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Articles by Peijun Zhang in JoVE

 JoVE Immunology and Infection

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

1Department of Structural Biology, University of Pittsburgh School of Medicine

JoVE 3041

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

 JoVE Bioengineering

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine

JoVE 50386

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Other articles by Peijun Zhang on PubMed

Amphi-Eomes/Tbr1: an Amphioxus Cognate of Vertebrate Eomesodermin and T-Brain1 Genes Whose Expression Reveals Evolutionarily Distinct Domain in Amphioxus Development

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode.

Beta-Catenin in Early Development of the Lancelet Embryo Indicates Specific Determination of Embryonic Polarity

The lancelet (amphioxus) embryo develops from a miolecithal egg and starts gastrulation when it is approximately 400 cells in size, in a fashion similar to that of some non-chordate deuterostomes. Throughout this type of gastrulation, the embryo develops characteristics such as the notochord and hollow nerve cord that commonly appear in chordates. beta-Catenin is an important factor in initiating body patterning. The behavior and developmental pattern of this protein in early lancelet development was examined in this study. Cytoplasmic beta-catenin was localized to the animal pole after fertilization and then was incorporated asymmetrically into the blastomeres during the first cleavage. Asymmetric distribution was observed at least until the 32-cell stage. The first nuclear localization was at the 64-cell stage, and involved all of the cells. At the initial gastrula stage, however, concentrated beta-catenin was found on the dorsal side. LiCl treatment affected the asymmetric pattern of beta-catenin during the first cleavage. LiCl also changed distribution of nuclear beta-catenin at the initial gastrula stage: distribution extended to cells on the animal side. Apparently associated with this change, expression domains of goosecoid, lhx3 and otx also changed to a radially symmetric pattern centered at the animal pole. However, LiCl-treated embryos were able to establish embryonic polarity. The present study suggests that in the lancelet embryo, polarity determination is independent of dorsal morphogenesis.

[Classification for One Pathogenic Vibrio Anguillarum Strain Isolated from Skin-ulcer Flounder]

A pathogenic bacterial strain M3 revealed gram negative rod shape, motile and translucent clone was studied. This bacterium was isolated from the skin-ulcer flounder in the fish farm of RongCheng City, Shandong Provice, and could not be identified by BIOLOG ID SYSTEM. Comparative 16S rDNA sequence analyses revealed that strain M3 was most related to the genus of Vibrio. The overall similarity value between strain M3 and Vibrio species were 94% to 98%. Phylogentic analysis showed that M3 strain exhibited the highest level of similarity to Vibrio anguillarum, and the biological features of strain M3 were very similar to that of Vibrio anguillarum. Based on the results obtained, strain M3 was identified as Vibrio anguillarum.

Haematopoietic Lineage Cell-specific Protein 1 (HS1) Promotes Actin-related Protein (Arp) 2/3 Complex-mediated Actin Polymerization

HS1 (haematopoietic lineage cell-specific gene protein 1), a prominent substrate of intracellular protein tyrosine kinases in haematopoietic cells, is implicated in the immune response to extracellular stimuli and in cell differentiation induced by cytokines. Although HS1 contains a 37-amino acid tandem repeat motif and a C-terminal Src homology 3 domain and is closely related to the cortical-actin-associated protein cortactin, it lacks the fourth repeat that has been shown to be essential for cortactin binding to filamentous actin (F-actin). In this study, we examined the possible role of HS1 in the regulation of the actin cytoskeleton. Immunofluorescent staining demonstrated that HS1 co-localizes in the cytoplasm of cells with actin-related protein (Arp) 2/3 complex, the primary component of the cellular machinery responsible for de novo actin assembly. Furthermore, recombinant HS1 binds directly to Arp2/3 complex with an equilibrium dissociation constant (K(d)) of 880 nM. Although HS1 is a modest F-actin-binding protein with a K(d) of 400 nM, it increases the rate of the actin assembly mediated by Arp2/3 complex, and promotes the formation of branched actin filaments induced by Arp2/3 complex and a constitutively activated peptide of N-WASP (neural Wiskott-Aldrich syndrome protein). Our data suggest that HS1, like cortactin, plays an important role in the modulation of actin assembly.

