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The "Information Age" is starting to have an impact on dental technology. Procera, Cerac and several other digital systems are commonly used by many technicians. Other systems are being developed and marketed. Computer Aided Design (CAD) can be used by dental technicians to create precise dental restorations. Solid modeling techniques can provide unique dental applications.
We have used chimeric IgG antibodies and their genetically engineered variants prepared by a combination of site-directed mutagenesis and exon exchange to define the structure(s) on IgG recognized by monoclonal rheumatoid factor (RF) autoantibodies from rheumatoid arthritis (RA) patients. Nineteen RF produced by EBV-transformed cell lines from the synovium or blood of RA patients were analyzed. Their binding patterns differ significantly from those seen with RF obtained from patients with Waldenstrom's macroglobulinemia (WMac). Half of the RA-derived RF bound IgG1, 2, and 4, but not 3 (Ga specificity), the common pattern in WMac. However, heterogeneity in fine specificity within the Ga reactivity pattern was observed. Moreover, seven others bound all four IgG subclasses, a pattern observed for only one WMac-derived RF from a patient who also had RA. Three RF had subclass specificities unlike any observed with WMac-derived RF. Most RA-derived RF bound IgG at a discontinuous epitope comprised of residues from both the CH2 and CH3 H chain constant regions. However, unlike any WMac-derived RF, one RA-derived RF bound IgG in CH2, another in CH3, and a third at an undetermined site outside of the CH2-CH3 interface. Some RA-derived RF bound aglycosylated IgG4 less well than glycosylated IgG4, suggesting that the carbohydrate moiety was important in establishing their binding epitope in CH2. These studies demonstrate that the repertoire of RF expressed by RA patients contains some unique binding specificities for IgG epitopes not found among our panel of WMac-derived RF. Our results therefore call into question whether WMac-derived RF with their limited diversity are appropriate models for disease-related RF. In addition, RF with their multiple specificities can serve as probes of antibody structure.
UNLABELLED: Laparoscopic surgical skills evaluation of surgery residents is usually a subjective process, carried out in the operating room by senior surgeons. By its nature, this process is performed using fuzzy criteria. The objective of the current study was to develop and assess an objective laparoscopic surgical skill scale using Hidden Markov Models (HMM) based on haptic information, tool/tissue interactions and visual task decomposition. METHODS: Eight subjects (six surgical trainees: first year surgical residents 2 x R1, third year surgical residents 2 x R3 fifth year surgical residents 2 x R5; and two expert laparoscopic surgeons: 2 x ES) performed laparoscopic cholecystectomy following a specific 7 steps protocol on a pig. An instrumented laparoscopic grasper equipped with a three-axis force/torque sensor located at the proximal end with an additional force sensor located on the handle, was used to measure the forces and torques. The hand/tool interface force/torque data was synchronized with a video of the tool operative maneuvers. A synthesis of frame-by-frame video analysis was used to define 14 different types of tool/tissue interactions, each one associated with unique force/torque (F/T) signatures. HMMs were developed for each subject representing the surgical skills by defining the various tool/tissue interactions as states and the associated F/T signatures as observations. The statistical distance between the HMMs representing residents at different levels of their training and the HMMs of expert surgeons were calculated in order to generate a learning curve of selected steps during laparoscopic cholecystectomy. RESULTS: Comparison of HMM's between groups showed significant differences between all skill levels, supporting the objective definition of a learning curve. The major differences between skill levels were: (i) magnitudes of F/T applied (ii) types of tool/tissue interactions used and the transition between them and (iii) time intervals spent in each tool/tissue interaction and the overall completion time. The objective HMM analysis showed that the greatest difference in performance was between R1 and R3 groups and then decreased as the level of expertise increased, suggesting that significant laparoscopic surgical capability develops between the first and the third years of their residency training. The power of the methodology using HMM for objective surgical skill assessment arises from the fact that it compiles enormous amount of data regarding different aspects of surgical skill into a very compact model that can be translated into a single number representing the distance from expert performance. Moreover, the methodology is not limited to in-vivo condition as demonstrated in the current study. It can be extended to other modalities such as measuring performance in surgical simulators and robotic systems.
