Regulation of HIV Gag conformation and complex formation

The results of this research will help in a better understanding of HIV morphogenesis, which will hopefully lead to a structure based drug design towards the virus.

Biosynthesis, localization, and macromolecular arrangement of the plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA+ ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The Saccharomyces cerevisiae RAD1 and human XPF genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An Arabidopsis thaliana gene (AtRAD1) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct AtRAD1 cDNAs from A. thaliana leaf tissue RNA. One of the isolates (AtRAD1-1) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (AtRAD1-2, AtRAD1-3) differ slightly in size due to variations at the 5′ end of exon 6 or the 3′ end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the AtRAD1-1 and AtRAD1-2 but not AtRAD1-3 transcripts in bud and root tissue, and to a fourth transcript (AtRAD1-4), having both alterations identified in AtRAD1-2 and AtRAD1-3, in root tissue. However, the low frequency of detection of AtRAD1-3 and AtRAD1-4 makes the significance of these tissue-specific patterns unclear. The predicted AtRad1-2, AtRad1-3 and AtRad1-4 proteins lack part of the region likely required for endonuclease complex formation. Expression of AtRAD1-2 and AtRAD1-3 in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

Synthesis and structure of 1,3,5-tris(diorganohydroxysilyl)benzenes. Novel building blocks in supramolecular silanol chemistry

The 1,3,5-tris(diorganohydroxysilyl)benzenes 1,3,5-(HOR₂Si)₃C₆H₃ (TMSB, R = Me; TPSB, R = Ph) have been prepared and fully characterized by X-ray crystallography. The crystal structure of TMSB features pairwise connected layers, in which the molecules are involved in interlayer hydrogen bonding. The supramolecular hydrogen bond motif may be described as a 12-membered ring that adopts a chair conformation. TPSB forms an equimolar inclusion complex with water, which is associated via hydrogen bonding and apparently fills a void in the crystal packing. In this case, the supramolecular hydrogen bond motif may be described as an eight-membered ring. Two of the water molecules are also associated, giving rise to a water dimer entrapped in the silanol matrix. Besides the hydrogen bonds, the crystal structure of the TPSB·H₂O complex reveals intra- and intermolecular C-H··· π stacking of most of the phenyl groups. Electrospray mass spectrometry shows that TPSB undergoes supramolecular complex formation with a variety of N-donors such as 4-(dimethylamino)pyridine, N,N,N',N'-tetramethylethylenediamine, imidazole, 2-(dimethylamino)pyridine, and 2,2'-dipyridylamine.

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Transcriptional regulation of the yghJ-pppA-yghG-gspCDEFGHIJKLM cluster, encoding the type II secretion pathway in Enterotoxigenic Escherichia coli

The gene cluster gspCDEFGHIJKLM codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic Escherichia coli (ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the gspCDEFGHIJKLM cluster and three other upstream genes, yghJ, pppA, and yghG, are cotranscribed and that a promoter located in the upstream region of yghJ plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the yghJ promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of yghJ was required for full repression. The yghJ promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of E. coli.

Interactions between fibroin and sericin proteins from antheraea pernyi and bombyx mori silk fibers

Silkworm silk fibers are core-shell composites of fibroin and sericin proteins. Studying the interactions between fibroin and sericin is essential for understanding the properties of these composites. It isobserved that compared to the domestic silk cocoon Bombyx mori (B. mori), the adhesion between fibroin and sericin from the wild silk cocoon, Antheraea pernyi (A. pernyi), is significantly stronger with a higherdegree of heterogeneity. The adsorption of A. pernyi sericin on its fibroin is almost twice the value for B.mori sericin on fibroin, both showing a monolayer Langmuir adsorption. 1H NMR and FTIR studiesdemonstrate on a molecular level the stronger interactions and the more intensive complex formation between A. pernyi fibroin and sericin, facilitated by the hydrogen bonding between glycine and serine.The findings of this study may help the design of composites with superior interfacial adhesion between different components.

The formation of complex microstructures after different deformation modes in advanced high-strength steels

The microstructure of transformation induced plasticity (TRIP) and dual phase (DP) multiphase steels after stamping of an industrial component at different strain levels was investigated using transmission electron microscopy. The TRIP steel microstructure showed a more complex dislocation substructure of ferrite at different strain levels than DP steel. The deformation microstructure of the stamped parts was compared to the deformation microstructure in these complex steels for different "equivalent" tensile strains. It was found that the microstructures are similar only at high levels of strain (>10 pct) for both steels. © 2014 The Minerals, Metals & Materials Society and ASM International.

