2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli.

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Cell-cell fusion induced by the Ig3 domain of receptor FGFRL1 in CHO cells.

FGFRL1 is a single-pass transmembrane protein with three extracellular Ig domains. When overexpressed in CHO cells or related cell types, it induces cell-cell fusion and formation of large, multinucleated syncytia. For this fusion-promoting activity, only the membrane-proximal Ig domain (Ig3) and the transmembrane domain are required. It does not matter whether the transmembrane domain is derived from FGFRL1 or from another receptor, but the distance of the Ig3 domain to the membrane is crucial. Fusion can be inhibited with soluble recombinant proteins comprising the Ig1-Ig2-Ig3 or the Ig2-Ig3 domains as well as with monoclonal antibodies directed against Ig3. Mutational analysis reveals a hydrophobic site in Ig3 that is required for fusion. If a single amino acid from this site is mutated, fusion is abolished. The site is located on a β-sheet, which is part of a larger β-barrel, as predicted by computer modeling of the 3D structure of FGFRL1. It is possible that this site interacts with a target protein of neighboring cells to trigger cell-cell fusion.

Differentiation of Diabetic Macular Edema From Pseudophakic Cystoid Macular Edema by Spectral-Domain Optical Coherence Tomography.

PURPOSE: To differentiate diabetic macular edema (DME) from pseudophakic cystoid macular edema (PCME) based solely on spectral-domain optical coherence tomography (SD-OCT). METHODS: This cross-sectional study included 134 participants: 49 with PCME, 60 with DME, and 25 with diabetic retinopathy (DR) and ME after cataract surgery. First, two unmasked experts classified the 25 DR patients after cataract surgery as either DME, PCME, or mixed-pattern based on SD-OCT and color-fundus photography. Then all 134 patients were divided into two datasets and graded by two masked readers according to a standardized reading-protocol. Accuracy of the masked readers to differentiate the diseases based on SD-OCT parameters was tested. Parallel to the masked readers, a computer-based algorithm was established using support vector machine (SVM) classifiers to automatically differentiate disease entities. RESULTS: The masked readers assigned 92.5% SD-OCT images to the correct clinical diagnose. The classifier-accuracy trained and tested on dataset 1 was 95.8%. The classifier-accuracy trained on dataset 1 and tested on dataset 2 to differentiate PCME from DME was 90.2%. The classifier-accuracy trained and tested on dataset 2 to differentiate all three diseases was 85.5%. In particular, higher central-retinal thickness/retinal-volume ratio, absence of an epiretinal-membrane, and solely inner nuclear layer (INL)-cysts indicated PCME, whereas higher outer nuclear layer (ONL)/INL ratio, the absence of subretinal fluid, presence of hard exudates, microaneurysms, and ganglion cell layer and/or retinal nerve fiber layer cysts strongly favored DME in this model. CONCLUSIONS: Based on the evaluation of SD-OCT, PCME can be differentiated from DME by masked reader evaluation, and by automated analysis, even in DR patients with ME after cataract surgery. The automated classifier may help to independently differentiate these two disease entities and is made publicly available.

Gradient-domain Path Tracing

We introduce gradient-domain rendering for Monte Carlo image synthesis.While previous gradient-domain Metropolis Light Transport sought to distribute more samples in areas of high gradients, we show, in contrast, that estimating image gradients is also possible using standard (non-Metropolis) Monte Carlo algorithms, and furthermore, that even without changing the sample distribution, this often leads to significant error reduction. This broadens the applicability of gradient rendering considerably. To gain insight into the conditions under which gradient-domain sampling is beneficial, we present a frequency analysis that compares Monte Carlo sampling of gradients followed by Poisson reconstruction to traditional Monte Carlo sampling. Finally, we describe Gradient-Domain Path Tracing (G-PT), a relatively simple modification of the standard path tracing algorithm that can yield far superior results.