A simple but highly integrated digital signal processing system is described for real time filtering of biomedical signals. It includes the necessary processing and communications hardware, the processing code itself and a high-level software interface that enables the user to design and download arbitrary finite impulse response filters to the run-time system. The filter coefficients are calculated using the frequency sampling method. Since the filters are realized using a finite impulse response, no phase distortion is introduced into the processed signals. A unique feature of the design is the manner in which the software and hardware components have been organized as an intelligent system, obviating on the part of the user a detailed knowledge of filter design theory or any abilities in processor architecture and assembly code programming.
Dimeric transcription factors of the NF-kappaB/Rel family are sequence-specific DNA-binding proteins that mediate the inducible expression of immunologically important eukaryotic genes by competing for kappaB sites. The kinetic and thermodynamic components of these interactions were probed by mutation of the subunit interface of the p50 homodimer, a paradigm for other family members. Guided by the crystal structure, we selected the side chains of five key residues (R255, Y270, L272, A311 and V313) for individual and combinatorial truncation, with the aim of generating a mutant panel. Homodimerisation was assessed indirectly by measurement of DNA binding with an optical biosensor in order to unmask the relative contributions of each residue. Surface plasmon resonance revealed that a unanimous bias for a palindromic kappaB site over an asymmetric one was mainly the result of a slower dissociation rate for the DNA/homodimer complex in the case of the palindromic kappaB site. Y270 and L272 were individually the most critical residues in homodimerisation. DNA binding was abolished when all five residues were substituted, which reinforces the notion that only a subset of residues contributes crucial dimer-forming contacts. The role of Y270 was unique, since its mutation to glycine dramatically slowed both the association and dissociation rates for DNA binding. Surprisingly, R255 was shown to be of little importance in the stability of the p50 homodimer, despite its apparent participation in a salt bridge at the dimer interface. Our results suggest that binding modes inferred from structural data should be treated cautiously.
The exocoelomic cavity was probably the last remaining physiological body fluid cavity to be explored in the human embryo. Its unique anatomical position has enabled us to study the protein metabolism of the early placenta and secondary yolk sac and to explore materno-embryonic transfer pathways. The exocoelomic cavity forms inside the extraembryonic mesoderm alongside the placental chorionic plate and is now believed to be an important transfer interface and a reservoir of nutrients for the embryo. Maternal or placental proteins filtered in the extraembryonic coelomic cavity are probably absorbed by the secondary yolk sac which is directly connected with the primitive digestive system throughout embryonic development. Protein electrophoresis has shown that the coelomic fluid results from an ultrafiltrate of maternal serum with the addition of specific placental and secondary yolk sac bioproducts demonstrating that the exocoelomic cavity is a physiological liquid extension of the early placenta. The selective sampling of fluid from the exocoelomic cavity has also offered a novel approach to the study of drug and toxin transfer across the early human placenta and as a unique tool to explore embryonic physiology in vivo. Further investigation should include a comparison between the coelomic fluid values of a molecule and its quantifiable presence in decidual, placental and fetal tissues.
A digital-electronic reconstruction system for MRI has been designed and demonstrated. The system is capable of reconstructing a 128 x 128 pixel image from complex-valued data in approximately 8 ms (122 frames per second) or a 256 x 256 pixel image in 32 ms (30 frames per second) using the standard 2D FFT reconstruction algorithm. Real-time MR imaging can be obtained when this reconstruction system is coupled with fast continuous echo-planar type data acquisition. This provides the unique potential for real-time monitoring of interventional procedures or for rapid patient positioning. The real-time reconstruction system presented here consists of four main subsystems: an analog to digital converter, an interface memory, the Fourier processor, and the display processor. The basic design of this reconstruction system is presented along with results, demonstrating the capability of the system.