Postcolonial bildungsroman: indigenous subjects and identity-formation

In the bildungsroman as it has conventionally been defined, individuals attain self-actualisation through a series of experiences whereby they accommodate their individuality to a social order.  Protagonists negotiate their transition from childhood to young-adulthood by way of educative experiences, trials of various kinds, and a search for identity, which is generally formulated as a fixed or stable essence which they must discover or accept.  In this paper I focus on two examples of bildungsroman by Indigenous Canadian and Australian authors: Jeannette Armstrong’s Slash (1985),and Richard J. Frankland’s Digger J. Jones (2007).  Both novels feature male protagonists whose stories play out against the background of Indigenous activism in the 1960s.  As they track the identity-formation of their protagonists, the two novels deconstruct simple or fixed ideas of national identity by pointing to the complex cross-cultural relationships which have characterised settler societies.  At the same time,these novels dramatise the power of socialising practices which promote white superiority and position Indigenous peoples as supplicants or victims.  Both novels draw on what Paul Havemann terms the "new politics of identity and cultural recognition" which characterise contemporary Indigenous activism, re-reading events and settings of the 1960s in the light of discourses of cultural recognition and self-determination.

Late Silurian trace fossils from the Melbourne Formation, Studley Park, Victoria, southeastern Australia

An ichnoassemblage of 10 ichnospecies is described for the first time from the Late Silurian Melbourne Formation at Studley Park, Victoria, southeastern Australia. The ichnofauna is preserved in a typical deep-water turbidite succession of alternating thin- to thick-bedded sandstone and thin- to medium-bedded mudrocks. Trace fossils observed within the study site have been assigned to three main ichnofacies. Ichnofacies 1 is best developed on the linguoid-rippled upper surface of thin sandstone beds and includes Laevicyclus, Aulichnites, Nereites, Helminthoidichnites, small Chondrites and possible Zoophycos. Ichnofacies 2 is very similar to Ichnofacies 1 in ichnospecies composition but instead contains large forms of Chondrites together with other thin burrow types usually poorly preserved and in very low abundance compared with Ichnofacies 1. Ichnofacies 3 is preserved mainly as casts on the underside of medium- to thick-bedded turbiditic sandstones, and has a very low diversity, with Planolites being the most common trace. A detailed analysis of the ichnofabrics and tiering structures of these ichnofacies suggest that Ichnofacies 1 and 3 represent "simple tiering’, in contrast to Ichnofacies 2, which is more characteristic of 'complex tiering’. Despite the differences in ichnospecies composition and ichnofabrics between the three recognized ichnofacies, the collective ichnoassemblage from the study site can be assigned confidently to the Nereites ichnofacies and is, therefore, interpreted to have formed in a distal submarine fan environment of lower bathyal to abyssal depth. Further, it is possible to recognize two main subenvironments within this deep-sea setting to account for the differences between the ichnofacies. Ichnofacies 1 and 2 are interpreted to represent a typical Nereites ichnofacies located on a level basin floor subenvironment of relatively low energy conditions at the distal end of a submarine fan deposit. In comparison, Ichnofacies 3 is dominated by Planolites with rare other facies-crossing trace fossil forms, and lacks Nereites. It is, therefore, best interpreted as representing a relatively high-energy environment, possibly a distributary channel near the distal end of the submarine fan system.

A probabilistic movement model for shortest path formation in virtual ant-like agents

We propose a probabilistic movement model for controlling ant-like agents foraging between two points. Such agents are all identical, simple, autonomous and can only communicate indirectly through the environment. These agents secrete two types of pheromone, one to mark trails towards the goal and another to mark trails back to the starting point. Three pheromone perception strategies are proposed (Strategy A, B and C). Agents that use strategy A perceive the desirability of a neighbouring location as the difference between levels of attractive and repulsive pheromone in that location. With strategy B, agents perceive the desirability of a location as the quotient of levels of attractive and repulsive pheromone. Agents using strategy C determine the product of the levels of attractive pheromone with the complement of levels of repulsive pheromone. We conduct experiments to confirm directionality as emergent property of trails formed by agents that use each strategy. In addition, we compare path formation speed and the quality of the formed path under changes in the environment. We also investigate each strategy's robustness in environments that contain obstacles. Finally, we investigate how adaptive each strategy is when obstacles are eventually removed from the scene and find that the best strategy of these three is strategy A. Such a strategy provides useful guidelines to researchers in further applications of swarm intelligence metaphors for complex problem solving.