Expression of Muscle-related Genes and Two MyoD Genes During Amphioxus Notochord Development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expre-ssion patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropo-myosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), Amphi-nTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expres-sed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

Automated Image Acquisition and Processing Using a New Generation of 4K X 4K CCD Cameras for Cryo Electron Microscopic Studies of Macromolecular Assemblies

We have previously reported the development of AutoEM, a software package for semi-automated acquisition of data from a transmission electron microscope. In continuing efforts to improve the speed of structure determination of macromolecular assemblies by electron microscopy, we report here on the performance of a new generation of 4 K CCD cameras for use in cryo electron microscopic applications. We demonstrate that at 120 kV, and at a nominal magnification of 67000 x, power spectra and signal-to-noise ratios for the new 4 K CCD camera are comparable to values obtained for film images scanned using a Zeiss scanner to resolutions as high as approximately 1/6.5A(-1). The specimen area imaged for each exposure on the 4 K CCD is about one-third of the area that can be recorded with a similar exposure on film. The CCD camera also serves the purpose of recording images at low magnification from the center of the hole to measure the thickness of vitrified ice in the hole. The performance of the camera is satisfactory under the low-dose conditions used in cryo electron microscopy, as demonstrated here by the determination of a three-dimensional map at 15 A for the catalytic core of the 1.8 MDa Bacillus stearothermophilus icosahedral pyruvate dehydrogenase complex, and its comparison with the previously reported atomic model for this complex obtained by X-ray crystallography.

The Stalk Region of Dynamin Drives the Constriction of Dynamin Tubes

The GTPase dynamin is essential for numerous vesiculation events including clathrin-mediated endocytosis. Upon GTP hydrolysis, dynamin constricts a lipid bilayer. Previously, a three-dimensional structure of mutant dynamin in the constricted state was determined by helical reconstruction methods. We solved the nonconstricted state by a single-particle approach and show that the stalk region of dynamin undergoes a large conformational change that drives tube constriction.

Developmental Expression of Amphioxus GABAA Receptor-associated Protein-like 2 Gene

A full-length amphioxus gamma-aminobutyric acid type-A receptor-associated protein-like 2 ( GABARAPL2) cDNA was isolated. Its sequence and developmental expression are first described in this paper. The phylogenetic analysis shows that the amphioxus GABARAPL2 and GABARAPL2 in vertebrates are highly homologous. The results of in situ hybridization show that the amphioxus GABARAPL2 gene is expressed in the neural tube, neurenteric canal, notochord, muscle and developing alimentary canal.

Visualization of Alpha-helical Features in a Density Map Constructed Using 9 Molecular Images of the 1.8 MDa Icosahedral Core of Pyruvate Dehydrogenase

Strategies to achieve the highest resolutions in structures of protein complexes determined by cryo-electron microscopy generally involve averaging information from large numbers of individual molecular images. However, significant limitations are posed by heterogeneity in image quality and in protein conformation that are inherent to large data sets of images. Here, we demonstrate that the combination of iterative refinement and stringent molecular sorting is an effective method to obtain substantial improvements in map quality of the 1.8 MDa icosahedral catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus. From a starting set of 42,945 images of the core complex, we show that using only the best 139 particles in the data set produces a map that is superior to those constructed with greater numbers of images, and that the location of many of the alpha-helices in the structure can be unambiguously visualized in a map constructed from as few as 9 particles.

Three-dimensional Electron Microscopic Imaging of Membrane Invaginations in Escherichia Coli Overproducing the Chemotaxis Receptor Tsr

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.