A structural model of the sushi domain of IL-15Ralpha was first obtained by homology modeling to study its interactions with IL-15 by means of molecular modeling, peptide scanning, and site-directed mutagenesis. From these experimental data, a putative interacting surface of IL-15Ralpha with a previously published IL-15 model was inferred: Leu25, Leu44, and Glu46 of IL-15 and Arg35 of IL-15Ralpha were found to be key interfacial residues and were subsequently used as filters for the construction of docking solutions. Human IL-15/IL-15Ralpha complexes were constructed in two stages, with a preliminary docking procedure, treating the two partners as rigid bodies and using these filters. In this first stage, two classes of docking solutions were characterized. From a topological point of view, each solution could be derived from the other by reverse orientation of one partner in relation to the other. In a second stage, several further energy refinements clearly favored one solution. Moreover, this unique docking solution was confirmed by molecular modeling of IL-15 mutants previously built and tested in our laboratory. Finally, this complex model, which is a useful tool to study the IL-15/IL-15Ralpha interface, was topologically compared to IL-2/IL-2Ralpha complexes (previous model in the literature and recent crystal structure).
Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.
The crystal structures of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) complexed with phenylalanine and phenylalaninyl-adenylate (PheOH-AMP), the synthetic analogue of phenylalanyl-adenylate, have been determined at 2.7A and 2.5A resolution, respectively. Both Phe and PheOH-AMP are engulfed in the active site cleft of the catalytic alpha-subunit of PheRS, and neither makes contact with the PheRS beta-subunit. The conformations and binding of Phe are almost identical in both complexes. The recognition of Phe by PheRS is achieved through a mixture of multiple van der Waals interactions and hydrogen bonds. The side-chain of the Phe substrate is sandwiched between the hydrophobic side-chains of Phealpha258 and Phealpha260 on one side, and the main-chain atoms of the two adjacent beta-strands on the other. The side-chains of Valalpha261 and Alaalpha314 form the back wall of the amino acid binding pocket. In addition, PheRS residues (Trpalpha149, Seralpha180, Hisalpha178, Argalpha204, Glnalpha218, and Glualpha220) form a total of seven hydrogen bonds with the main-chain atoms of Phe. The conformation of PheOH-AMP and the network of interactions of its AMP moiety with PheRS are reminiscent of the other class II synthetases. The structural similarity between PheRS and histidyl-tRNA synthetase extends to the amino acid binding site, which is normally unique for each enzyme. The complex structures suggest that the PheRS beta-subunit may affect the first step of the reaction (formation of phenylalanyl-adenylate) through the metal-mediated conserved alpha/beta-subunit interface. The modeling of tyrosine in the active site of PheRS revealed no apparent close contacts between tyrosine and the PheRS residues. This result implies that the proofreading mechanism against activated tyrosine, rather than direct recognition, may play the major role in the PheRS specificity.
Myosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser.
Invasive EEG recordings with depth and/or subdural electrodes are occasionally necessary for the surgical management of patients with epilepsy refractory to medications. In addition to their vital clinical utility, electrocorticographic (ECoG) recordings provide an unprecedented opportunity to study the electrophysiological correlates of functional brain activation in greater detail than non-invasive recordings. The proximity of ECoG electrodes to the cortical sources of EEG activity enhances their spatial resolution, as well as their sensitivity and signal-to-noise ratio, particularly for high-frequency EEG activity. ECoG recordings have, therefore, been used to study the event-related dynamics of brain oscillations in a variety of frequency ranges, and in a variety of functional-neuroanatomic systems, including somatosensory and somatomotor systems, visual and auditory perceptual systems, and cortical networks responsible for language. These ECoG studies have confirmed and extended the original non-invasive observations of ERD/ERS phenomena in lower frequencies, and have discovered novel event-related responses in gamma frequencies higher than those previously observed in non-invasive recordings. In particular, broadband event-related gamma responses greater than 60 Hz, extending up to approximately 200 Hz, have been observed in a variety of functional brain systems. The observation of these "high gamma" responses requires a recording system with an adequate sampling rate and dynamic range (we use 1000 Hz at 16-bit A/D resolution) and is facilitated by event-related time-frequency analyses of the recorded signals. The functional response properties of high-gamma activity are distinct from those of ERD/ERS phenomena in lower frequencies. In particular, the timing and spatial localization of high-gamma ERS often appear to be more specific to the putative timing and localization of functional brain activation than alpha or beta ERD/ERS. These findings are consistent with the proposed role of synchronized gamma oscillations in models of neural computation, which have in turn been inspired by observations of gamma activity in animal preparations, albeit at somewhat lower frequencies. Although ECoG recordings cannot directly measure the synchronization of action potentials among assemblies of neurons, they may demonstrate event-related interactions between gamma oscillations in macroscopic local field potentials (LFP) generated by different large-scale populations of neurons engaged by the same functional task. Indeed, preliminary studies suggest that such interactions do occur in gamma frequencies, including high-gamma frequencies, at latencies consistent with the timing of task performance. The neuronal mechanisms underlying high-gamma activity and its unique response properties in humans are still largely unknown, but their investigation through invasive methods is expected to facilitate and expand their potential clinical and research applications, including functional brain mapping, brain-computer interfaces, and neurophysiological studies of human cognition.
