Domain Exchange Research Articles

Structural Basis for Enhanced Hiv-1 Neutralization by a Dimeric Immunoglobulin G Form of the Glycan-Recognizing Antibody 2G12

The human immunoglobulin G (IgG) 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1) envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc) regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab)2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and SPR binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12. In addition, we present this as the first known crystal structure of an IgG dimer.

The Humanitarian eXchange Language: Coordinating disaster response with semantic web technologies

The Humanitarian eXchange Language (HXL) is a project by the United Nations Office for the Coordination of Humanitarian Affairs that aims at refining data management and exchange for disaster response. Data exchange in this field, which often has to deal with chaotic environments heavily affected by an emergency such as a natural disaster or an armed conflict, still happens mostly manually. The goal of HXL is to contribute to the automatization of many of these processes, saving valuable time for staff in the field and improving the information flow for decision makers who have to allocate resources for response activities. This paper gives an overview of this initiative, which is set to significantly improve information exchange in the humanitarian domain. We introduce the HXL vocabulary, which provides a formal definition of the terminology used in this domain, and an initial set of tools and services that produce and consume HXL data. The HXL system infrastructure is introduced, along with its data management principles. The paper concludes with an outlook on the future of HXL and its role in the humanitarian ecosystem.

Putative domain-domain interactions in the vesicular stomatitis virus L polymerase protein appendage region.

The multidomain polymerase protein (L) of nonsegmented negative-strand (NNS) RNA viruses catalyzes transcription and replication of the virus genome. The N-terminal half of the protein forms a ring-like polymerase structure, while the C-terminal half encoding viral mRNA transcript modifications consists of a flexible appendage with three distinct globular domains. To gain insight into putative transient interactions between L domains during viral RNA synthesis, we exchanged each of the four distinct regions encompassing the appendage region of vesicular stomatitis virus (VSV) Indiana serotype L protein with their counterparts from VSV New Jersey and analyzed effects on virus polymerase activity in a minigenome system. The methyltransferase domain exchange yielded a fully active polymerase protein, which functioned as well as wild-type L in the context of a recombinant virus. Exchange of the downstream C-terminal nonconserved region abolished activity, but coexchanging it with the methyltransferase domain generated a polymerase favoring replicase over transcriptase activity, providing strong evidence of interaction between these two regions. Exchange of the capping enzyme domain or the adjacent nonconserved region thought to function as an "unstructured" linker also abrogated polymerase activity even when either domain was coexchanged with other appendage domains. Further probing of the putative linker segment using in-frame enhanced green fluorescent protein (EGFP) insertions similarly abrogated activity. We discuss the implications of these findings with regard to L protein appendage domain structure and putative domain-domain interactions required for polymerase function. NNS viruses include many well-known human pathogens (e.g., rabies, measles, and Ebola viruses), as well as emerging viral threats (e.g., Nipah and Hendra viruses). These viruses all encode a large L polymerase protein similarly organized into multiple domains that work in concert to enable virus genome transcription and replication. But how the unique L protein carries out the multiplicity of individual steps in these two distinct processes is poorly understood. Using two different approaches, i.e., exchanging individual domains in the C-terminal appendage region of the protein between two closely related VSV serotypes and inserting unrelated protein domains, we shed light on requirements for domain-domain interactions and domain contiguity in polymerase function. These findings further our understanding of the conformational dynamics of NNS L polymerase proteins, which play an essential role in the pathogenic properties of these viruses and represent attractive targets for the development of antiviral measures.

Heterogeneous dynamics in ionic liquids at the glass transition: Fluorescence recovery after photobleaching measurements of probe rotational motion from Tg − 6 K to Tg + 4 K