Comparative Expression Analysis of Transcription Factor Genes in the Endostyle of Invertebrate Chordates

The endostyle of invertebrate chordates is a pharyngeal organ that is thought to be homologous with the follicular thyroid of vertebrates. Although thyroid-like features such as iodine-concentrating and peroxidase activities are located in the dorsolateral part of both ascidian and amphioxus endostyles, the structural organization and numbers of functional units are different. To estimate phylogenetic relationships of each functional zone with special reference to the evolution of the thyroid, we have investigated, in ascidian and amphioxus, the expression patterns of thyroid-related transcription factors such as TTF-2/FoxE4 and Pax2/5/8, as well as the forkhead transcription factors FoxQ1 and FoxA. Comparative gene expression analyses depicted an overall similarity between ascidians and amphioxus endostyles, while differences in expression patterns of these genes might be specifically related to the addition or elimination of a pair of glandular zones. Expressions of Ci-FoxE and BbFoxE4 suggest that the ancestral FoxE class might have been recruited for the formation of thyroid-like region in a possible common ancestor of chordates. Furthermore, coexpression of FoxE4, Pax2/5/8, and TPO in the dorsolateral part of both ascidian and amphioxus endostyles suggests that genetic basis of the thyroid function was already in place before the vertebrate lineage.

Electron Tomography of Degenerating Neurons in Mice with Abnormal Regulation of Iron Metabolism

Previous studies have shown that IRP1(+/-) IRP2(-/-) knockout mice develop progressive neurodegenerative symptoms similar to those observed in human movement disorders such as Parkinson's disease. Histological investigations using optical microscopy show that these IRP knockout mice display accumulation of ferritin in axonal tracts in the brain, suggesting a possible role for excess ferritin in mediating axonal degeneration. Direct observation of the 3D distribution of ferritin by electron tomography indicates that ferritin amounts are increased by 3- to 4-fold in selected regions of the brain, and structural damage is observed within the axon as evidenced by the loss of the internal network of filaments, and the invaginations of neighboring oligodendrocyte membranes into the axonal medium. While optical microscopic investigations suggest that there is a large increase in ferritin in the presumptive axonal regions of the IRP knockout mice, electron tomographic studies reveal that most of the excess ferritin is localized to double-walled vesicular compartments which are present in the interior of the axon and appear to represent invaginations of the oligodendrocyte cells into the axon. The amount of ferritin observed in the axonal space of the knockout mice is at least 10-fold less than the amount of ferritin observed in wild-type mouse axons. The surprising conclusion from our analysis, therefore, is that despite the overall increase in ferritin levels in the knockout mouse brain, ferritin is absent from axons of degenerating neurons, suggesting that trafficking is compromised in early stages of this type of neuronal degeneration.

Cortactin Mediated Morphogenic Cell Movements During Zebrafish (Danio Rerio) Gastrulation

Cell migration is essential to direct embryonic cells to specific sites at which their developmental fates are ultimately determined. However, the mechanism by which cell motility is regulated in embryonic development is largely unknown. Cortactin, a filamentous actin binding protein, is an activator of Arp2/3 complex in the nucleation of actin cytoskeleton at the cell leading edge and acts directly on the machinery of cell motility. To determine whether cortactin and Arp2/3 mediated actin assembly plays a role in the morphogenic cell movements during the early development of zebrafish, we initiated a study of cortactin expression in zebrafish embryos at gastrulating stages when massive cell migrations occur. Western blot analysis using a cortactin specific monoclonal antibody demonstrated that cortactin protein is abundantly present in embryos at the most early developmental stages. Immunostaining of whole-mounted embryo showed that cortactin immunoreactivity was associated with the embryonic shield, predominantly at the dorsal side of the embryos during gastrulation. In addition, cortactin was detected in the convergent cells of the epiblast and hypoblast, and later in the central nervous system. Immunofluorescent staining with cortactin and Arp3 antibodies also revealed that cortactin and Arp2/3 complex colocalized at the periphery and many patches associated with the cell-to-cell junction in motile embryonic cells. Therefore, our data suggest that cortactin and Arp2/3 mediated actin polymerization is implicated in the cell movement during gastrulation and perhaps the development of the central neural system as well.

