A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete–scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of Gαi subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein Gαi3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [35S]methionine, two pools of GAIP—a soluble and a membrane-anchored pool—were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [3H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the Gαi subfamily, Gαi1, Gαi2, Gαi3, Gαz, and Gαo, but not with members of other Gα subfamilies, Gαs, Gαq, and Gα12/13. The C terminus of Gαi3 is important for binding because a 10-aa C-terminal truncation and a point mutant of Gαi3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of Gαi3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP’s possible involvement in membrane trafficking.

CTXφ contains a hybrid genome derived from tandemly integrated elements

CTXφ is a filamentous, temperate bacteriophage whose genome includes ctxAB, the genes that encode cholera toxin. In toxigenic isolates of Vibrio cholerae, tandem arrays of prophage DNA, usually interspersed with the related genetic element RS1, are integrated site-specifically within the chromosome. We have discovered that these arrays routinely yield hybrid virions, composed of DNA from two adjacent prophages or from a prophage and a downstream RS1. Coding sequences are always derived from the 5′ prophage whereas most of an intergenic sequence, intergenic region 1, is always derived from the 3′ element. The presence of tandem elements is required for production of virions: V. cholerae strains that contain a solitary prophage rarely yield CTX virions, and the few virions detected result from imprecise excision of prophage DNA. Thus, generation of the replicative form of CTXφ, pCTX, a step that precedes production of virions, does not depend on reversal of the process for site-specific integration of CTXφ DNA into the V. cholerae chromosome. Production of pCTX also does not depend on RecA-mediated homologous recombination between adjacent prophages. We hypothesize that the CTXφ-specific proteins required for replication of pCTX can also function on a chromosomal substrate, and that, unlike the processes used by other integrating phages, production of pCTX and CTXφ does not require excision of the prophage from the chromosome. Use of this replication strategy maximizes vertical transmission of prophage DNA while still enabling dissemination of CTXφ to new hosts.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3.

To identify proteins that regulate the transcriptional activity of c-Jun, we have used the yeast two-hybrid screen to detect mammalian polypeptides that might interact functionally with the N-terminal segment of c-Jun, a known regulatory region. Among the proteins identified is a short form of Stat3 (designated Stat3 beta). Stat3 beta is missing the 55 C-terminal amino acid residues of the long form (Stat3 alpha) and has 7 additional amino acid residues at its C terminus. In the absence of added cytokines, expression of Stat3 beta (but not Stat3 alpha) in transfected cells activated a promoter containing the interleukin 6 responsive element of the rat alpha 2-macroglobulin gene; coexpression of Stat3 beta and c-Jun led to enhanced cooperative activation of the promoter. Nuclear extracts of cells transfected with a Stat3 beta expression plasmid formed a complex with an oligonucleotide containing a Stat3 binding site, whereas extracts of cells transfected with a Stat3 alpha plasmid did not. We conclude that there is a short form of Stat3 (Stat3 beta), that Stat3 beta is transcriptionally active under conditions where Stat3 alpha is not, and that Stat3 beta and c-Jun are capable of cooperative activation of certain promoters.

The Hybrid Spirit Animating Chinese Pentecostals in Malaysia

This dissertation interrogates the assertion in postcolonial scholarship, especially from the work of Homi Bhabha that the construction and performance of hybrid identities act as a form of resistance for marginalized communities against structures of oppression. While this study supports this assertion, it also critiques how hybridity fails to address issues of unequal power relations. This has led to an uncritical use of hybridity that reproduces the very idea of static identity which its proponents claim to transcend. Through qualitative study of Chinese members of a Pentecostal church in Malaysia, this study argues that church members engage in "unequal belonging" where they privilege certain elements of their identities over others. In concert with Pierre Bourdieu's conceptions of habitus, field, and capital, unequal belonging highlights how hybridity fails to capture the intersecting and competing loyalties, strategies, and complexities of identity formation on a contextual level. Unequal belonging challenges postcolonial scholars to locate the subtle workings of power and privilege that manifest even among marginalized communities. The study first situates the Chinese through an analysis of the historical legacy of British colonialism that has structured the country's current socio-political configuration along bounded categories of identification. The habitus constrains church members to accept certain Chinese ethnic markers as "givens." Although they face continuous marginalization, interviewee data demonstrates that church members negotiate their Chineseness and construct a "Modern Chinese" ethnic identity as a strategic move away from Chinese stereotypes. Moreover, conversion to Christianity affords church members access to cultural capital. Yet, it is limited and unequal capital. In particular, the "Chinese Chinese," who church members have demarcated as backward and traditional, are unable to gain access to this capital because they lack fluency in English and knowledge in modern, westernized worldviews. Unequal belonging nuances monolithic conceptions of hybridity. It demonstrates how church members' privilege of Christianity over Chineseness exposes the complex processes of power and privilege that makes westernized-English-speaking Chinese Christians culturally "higher" than non-English-speaking, non-Christian, Chinese. This study provides significant contribution to the complex aspect of hybridity where it is both a site of resistance and oppression.