The rotational dynamics of tetracene and rubrene in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([C4C1im][PF6]) at the glass transition (Tg=196K), from Tg−6K to Tg+4K, were measured using the technique of fluorescence recovery after photobleaching. The rotational anisotropy decays of these probes in [C4C1im][PF6] were found to be non-exponential and well fit by the Kohlrausch–Williams–Watts (KWW) function with the stretching parameter βKWW equal to 0.70±0.03 for tetracene and 0.88±0.04 for rubrene in the temperature range of the measurements. The rotational correlation time τc at Tg is equal to 19±1s for the smaller probe tetracene and 180±40s for the larger probe rubrene. Below Tg, τc shows a slight decoupling from the extrapolation of fits of the Vogel–Fulcher–Tammann equation to the viscosity η. This decoupling is characterized by a fractional Debye–Stokes–Einstein relation, τc ∝ ηξ/T, with ξ equal to 0.78±0.02 for rubrene and 0.85±0.01 for tetracene. The dependence of βKWW on probe size is consistent with the dynamics in [C4C1im][PF6] being heterogeneous and is rationalized in terms of the time scale of the probe rotational motion compared to the domain exchange time.

Intelligent Architecture for Comparative Analysis of Public Companies Using Semantics and XBRL Data

"The new source of power is not money in the hands of a few, but information in the hands of many." The aforementioned quote from John Naisbitt seems to be even more relevant in the world of finance at this very moment. Many financial decisions come from watching the information stream, selecting relevant data, analyzing it and acting accordingly. With the increasing global competition, the need for swift data analysis, high accuracy and quality becomes a must. XBRL (Extensible Business Reporting Language)XBRL: http://www.xbrl.org/ standard was proposed to improve efficiency of data exchange in the financial domain. However; it is still struggling with interoperability problems, not to mention comparability of data or multisource data integration. This paper presents the FLORA intelligent platform: an approach for dealing with current financial information shortcomings and achieving more effective way of processing financial data based on the Linked Data principles. The article also explains the process of data extraction and semantic modeling which are the cornerstones of efficient financial data analysis. As a result, the FLORA architecture facilitates effective, data-driven, financial analyses and Web-scale integration between financial applications and platforms.

Structural basis of Rad53 kinase activation by dimerization and activation segment exchange

The protein kinase Rad53 is a key regulator of the DNA damage checkpoint in budding yeast. Its human ortholog, CHEK2, is mutated in familial breast cancer and mediates apoptosis in response to genotoxic stress. Autophosphorylation of Rad53 at residue Thr354 located in the kinase activation segment is essential for Rad53 activation. In this study, we assessed the requirement of kinase domain dimerization and the exchange of its activation segment during the Rad53 activation process. We solved the crystal structure of Rad53 in its dimeric form and found that disruption of the observed head-to-tail, face-to-face dimer structure decreased Rad53 autophosphorylation on Thr354 in vitro and impaired Rad53 function in vivo. Moreover, we provide critical functional evidence that Rad53 trans-autophosphorylation may involve the interkinase domain exchange of helix αEF via an invariant salt bridge. These findings suggest a mechanism of autophosphorylation that may be broadly applicable to other protein kinases.

Modeling ZO‐1‐C‐terminal domain exchange at the membrane as a diffusion problem (1111.4)

Zonula occludens‐1 (ZO‐1) is a membrane‐associated tight junction protein that has been observed to participate in ATP‐dependent exchange via an intracellular pool. We hypothesized that a diffusion model can explain the ZO‐1‐C‐terminal domain exchange dynamics at the tight junction. We developed stable cell lines with GFP‐ZO‐1 carboxy‐terminus fusion constructs in Madin‐Darby canine kidney cells. Fluorescence recovery after photobleaching (FRAP) experiments were conducted using a Nikon A1R laser scanning confocal microscope to photobleach the localized construct and acquire intensity data over time. Recovery was analyzed by partitioning the bleach region of interest into one‐square micron sections or the entire bleach ROI. The local fluorescence recovery in the photobleached area yields curves that are modeled to be solutions of the diffusion process. Fitting experimental data to a one‐ or two‐dimensional diffusion model will quantitatively reveal if the exchange occurs within the membrane or via an intracellular pool, respectively. Future research will compare the behavior of different ZO‐1 constructs and relate the quantity of ZO‐1 expression to its recovery behavior.Grant Funding Source: Supported in part by NSF‐MRI #1229702 and NSF Award #0926702.

Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model.