A novel photoresist (PR) that can be processed under mild aqueous conditions was synthesized and used to create photogenerated polyelectrolyte bilayers. Thin films of the PR cast on polycation-coated substrates were exposed to UV irradiation to generate carboxylate groups in the photoresist. The bulk of the UV-exposed PR film was dissolved by rinsing with pH 7.4 phosphate-buffered saline, but a polyelectrolyte bilayer formed in situ at the PR/polycation interface on exposure remained bound to the substrate. The UV-exposed photoresist also exhibited pH-dependent solubility; it was soluble in water above pH 6.6, but insoluble at lower pHs. Using these unique properties, two-component protein patterning was achieved using biotinylated PR films under conditions that avoid exposing the proteins to conditions outside the narrow range of physiological pH, ionic strength, and temperature where their stability is greatest.
We recently identified the gold compound aurothiomalate (ATM) as a potent inhibitor of the Phox and Bem1p (PB1)-PB1 domain interaction between protein kinase C (PKC) iota and the adaptor molecule Par6. ATM also blocks oncogenic PKCiota signaling and the transformed growth of human lung cancer cells. Here we demonstrate that ATM is a highly selective inhibitor of PB1-PB1 domain interactions between PKCiota and the two adaptors Par6 and p62. ATM has no appreciable inhibitory effect on other PB1-PB1 domain interactions, including p62-p62, p62-NBR1, and MEKK3-MEK5 interactions. ATM can form thio-gold adducts with cysteine residues on target proteins. Interestingly, PKCiota (and PKCzeta) contains a unique cysteine residue, Cys-69, within its PB1 domain that is not present in other PB1 domain containing proteins. Cys-69 resides within the OPR, PC, and AID motif of PKCiota at the binding interface between PKCiota and Par6 where it interacts with Arg-28 on Par6. Molecular modeling predicts formation of a cysteinyl-aurothiomalate adduct at Cys-69 that protrudes into the binding cleft normally occupied by Par6, providing a plausible structural explanation for ATM inhibition. Mutation of Cys-69 of PKCiota to isoleucine or valine, residues frequently found at this position in other PB1 domains, has little or no effect on the affinity of PKCiota for Par6 but confers resistance to ATM-mediated inhibition of Par6 binding. Expression of the PKCiota C69I mutant in human non-small cell lung cancer cells confers resistance to the inhibitory effects of ATM on transformed growth. We conclude that ATM inhibits cellular transformation by selectively targeting Cys-69 within the PB1 domain of PKCiota.
MOTIVATION: The AGENT project established a network of actively cooperating European genebanks, integrating genomic and phenotypic data from accessions of wheat and barley. Due to specific storage demands for phenotypic and genotypic data, the project used separate database instances and backend technologies to manage integrated phenotypic and genotypic data. RESULTS: We discuss the challenges encountered when integrating dispersed data to serve through a single interface such as the Plant Breeding Application Programming Interface, BrAPI. We examine how the consistent mappability of genebank data to the BrAPI model can enable the implementation of effective services. The advantages of BrAPI in transparently linking distributed data entities through embedded, unique identifiers are highlighted. We present a technical solution involving a BrAPI proxy, which combines and merges separate BrAPI endpoints. Finally, we demonstrate the AGENT BrAPI implementation with an illustrative example that validates a suggested SNP for a trait from the literature by linking phenotypic, genotypic and passport data. AVAILABILITY AND IMPLEMENTATION: The BrAPI proxy implementation and documentation is available at the Python Package Index (https://pypi.org/project/brapi-proxy) and archived in Zenodo (doi: 10.5281/zenodo.19436445). SUPPLEMENTARY INFORMATION: A Jupyter Notebook file for the validation example using a marker-trait relationship found in the literature.