Tracing Hybrid Collectives of Illness, War, and Medicine in Twentieth- and Twenty-First Century Narratives of Illness

Thesis (Ph.D.)--University of Washington, 2016-06

New hybrid system:poly (ethylene glycol) hydrogel with covalently bonded pegylated nanotubes

Hydrogels containing carbon nanotubes (CNTs) are expected to be promising conjugates because they might show a synergic combination of properties from both materials. Most of the hybrid materials containing CNTs only entrap them physically, and the covalent attachment has not been properly addressed yet. In this study, single-walled carbon nanotubes (SWNTs) were successfully incorporated into a poly(ethylene glycol) (PEG) hydrogel by covalent bonds to form a hybrid material. For this purpose, SWNTs were functionalized with poly(ethylene glycol) methacrylate (PEGMA) to obtain water-soluble pegylated SWNTs (SWNT–PEGMA). These functionalized SWNTs were covalently bonded through their PEG moieties to a PEG hydrogel. The hybrid network was obtained from the crosslinking reaction of poly(ethylene glycol) diacrylate prepolymer and the SWNT–PEGMA by dual photo-UV and thermal initiations. The mechanical and swelling properties of the new hybrid material were studied. In addition, the material and lixiviates were analyzed to elucidate any kind of SWNT release and to evaluate a possible in vitro cytotoxic effect. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011.

A Proposed Structure of Knowledge-Based Hybrid Intelligent Systems for Sophisticated Environments

The paper deals with a problem of intelligent system’s design for complex environments. There is discussed a possibility to integrate several technologies into one basic structure. One possible structure is proposed in order to form a basis for intelligent system that would be able to operate in complex environments. The basic elements of the proposed structure have found their implemented in software system. This software system is shortly presented in the paper. The most important results of experiments are outlined and discussed at the end of the paper. Some possible directions of further research are sketched.

Photonic gas sensors exploiting directly the optical properties of hybrid carbon nanotube localized surface plasmon structures

We investigate the modification of the optical properties of carbon nanotubes (CNTs) resulting from a chemical reaction triggered by the presence of a specific compound (gaseous carbon dioxide (CO2)) and show this mechanism has important consequences for chemical sensing. CNTs have attracted significant research interest because they can be functionalized for a particular chemical, yielding a specific physical response which suggests many potential applications in the fields of nanotechnology and sensing. So far, however, utilizing their optical properties for this purpose has proven to be challenging. We demonstrate the use of localized surface plasmons generated on a nanostructured thin film, resembling a large array of nano-wires, to detect changes in the optical properties of the CNTs. Chemical selectivity is demonstrated using CO2 in gaseous form at room temperature. The demonstrated methodology results additionally in a new, electrically passive, optical sensing configuration that opens up the possibilities of using CNTs as sensors in hazardous/explosive environments.

All-fiber loading sensor based on a hybrid 45° and 81° tilted fiber grating structure

We experimentally demonstrate an all-fiber loading sensor system based on a 45° and an 81° tilted fiber grating (TFG). We have fabricated two TFGs adjacent to each other in a single fiber to form a hybrid structure. When the transverse load applied to the 81° TFG, the light coupling to the two orthogonally polarized modes will interchange the power according to the load applied to the fiber, which provides a solution to measure the load. For real applications, we further investigated the interrogation of this all-fiber loading sensor system using a low-cost and compact-size single wavelength source and a power meter. The experimental results have clearly shown that a low-cost high-sensitivity loading sensor system can be developed based on the proposed TFG configuration.

Building the hybrid organisation through ERP and enterprise social software

This research traces the implementation of an information system in the form of ERP modules covering tenant and contract management in a Chinese service company. Misalignments between the ERP system specification and user needs led to the adoption of informal processes within the organisation. These processes are facilitated within an informal organisational structure and are based on human interactions undertaken within the formal organisation. Rather than to attempt to suppress the emergence of the informal organisation the company decided to channel the energies of staff involved in informal processes towards organisational goals. The company achieved this by harnessing the capabilities of what we term a hybrid ERP system, combining the functionality of a traditional (formal) ERP installation with the capabilities of Enterprise Social Software (ESS). However the company recognised that the successful operation of the hybrid ERP system would require a number of changes in organisational design in areas such as reporting structures and communication channels. A narrative provided by interviews with company personnel is thematised around the formal and informal characteristics of the organisation as defined in the literature. This leads to a definition of the characteristics of the hybrid organisation and strategies for enabling a hybrid organisation, facilitated by a hybrid ERP system, which directs formal and informal behaviour towards organisational goals and provides a template for future hybrid implementations.