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Overlap coincidence to strong coincidence in substitution tiling dynamics

Overlap coincidence is an equivalent criterion to pure discrete spectrum of the dynamics of self-affine tilings in Rd. In the case of d=1, strong coincidence on m-letter irreducible substitution has been introduced in Dekking (1978) and Arnoux and Ito (2001) which implies that the system is metrically conjugate to a domain exchange in Rm−1. However being a domain exchange does not imply the property of pure discrete spectrum of the tiling dynamics. The relation between two coincidences has not been established completely. In this paper we generalize strong coincidence to higher dimensions and show the implication from overlap coincidence to the new strong coincidence when the associated height group is trivial. Furthermore we introduce a new criterion ‘simultaneous coincidence’ and show the implication from overlap coincidence to the simultaneous coincidence. The triviality of the height group is shown in Barge and Kwapisz (2006) and Sing (2006) for 1-dimension irreducible Pisot substitutions.

Stepped surfaces and Rauzy fractals induced from automorphisms on the free group of rank 2

From irreducible unimodular Pisot substitutions of rank 2, we can construct the discrete approximations of the lines, the so-called stepped surfaces, and moreover, we obtain Rauzy fractals as geometric realizations of the symbolic dynamical systems. In this paper, we study a particular infinite family of automorphisms on the free group of rank 2. We see that for every hyperbolic quadratic unit integer λ, there is a corresponding automorphism in the infinite family whose incidence matrix has eigenvalue λ; and the automorphism is conjugate to a substitution or “an alternating substitution”. And we construct stepped surfaces and Rauzy fractals induced from automorphisms in the family by using tiling substitutions. We also discuss the domain exchange transformations on Rauzy fractals.

Rational Design of Modular Polyketide Synthases: Morphing the Aureothin Pathway into a Luteoreticulin Assembly Line

The unusual nitro-substituted polyketides aureothin, neoaureothin (spectinabilin), and luteoreticulin, which are produced by diverse Streptomyces species, point to a joint evolution. Through rational genetic recombination and domain exchanges we have successfully reprogrammed the modular (type I) aur polyketide synthase (PKS) into a synthase that generates luteoreticulin. This is the first rational transformation of a modular PKS to produce a complex polyketide that was initially isolated from a different bacterium. A unique aspect of this synthetic biology approach is that we exclusively used genes from a single biosynthesis gene cluster to design the artificial pathway, an avenue that likely emulates natural evolutionary processes. Furthermore, an unexpected, context-dependent switch in the regiospecificity of a pyrone methyl transferase was observed. We also describe an unprecedented scenario where an AT domain iteratively loads an extender unit onto the cognate ACP and the downstream ACP. This aberrant function is a novel case of non-colinear behavior of PKS domains.

The geometry of non-unit Pisot substitutions

It is known that with a non-unit Pisot substitution σ one can associate certain fractal tiles, so-called Rauzy fractals. In our setting, these fractals are subsets of a certain open subring of the adèle ring of the associated Pisot number field. We present several approaches on how to define Rauzy fractals and discuss the relations between them. In particular, we consider Rauzy fractals as the natural geometric objects of certain numeration systems, in terms of the dual of the one-dimensional realization of σ, and in the context of model sets for particular cut and project schemes. We also define stepped surfaces suited for non-unit Pisot substitutions. We provide basic topological and geometric properties of the Rauzy fractals, prove some tiling results for them, and provide relations to subshifts defined in terms of the periodic points of σ, to adic transformations, and a domain exchange.

Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange

Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.

Kinetics of Interactions between LOV Domains from Chlamydomonas Reinhardtii

Kinetics of Interactions between LOV domains from Chlamydomonas reinhardtiiCarey K. Johnson, Kathrin Magerl, Katee Wyant, Ashley McDade, Will Newhart, David C. Arnett, and Bernhard Dick.We have investigated the kinetics of interactions between Light-Oxygen-Voltage (LOV) domains of the light-sensing protein phototropin from Chlamydomonas reinhardtii. The photochemical response of phototropin to blue light involves adduct formation between a flavin mononucleotide cofactor and a nearby Cys residue. Interactions between LOV domains, either domains LOV1 and LOV2 within the same phototropin, or between LOV domains from different phototropin molecules, are thought to play a role in the subsequent activation of the phototropin kinase domain. We studied the kinetics of exchange of LOV domains between complexes (dimers or higher order oligomers) by stop-flow FRET measurements. A biphasic response with time constants from tens to hundreds of seconds was sensitive to prior exposure of LOV domains to blue light. LOV-LOV interactions have also been detected at the single-molecule level by TIRF imaging, providing further information about the interaction kinetics.