Sequence Analysis and Functional Study of Thymidylate Synthase from Zebrafish, Danio Rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K(m) value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Identification and Immunogenicity of an Immunodominant Mimotope of Avibacterium Paragallinarum from a Phage Display Peptide Library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Electron Tomography of Bacterial Chemotaxis Receptor Assemblies

Direct Visualization of Escherichia Coli Chemotaxis Receptor Arrays Using Cryo-electron Microscopy

Signal transduction in bacterial chemotaxis is initiated by the binding of extracellular ligands to a specialized family of methyl-accepting chemoreceptor proteins. Chemoreceptors cluster at distinct regions of the cell and form stable ternary complexes with the histidine autokinase CheA and the adapter protein CheW. Here we report the direct visualization and spatial organization of chemoreceptor arrays in intact Escherichia coli cells by using cryo-electron tomography and biochemical techniques. In wild-type cells, ternary complexes are arranged as an extended lattice, which may or may not be ordered, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. At disproportionately high receptor levels, membrane invaginations containing nonfunctional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be reestablished by increasing cellular levels of CheA and CheW. Our results demonstrate that chemotaxis in E. coli requires the presence of chemoreceptor arrays and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW.

The Expression of AmphiTCTP, a TCTP Orthologous Gene in Amphioxus Related to the Development of Notochord and Somites

The translationally controlled tumor protein (TCTP) is highly conserved and has been widely found in eukaryotic organisms. Here, we report the phylogenetic analysis and developmental expression of AmphiTCTP, a TCTP homologous gene in cephalochordate amphioxus. Phylogenetic analysis indicates that the putative protein of AmphiTCTP is close to its vertebrate orthologs. The mRNA of AmphiTCTP is found in fertilized eggs, early cleavage embryo and most of the early developmental stages by in situ hybridization and RT-PCR, but its expression is not detectable from late cleavage stage to mid-gastrula. The expression of AmphiTCTP in zygotes and early cleavage stages shows that AmphiTCTP may be a maternal gene. From the early neurula stage onward, AmphiTCTP transcript is localized in the presumptive notochord, presomitic mesoderm, and nascent somites. However, its expression is gradually down-regulated after the notochord and somites have been formed. The expression pattern of AmphiTCTP thus coincides with the differentiation of the notochord and somites, this suggests that AmphiTCTP may not be a housekeeping gene and may play an important role in mesoderm development.

A New Peptide-based Method for the Design and Synthesis of Nanoparticle Superstructures: Construction of Highly Ordered Gold Nanoparticle Double Helices

Left-handed gold nanoparticle double helices were prepared using a new method that allows simultaneous synthesis and assembly of discrete nanoparticles. This method involves coupling the processes of peptide self-assembly of and peptide-based biomineralization of nanoparticles. In this study, AYSSGAPPMPPF (PEPAu), an oligopeptide with an affinity for gold surfaces, was modified with an aliphatic tail to generate C12-PEPAu. In the presence of buffers and gold salts, amphiphilic C12-PEPAu was used to both control the formation of monodisperse gold nanoparticles and simultaneously direct their assembly into left-handed gold nanoparticle double helices. The gold nanoparticle double helices are highly regular, spatially complex, and they exemplify the utility of this methodology for rationally controlling the topology of nanoparticle superstructures and the stereochemical organization of discrete nanoparticles within these structures.

Role of HAMP Domains in Chemotaxis Signaling by Bacterial Chemoreceptors

Bacterial chemoreceptors undergo conformational changes in response to variations in the concentration of extracellular ligands. These changes in chemoreceptor structure initiate a series of signaling events that ultimately result in regulation of rotation of the flagellar motor. Here we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We demonstrate that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the "expanded" conformation and chemoreceptor methylation favoring the "compact" conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the "kinase-on" and "kinase-off" states of chemoreceptor trimers, respectively.