The expression and function of the human major histocompatibility complex (MHC) class Ia genes, human leukocyte antigen (HLA)-A, -B, and -C, is well-established; they are expressed in most nucleated cells and present endogenous peptides to CD8+ T cells. However, MHC class Ib genes are poorly characterized and have unknown functions. In humans, the best-characterized class Ib gene is HLA-G. This gene has a restricted tissue expression of the mRNA and a unique pattern of protein expression; it is expressed mainly in the extravillous cytotrophoblast cells in the placenta. The function of HLA-G is not clear, but its presence at the maternal-fetal interface suggests a role in protection of the semiallogeneic fetus. Whereas functional studies using in vitro models and transgenic mice provide useful insights regarding the potential function of this molecule, in vivo studies cannot be performed in humans. Nonhuman primates that are closely related to humans phylogenetically contain homologues of HLA-G. The MHC-G loci in nonhuman primates appear to have diverged from the human HLA-G. However, in the rhesus monkey (Macaca mulatta) and olive baboon (Papio anubis), a novel class Ia-related locus has been described. This gene encodes glycoproteins with characteristics that resemble those of HLA-G, including restricted tissue distribution, alternative splicing of mRNA, truncated cytoplasmic domain, and limited polymorphism. Thus, this molecule may be the functional homologue of HLA-G, and these two species may comprise appropriate models for elucidating the function of HLA-G.
To identify molecular genetic markers of resistance or susceptibility to dermatophilosis in cattle, we used a functional candidate gene approach to analyze the DNA polymorphisms of targeted genes encoding molecules implicated in known mechanisms of both nonspecific and specific immune responses existing in the pathogen/host interface mechanisms. The most significant results were obtained within the Major Histocompatibility Complex (MHC) where the BoLA-DRB3 and DQB genes encode molecules involved in the antigen presentation to T cell receptors. A unique BoLA class II haplotype, made up of one DRB3 exon 2 allele and one DQB allele, highly correlates with the susceptibility character (P < 0.001). This haplotype marker of susceptibility was also found and validated in other bovine populations. A eugenic marker-assisted selection was developed in the field by eliminating only the animals having this haplotype. The disease prevalence was thereby reduced from 0.76 to 0.02 over 5 years. A crossbreeding plan is in progress to study the genetic transmission of the genotypic and phenotypic characters of susceptibility to dermatophilosis. In conclusion, we discuss several hypotheses at the molecular and cellular levels to better define the exact role of the MHC molecules in disease control and to answer the question: How is MHC diversity selectively maintained by natural selection imposed by pathogens?
Epilithic microbial communities, ubiquitously found in biofilms on submerged granite, limestone, and sandstone, as well as on the concrete support pillars of bridges, were examined in the Speed River, Ontario, Canada. Transmission electron microscopy showed that attached bacteria (on all substrata) were highly mineralized, ranging from Fe-rich capsular material to fine-grained (<1 mum) authigenic (primary) mineral precipitates. The authigenic grains exhibited a wide range of morphologies, from amorphous gel-like phases to crystalline structures. Energy-dispersive X-ray spectroscopy indicated that the most abundant mineral associated with epilithic bacteria was a complex (Fe, Al) silicate of variable composition. The gel-like phases were similar in composition to a chamositic clay, whereas the crystalline structures were more siliceous and had compositions between those of glauconite and kaolinite. The consistent formation of (Fe, Al) silicates by all bacterial populations, regardless of substratum lithology, implies that biomineralization was a surface process associated with the anionic nature of the cell wall. The adsorption of dissolved constituents from the aqueous environment contributed significantly to the mineral formation process. In this regard, it appears that epilithic microbial biofilms dominate the reactivity of the rock-water interface and may determine the type of minerals formed, which will ultimately become part of the riverbed sediment. Because rivers typically contain high concentrations of dissolved iron, silicon, and aluminum, these findings provide a unique insight into biogeochemical activities that are potentially widespread in natural waters.