Architecture of macromolecular network of soft functional materials : from structure to function

An enhanced macromolecular nanofiber network and its implications have been developed by employing the understanding of its formation with an emphasis on its topological aspect. Using agarose aqueous solution as a typical example, the macromolecular nanofiber network of soft functional materials has been clearly visualized for the first time using the developed technique of field emission scanning electronic microscopy coupled with flash-freeze-drying. Both the systematic kinetic study and the image evidence indicates that the nanofiber network in soft functional materials such as agarose turns out to form through a self-expitaxial nucleation-controlled process. This new understanding enables us to engineer ultra functions of soft materials via nanofiber network architecture, which in turn opens up a new direction in nano fabrication.

The role of the yeast cleavage and polyadenylation factor subunit Ydh1p/Cft2p in pre-mRNA 3' end formation

Cleavage and polyadenylation factor (CPF) is a multi‐protein complex that functions in pre‐mRNA 3′‐end formation and in the RNA polymerase II (RNAP II) transcription cycle. Ydh1p/Cft2p is an essential component of CPF but its precise role in 3′‐end processing remained unclear. We found that mutations in YDH1 inhibited both the cleavage and the polyadenylation steps of the 3′‐end formation reaction in vitro. Recently, we demonstrated that an important function of CPF lies in the recognition of poly(A) site sequences and RNA binding analyses suggesting that Ydh1p/Cft2p interacts with the poly(A) site region. Here we show that mutant ydh1 strains are deficient in the recognition of the ACT1 cleavage site in vivo. The C‐terminal domain (CTD) of RNAP II plays a major role in coupling 3′‐end processing and transcription. We provide evidence that Ydh1p/Cft2p interacts with the CTD of RNAP II, several other subunits of CPF and with Pcf11p, a component of CF IA. We propose that Ydh1p/Cft2p contributes to the formation of important interaction surfaces that mediate the dynamic association of CPF with RNAP II, the recognition of poly(A) site sequences and the assembly of the polyadenylation machinery on the RNA substrate.

Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation

The Saccharomyces cerevisiae WD-40 repeat protein Swd2p associates with two functionally distinct multiprotein complexes: the cleavage and polyadenylation factor (CPF) that is involved in pre-mRNA and snoRNA 3′ end formation and the SET1 complex (SET1C) that methylates histone 3 lysine 4. Based on bioinformatic analysis we predict a seven-bladed β-propeller structure for Swd2p proteins. Northern, transcriptional run-on and in vitro 3′ end cleavage analyses suggest that temperature sensitive swd2 strains were defective in 3′ end formation of specific mRNAs and snoRNAs. Protein–protein interaction studies support a role for Swd2p in the assembly of 3′ end formation complexes. Furthermore, histone 3 lysine 4 di-and tri-methylation were adversely affected and telomeres were shortened in swd2 mutants. Underaccumulation of the Set1p methyltransferase accounts for the observed loss of SET1C activity and suggests a requirement for Swd2p for the stability or assembly of this complex. We also provide evidence that the roles of Swd2p as component of CPF and SET1C are functionally independent. Taken together, our results establish a dual requirement for Swd2p in 3′ end formation and histone tail modification.

Recruitment of class I hydrophobins to the air : water interface initiates a multi-step process of functional amyloid formation

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.

Synthesis and mechanistic insights of macromolecular assemblies from ABA triblock copolymer blends

Macromolecular assembly of block copolymers into numerous nanostructures resembles self-organization of proteins and cellular components found in nature. In order to mimic nature’s assemblies either to cure a disease or construct functional devices, the organization principles underpinning the emergence of complex shapes need to be understood. In the same vein, this study aimed at understanding morphology evolution in a triblock copolymer blend in aqueous solution. An ABA type amphiphilic triblock copolymer (polystyrene-b-polyethylene oxide-b-polystyrene, PS-b-PEO-b-PS) was synthesized at different compositions via atom transfer radical polymerization (ATRP) and self-assembly behavior of a binary mixture in aqueous solution was studied. Block copolymers that form worms and vesicles in its pristine state was shown to form complex morphologies such as fused rings, “jellyfish”, toroid vesicles, large compound vesicles and large lamellae after blending. The tendency of vesicle-forming block copolymer to form bilayers may be responsible for triggering complex morphologies when mixed with a worm or micelle-forming polymer. In other words, the interplay between curvature effects produced by two distinct polymers with different hydrophobic block lengths results in complex morphologies due to chain segregation within the nanostructure.