[Identification and Phylogenetic Analysis on the AroA Gene of Haemophilus Parasuis]

We clarified the genetic structure and phylogenetic relationship of the aroA gene in Haemophilus parasuis, a gene encoding 5'-enolpyruvylshikimate-3-phosphate synthetase.

Expression Pattern of Dmrt4 from Olive Flounder (Paralichthys Olivaceus) in Adult Gonads and During Embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Genetic Characterization of Asymmetric Reciprocal Hybridization Between the Flatfishes Paralichthys Olivaceus and Paralichthys Dentatus

Interspecific reciprocal crosses between the two flatfishes Paralichthys olivaceus and P. dentatus yielded hybrids with different viabilities. Specifically, the hybrids of P. olivaceus female and P. dentatus male (HI) were found to be viable, while the reciprocal hybrids from P. dentatus female and P. olivaceus male (HII) were completely inviable. All the HII individuals showed morphological deformities and died before first feeding. The chromosome analysis showed that HI individuals had the same chromosome number as parents. However, two chromosomes were missing in HII offspring indicating that the latter were aneuploids. Genomic inheritance from the parents to F(1) progeny was also examined by amplified fragment length polymorphism (AFLP) analyses, and the results showed differences between reciprocal hybrids. Almost all AFLP bands (97.71%) observed in parents were passed on to HI individuals. In contrast, only 86.64% of the AFLP bands from parents were scored in HII individuals. Frequency of lost parental bands was thus significantly higher in HII than that in HI and intraspecific crosses, which was probably associated with chromosomal elimination. In addition, higher segregation distortions were found in hybrids than in controls, although these differences were not significant. The present study indicates that chromosomal elimination and loss of AFLP loci occurred in inviable HII individuals, while such genomic changes were not found in viable HI individuals. Possible implications of such difference on genomic changes for asymmetric viability in reciprocal hybrids are discussed.

Structural Convergence Between Cryo-EM and NMR Reveals Intersubunit Interactions Critical for HIV-1 Capsid Function

Mature HIV-1 particles contain conical-shaped capsids that enclose the viral RNA genome and perform essential functions in the virus life cycle. Previous structural analysis of two- and three-dimensional arrays of the capsid protein (CA) hexamer revealed three interfaces. Here, we present a cryoEM study of a tubular assembly of CA and a high-resolution NMR structure of the CA C-terminal domain (CTD) dimer. In the solution dimer structure, the monomers exhibit different relative orientations compared to previous X-ray structures. The solution structure fits well into the EM density map, suggesting that the dimer interface is retained in the assembled CA. We also identified a CTD-CTD interface at the local three-fold axis in the cryoEM map and confirmed its functional importance by mutagenesis. In the tubular assembly, CA intermolecular interfaces vary slightly, accommodating the asymmetry present in tubes. This provides the necessary plasticity to allow for controlled virus capsid dis/assembly.

Effective Bio-treatment of Fresh Leachate from Pretreated Municipal Solid Waste in an Expanded Granular Sludge Bed Bioreactor

This research investigated the anaerobic biodegradation of fresh leachate from pretreated municipal solid waste (MSW) in an expanded granular sludge bed (EGSB) bioreactor under mesophilic conditions. The observations showed that this bioreactor, inoculated with anaerobic granular sludge, could be readily activated. The chemical oxygen demand (COD) removal efficiency varied between 88% and 97% under normal operation conditions, and was kept at 94-96% under the proposed optimal conditions. We noted that 60-80% of the produced biogas was methane that was yielded at a rate depending on the organic loading rate (OLR) and the liquid up-flow velocity (Vup). Significantly, 80% of loaded COD or 83% of biodegraded COD was converted to methane under the proposed optimal conditions. These findings indicate that the fresh leachate from pretreated MSW can be efficiently treated in the EGSB bioreactor, and moreover, methane, a renewable energy, can be continuously generated.