Conditional functional dependencies validation for XML data: an approach based on attribute grammar

The representation and the exchange of information originating from different data sources is an increasingly common need for companies and industries to integrate their operations and also to publish and trade information with government and other enterprises. For this purpose, there are many standards based on XML language that were created to allow effective data communication and exchange in a particular domain. In order to ensure data quality for XML data, this paper presents an approach based on conditional functional dependencies verification. Conditional dependencies are an extension of traditional database dependencies with the ability to enforce bindings of semantically related data values. The basis of our verification method is a generic grammarware for validating XML integrity constraints in one tree traversal. We use an attribute grammar to describe XML documents and constraints. Keywords: conditional functional dependencies, data quality, XML integrity constraints.

Structural Basis for Enhanced HIV-1 Neutralization by a Dimeric Immunoglobulin G Form of the Glycan-Recognizing Antibody 2G12

The human immunoglobulin G (IgG) 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1) envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc) regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab)2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.

Automatic control of farming operations based on spatial web services

Field operations relating to arable farming are often very data intensive tasks. An increasing number of regulations have been set to ensure food safety and environmental aspects. Also, the number of tools for the best practice management applied in precision agriculture is growing. However, there are yet no standardized, automated methods for a compliance management used in situations where circumstances change and are dependent on the specific location. Therefore compliance checks during the work progress online or on demand are difficult to achieve and the temporal accuracy can be very poor. In this work, we have developed a task controller (TC) prototype with an ISOBUS-compatible process data messages to be able to utilize multiple external services such as WFS (Web Feature Service) during a spraying operation. The WFS was set up in Germany to provide geodata while the actual task execution was performed in Finland. We developed a possibility to use and integrate external data from different sources in the TC on the tractor. Methods presented in this article serve as the basis for the development of multiple tools that can be used for improving farming system development, the environmental risk reduction of agricultural production and compliance checks. Existing information sources such as on board sensors, weather and forecast information, disease pressure, spatial environmental risks and real time remote sensing can be combined for new solutions of this kind. The development of technical standards for the seamless data exchange in the agricultural domain is therefore crucial. In this work, we are focussing on spatial data exchange between heterogeneous IT systems as a component of on-field machinery used in precision management.

Analytical and experimental analysis of solute transport in heterogeneous porous media

Knowledge of solute transport in heterogeneous porous media is crucial to monitor contaminant fate and transport in soil and groundwater systems. In this study, we present new findings from experimental and mathematical analysis to improve current understanding of solute transport in structured heterogeneous porous media. Three saturated columns packed with different sand combinations were used to examine the breakthrough behavior of bromide, a conservative tracer. Experimental results showed that bromide had different breakthrough responses in the three types of sand combinations, indicating that heterogeneity in hydraulic conductivity has a significant effect on the solute transport in structured heterogeneous porous media. Simulations from analytical solutions of a two-domain solute transport model matched experimental breakthrough data well for all the experimental conditions tested. Experimental and model results show that under saturated flow conditions, advection dominates solute transport in both fast-flow and slow-flow domains. The sand with larger hydraulic conductivity provided a preferential flow path for solute transport (fast-flow domain) that dominates the mass transfer in the heterogeneous porous media. Importantly, the transport in the slow-flow domain and mass exchange between the domains also contribute to the flow and solute transport processes and thus must be considered when investigating contaminant transport in heterogeneous porous media.

Identity Management--In Privacy We Trust: Bridging the Trust Gap in eHealth Environments

Healthcare systems are moving to decentralized scenarios, forming a mesh of mobile services and care providers. A rapidly growing number of users aren't shy about using new services on their home computers, mobile phones, and laptops. Identity management solutions, deployed for ease of use, control the data that users provide to individual healthcare services. These solutions raise trust and privacy concerns about who owns users' data, how to control its spread, and how to build trustworthy associations between healthcare providers. Reputation systems can enhance eHealth systems by bridging the gap between strong contractual agreements and first-time domain exchanges.