Seismic performance of hybrid fiber reinforced polymer-concrete pier frame systems

As an alternative to transverse spiral or hoop steel reinforcement, fiber reinforced polymers (FRPs) were introduced to the construction industry in the 1980’s. The concept of concrete-filled FRP tube (CFFT) has raised great interest amongst researchers in the last decade. FRP tube can act as a pour form, protective jacket, and shear and flexural reinforcement for concrete. However, seismic performance of CFFT bridge substructure has not yet been fully investigated. Experimental work in this study included four two-column bent tests, several component tests and coupon tests. Four 1/6-scale bridge pier frames, consisting of a control reinforced concrete frame (RCF), glass FRP-concrete frame (GFF), carbon FRP-concrete frame (CFF), and hybrid glass/carbon FRP-concrete frame (HFF) were tested under reverse cyclic lateral loading with constant axial loads. Specimen GFF did not show any sign of cracking at a drift ratio as high as 15% with considerable loading capacity, whereas Specimen CFF showed that lowest ductility with similar load capacity as in Specimen GFF. FRP-concrete columns and pier cap beams were then cut from the pier frame specimens, and were tested again in three point flexure under monotonic loading with no axial load. The tests indicated that bonding between FRP and concrete and yielding of steel both affect the flexural strength and ductility of the components. The coupon tests were carried out to establish the tensile strength and elastic modulus of each FRP tube and the FRP mold for the pier cap beam in the two principle directions of loading. A nonlinear analytical model was developed to predict the load-deflection responses of the pier frames. The model was validated against test results. Subsequently, a parametric study was conducted with variables such as frame height to span ratio, steel reinforcement ratio, FRP tube thickness, axial force, and compressive strength of concrete. A typical bridge was also simulated under three different ground acceleration records and damping ratios. Based on the analytical damage index, the RCF bridge was most severely damaged, whereas the GFF bridge only suffered minor repairable damages. Damping ratio was shown to have a pronounced effect on FRP-concrete bridges, just the same as in conventional bridges. This research was part of a multi-university project, which is founded by the National Science Foundation (NSF) - Network for Earthquake Engineering Simulation Research (NEESR) program.

Interdigitating organic bilayers direct the short interlayer spacing in hybrid organic–inorganic layered vanadium oxide nanostructures

Layered metal oxides provide a single-step route to sheathed superlattices of atomic layers of a variety of inorganic materials, where the interlayer spacing and overall layered structure forms the most critical feature in the nanomaterials’ growth and application in electronics, health, and energy storage. We use a combination of computer simulations and experiments to describe the atomic-scale structure, dynamics and energetics of alkanethiol-intercalated layered vanadium oxide-based nanostructures. Molecular dynamics (MD) simulations identify the unusual substrate-constrained packing of the alkanethiol surfactant chains along each V2O5 (010) face that combines with extensive interdigitation between chains on opposing faces to maximize three-dimensional packing in the interlayer regions. The findings are supported by high resolution electron microscopy analyses of synthesized alkanethiol-intercalated vanadium oxide nanostructures, and the preference for this new interdigitated model is clarified using a large set of MD simulations. This dependency stresses the importance of organic–inorganic interactions in layered material systems, the control of which is central to technological applications of flexible hybrid nanomaterials.

Seismic Performance of Hybrid Fiber Reinforced Polymer-Concrete Pier Frame Systems

As an alternative to transverse spiral or hoop steel reinforcement, fiber reinforced polymers (FRPs) were introduced to the construction industry in the 1980's. The concept of concrete-filled FRP tube (CFFT) has raised great interest amongst researchers in the last decade. FRP tube can act as a pour form, protective jacket, and shear and flexural reinforcement for concrete. However, seismic performance of CFFT bridge substructure has not yet been fully investigated. Experimental work in this study included four two-column bent tests, several component tests and coupon tests. Four 1/6-scale bridge pier frames, consisting of a control reinforced concrete frame (RCF), glass FRP-concrete frame (GFF), carbon FRP-concrete frame (CFF), and hybrid glass/carbon FRP-concrete frame (HFF) were tested under reverse cyclic lateral loading with constant axial loads. Specimen GFF did not show any sign of cracking at a drift ratio as high as 15% with considerable loading capacity, whereas Specimen CFF showed that lowest ductility with similar load capacity as in Specimen GFF. FRP-concrete columns and pier cap beams were then cut from the pier frame specimens, and were tested again in three point flexure under monotonic loading with no axial load. The tests indicated that bonding between FRP and concrete and yielding of steel both affect the flexural strength and ductility of the components. The coupon tests were carried out to establish the tensile strength and elastic modulus of each FRP tube and the FRP mold for the pier cap beam in the two principle directions of loading. A nonlinear analytical model was developed to predict the load-deflection responses of the pier frames. The model was validated against test results. Subsequently, a parametric study was conducted with variables such as frame height to span ratio, steel reinforcement ratio, FRP tube thickness, axial force, and compressive strength of concrete. A typical bridge was also simulated under three different ground acceleration records and damping ratios. Based on the analytical damage index, the RCF bridge was most severely damaged, whereas the GFF bridge only suffered minor repairable damages. Damping ratio was shown to have a pronounced effect on FRP-concrete bridges, just the same as in conventional bridges. This research was part of a multi-university project, which is founded by the National Science Foundation (NSF) Network for Earthquake Engineering Simulation Research (NEESR) program.