Dual-Domain Filtering

We propose dual-domain filtering, an image processing paradigm that couples spatial domain with frequency domain filtering. Our dual-domain defined filter removes artifacts like residual noise of other image denoising methods and compression artifacts. Moreover, iterating the filter achieves state-of-the-art image denoising results, but with a much simpler algorithm than competing approaches. The simplicity and versatility of the dual-domain filter makes it an attractive tool for image processing.

Regularizing Image Reconstruction for Gradient-Domain Rendering with Feature Patches

We present a novel algorithm to reconstruct high-quality images from sampled pixels and gradients in gradient-domain rendering. Our approach extends screened Poisson reconstruction by adding additional regularization constraints. Our key idea is to exploit local patches in feature images, which contain per-pixels normals, textures, position, etc., to formulate these constraints. We describe a GPU implementation of our approach that runs on the order of seconds on megapixel images. We demonstrate a significant improvement in image quality over screened Poisson reconstruction under the L1 norm. Because we adapt the regularization constraints to the noise level in the input, our algorithm is consistent and converges to the ground truth.

Private Property, Public Use, and Just Compensation: The Economics of Eminent Domain

The eminent domain clause of the U.S. Constitution concerns the limits of the government's right to take private property for public use. The economic literature on this issue has examined (1) the proper scope of this power as embodied by the 'public use' requirement, (2) the appropriate definition, and implications, of 'just compensation,' and (3) the impact of eminent domain on land use incentives of owners whose land is subject to a taking risk. This essay reviews this literature and draws implications for our understanding of eminent domain law.

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

Xp95 is phosphorylated within the proline rich domain during Xenopus oocyte maturation

Xp95 is the Xenopus ortholog of a conserved family of scaffold proteins that have in common an N-terminal Bro1 domain and a C-terminal proline rich domain (PRD). The regulation of this protein family is poorly understood. We previously showed that Xp95 undergoes a phosphorylation-dependant gel mobility shift during meiotic maturation of Xenopus oocytes, the only natural biological system in which post-translational modifications of this family has been demonstrated. Here we characterized Xp95 phosphorylation via two approaches. First, we tested a series of Xp95 fragments for the ability to gel-shift during oocyte maturation, and found that a fragment containing amino acids 705-786 is sufficient to cause a gel-shift. This fragment is within the N-terminal region of Xp95's PRD (N-PRD). Second, we purified phosphorylated Xp95 and by mass spectrometry found that a 5080 Da peptide which maps to N-PRD (amino acids 706-756) contains two phosphorylation sites, one of which is T745, within the conserved CIN85 binding motif. By in vitro protein interaction assays, we that T745 is critical for CIN85/Xp95 interaction, and that Xp95 phosphorylation correlates with loss of binding to CIN85. We also show that an Alix fragment (amino acids 604-789) also undergoes a gel-shift during oocyte maturation and during colcemid-induced mitotic arrest of HeLa cells. These findings indicate that Xp95/Alix is phosphorylated on the PRD during M phase induction and that the PRD phosphorylation regulates partner protein interaction. ^

Lethal giant discs, a novel C2 domain protein, restricts Notch activation during endocytosis

The Notch signaling pathway plays a central role in metazoan growth and patterning, and its deregulation leads to many human diseases, including cancer. It is therefore important to understand the modes of Notch signaling regulation. Recent discoveries have demonstrated that mutations in conserved endosomal pathway components such as Erupted and Vps25 can ectopically activate Notch signaling in Drosophila. Mutations in the tumor suppressor lethal giant discs (lgd) display similar but even stronger and more specific Notch activation than in the erupted and vps25 mutant animals. This Notch activation in lgd mutant tissues causes hyperplastic overgrowth of the Drosophila imaginal discs, and the eventual lethality of the animal. However, the gene that encodes Lgd, and its function in the Notch pathway have not yet been identified. ^ I have found that Lgd is a novel, conserved C2 domain protein that regulates Notch trafficking. Lgd cell-autonomously restricts Notch signaling in the Drosophila wing disc to the target cells in the D/V boundary. The function of Lgd lies at or upstream of Notch S3 activation, but Lgd doesn't affect the binding affinities between Notch and Delta. Lgd is also not required for cis-inhibition of Notch signaling by ligands. Notch accumulates on the early endosome in lgd mutant cells and signals in a ligand-independent manner, a result that has previously been seen in endosomal pathway mutants. Interestingly, Notch activation in lgd mutant cells is dependent on the endosomal protein Hrs, and Lgd activity appears to be downstream of Hrs function in endocytosis. Taken together, my data identify Lgd as a novel tumor suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling. ^

Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^

Formalizing a Conceptual Framework of Work Domain Knowledge

Background: The failure rate of health information systems is high, partially due to fragmented, incomplete, or incorrect identification and description of specific and critical domain requirements. In order to systematically transform the requirements of work into real information system, an explicit conceptual framework is essential to summarize the work requirements and guide system design. Recently, Butler, Zhang, and colleagues proposed a conceptual framework called Work Domain Ontology (WDO) to formally represent users’ work. This WDO approach has been successfully demonstrated in a real world design project on aircraft scheduling. However, as a top level conceptual framework, this WDO has not defined an explicit and well specified schema (WDOS) , and it does not have a generalizable and operationalized procedure that can be easily applied to develop WDO. Moreover, WDO has not been developed for any concrete healthcare domain. These limitations hinder the utility of WDO in real world information system in general and in health information system in particular. Objective: The objective of this research is to formalize the WDOS, operationalize a procedure to develop WDO, and evaluate WDO approach using Self-Nutrition Management (SNM) work domain. Method: Concept analysis was implemented to formalize WDOS. Focus group interview was conducted to capture concepts in SNM work domain. Ontology engineering methods were adopted to model SNM WDO. Part of the concepts under the primary goal “staying healthy” for SNM were selected and transformed into a semi-structured survey to evaluate the acceptance, explicitness, completeness, consistency, experience dependency of SNM WDO. Result: Four concepts, “goal, operation, object and constraint”, were identified and formally modeled in WDOS with definitions and attributes. 72 SNM WDO concepts under primary goal were selected and transformed into semi-structured survey questions. The evaluation indicated that the major concepts of SNM WDO were accepted by 41 overweight subjects. SNM WDO is generally independent of user domain experience but partially dependent on SNM application experience. 23 of 41 paired concepts had significant correlations. Two concepts were identified as ambiguous concepts. 8 extra concepts were recommended towards the completeness of SNM WDO. Conclusion: The preliminary WDOS is ready with an operationalized procedure. SNM WDO has been developed to guide future SNM application design. This research is an essential step towards Work-Centered Design (WCD).

CONFORMATIONAL DYNAMICS OF K-RAS AND H-RAS PROTEINS: IS THERE FUNCTIONAL SPECIFICITY AT THE CATALYTIC DOMAIN?

Ras proteins serve as crucial signaling modulators in cell proliferation through their ability to hydrolyze GTP and exist in a GTP “on” state and GTP “off” state. There are three different human Ras isoforms: H-ras, N-ras and K-ras (4A and 4B). Although their sequence identity is very high at the catalytic domain, these isoforms differ in their ability to activate different effectors and hence different signaling pathways. Much of the previous work on this topic has attributed this difference to the hyper variable region of Ras proteins, which contains most of the sequence variance among the isoforms and encodes specificity for differential distribution in the membrane. However, we hypothesize that sequence variation on lobe II of Ras catalytic domain alters dynamics and leads to differential preference for different effectors or modulators. In this work, we used all atom molecular dynamics to analyze the dynamics in the catalytic domain of H-ras and K-ras. We have also analyzed the dynamics of a transforming mutant of H-ras and K-ras and further studied the dynamics of an effectorselective mutant of H-ras. Collectively we have determined that wild type K-ras is more dynamic than H-ras and that the structure of the effector binding loop more closely resembles that of the T35S Raf-selective mutant, possibly giving us a new view and insight into the v mode of effector specificity. Furthermore we have determined that specific mutations at the same location perturb the conformational equilibrium differently in H-ras and K-ras and that an enhanced oncogenic potential may arise from different structural perturbations for each point mutation of a specific isoform.

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^