent determinations during disease progression. Other variables were measured simultaneously for future modeling of ALS-specific equations to predict TDEE. In the NIPPV arm, rates of progression to the criteria for NIPPV intervention (80% or 50% predicted FVC) were computed. Additional factors were obtained longitudinally to develop indices of early ventilatory insufficiency. The results of this study will allow us to model equations to predict the energy requirements in ALS and to plan a study of early intervention with NIPPV.

3D chest tomosynthesis using a stationary flat panel source array and a stationary detector: a Monte Carlo proof of concept.

3D imaging modalities such as computed tomography and digital tomosynthesis typically scan the patient from different angles with a lengthy mechanical movement of a single x-ray tube. Therefore, millions of 3D scans per year require expensive mechanisms to support a heavy x-ray source and have to compensate for machine vibrations and patient movements. However, recent developments in cold-cathode field emission technology allow the creation of compact, stationary arrays of emitters. Adaptix Ltd has developed a novel, low-cost, square array of such emitters and demonstrated 3D digital tomosynthesis of human extremities and small animals. The use of cold-cathode field emitters also makes the system compact and lightweight. This paper presents Monte Carlo simulations of a concept upgrade of the Adaptix system from the current 60 kVp to 90 kVp and 120 kVp which are better suited for chest imaging. Between 90 kVp and 120 kVp, 3D image quality appears insensitive to voltage and at 90 kVp the photon yield is reduced by 40%-50% while effective dose declines by 14%. A square array of emitters can adequately illuminate a subject for tomosynthesis from a shorter source-to-image distance, thereby reducing the required input power, and offsetting the 28%-50% more input power that is required for operation at 90 kVp. This modelling suggests that lightweight, stationary cold-cathode x-ray source arrays could be used for chest tomosynthesis at a lower voltage, with less dose and without sacrificing image quality. This will reduce weight, size and cost, enabling 3D imaging to be brought to the bedside.

Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism.

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/ß hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

Tibial tray debonding from the cement mantle is associated with deformation of the backside of polyethylene tibial inserts.

AIMS: The aim of this study was to investigate whether wear and backside deformation of polyethylene (PE) tibial inserts may influence the cement cover of tibial trays of explanted total knee arthroplasties (TKAs). METHODS: At our retrieval centre, we measured changes in the wear and deformation of PE inserts using coordinate measuring machines and light microscopy. The amount of cement cover on the backside of tibial trays was quantified as a percentage of the total surface. The study involved data from the explanted fixed-bearing components of four widely used contemporary designs of TKA (Attune, NexGen, Press Fit Condylar (PFC), and Triathlon), revised for any indication, and we compared them with components that used previous generations of PE. Regression modelling was used to identify variables related to the amount of cement cover on the retrieved trays. RESULTS: A total of 114 explanted fixed-bearing TKAs were examined. This included 76 used with contemporary PE inserts which were compared with 15 used with older generation PEs. The Attune and NexGen (central locking) trays were found to have significantly less cement cover than Triathlon and PFC trays (peripheral locking group) (p = 0.001). The median planicity values of the PE inserts used with central locking trays were significantly greater than of those with peripheral locking inserts (205 vs 85 microns; p < 0.001). Attune and NexGen inserts had a characteristic pattern of backside deformation, with the outer edges of the PE deviating inferiorly, leaving the PE margins as the primary areas of articulation. CONCLUSION: Explanted TKAs with central locking mechanisms were significantly more likely to debond from the cement mantle. The PE inserts of these designs showed characteristic patterns of deformation, which appeared to relate to the manufacturing process and may be exacerbated in vivo. This pattern of deformation was associated with PE wear occurring at the outer edges of the articulation, potentially increasing the frictional torque generated at this interface. Cite this article: Bone Joint J 2021;103-B(12):1791-1801.