Identification of Putative Virulence-associated Genes of Haemophilus Parasuis Through Suppression Subtractive Hybridization

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki (H. parasuis serovar 5 reference strain, highly virulent) and SW114 (H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.

Protection of Chickens, with or Without Maternal Antibodies, Against IBDV Infection by a Recombinant IBDV-VP2 Protein

The use of avian herpesviruses (Marek's disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 10(3)PFU/chicken, whereas 10(4)PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens.

Expeditious Synthesis and Assembly of Sub-100 Nm Hollow Spherical Gold Nanoparticle Superstructures

Sub-100 nm hollow gold nanoparticle superstructures were prepared in a direct one-pot reaction. A gold-binding peptide conjugate, C(6)-AA-PEP(Au) (PEP(Au) = AYSSGAPPMPPF), was constructed and used to direct the simultaneous synthesis and assembly of gold nanoparticles. Transmission electron microscopy and electron tomography revealed that the superstructures are uniform and consist of monodisperse gold nanoparticles arranged into a spherical monolayer shell.

Structure of the HIV-1 Full-length Capsid Protein in a Conformationally Trapped Unassembled State Induced by Small-molecule Binding

The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts. We report the crystal structure of the HIV-1 CA, revealing the domain organization in the context of the wild-type full-length (FL) unassembled CA. The FL CA adopts an antiparallel dimer configuration, exhibiting a domain organization sterically incompatible with capsid assembly. A small compound, generated in situ during crystallization, is bound tightly at a hinge site ("H site"), indicating that binding at this interdomain region stabilizes the ADP conformation. Electron microscopy studies on nascent crystals reveal both dimeric and hexameric lattices coexisting within a single condition, in agreement with the interconvertibility of oligomeric forms and supporting the feasibility of promoting assembly-incompetent dimeric states. Solution characterization in the presence of the H-site ligand shows predominantly unassembled dimeric CA, even under conditions that promote assembly. Our structure elucidation of the HIV-1 FL CA and characterization of a potential allosteric binding site provides three-dimensional views of an assembly-defective conformation, a state targeted in, and thus directly relevant to, inhibitor development. Based on our findings, we propose an unprecedented means of preventing CA assembly, by "conformationally trapping" CA in assembly-incompetent conformational states induced by H-site binding.

The Cullin-RING E3 Ubiquitin Ligase CRL4-DCAF1 Complex Dimerizes Via a Short Helical Region in DCAF1

The cullin4A-RING E3 ubiquitin ligase (CRL4) is a multisubunit protein complex, comprising cullin4A (CUL4), RING H2 finger protein (RBX1), and DNA damage-binding protein 1 (DDB1). Proteins that recruit specific targets to CRL4 for ubiquitination (ubiquitylation) bind the DDB1 adaptor protein via WD40 domains. Such CRL4 substrate recognition modules are DDB1- and CUL4-associated factors (DCAFs). Here we show that, for DCAF1, oligomerization of the protein and the CRL4 complex occurs via a short helical region (residues 845-873) N-terminal to DACF1's own WD40 domain. This sequence was previously designated as a LIS1 homology (LisH) motif. The oligomerization helix contains a stretch of four Leu residues, which appear to be essential for α-helical structure and oligomerization. In vitro reconstituted CRL4-DCAF1 complexes (CRL4(DCAF1)) form symmetric dimers as visualized by electron microscopy (EM), and dimeric CRL4(DCAF1) is a better E3 ligase for in vitro ubiquitination of the UNG2 substrate compared to a monomeric complex.

Rhesus TRIM5α Disrupts the HIV-1 Capsid at the Inter-hexamer Interfaces

TRIM proteins play important roles in the innate immune defense against retroviral infection, including human immunodeficiency virus type-1 (HIV-1). Rhesus macaque TRIM5α (TRIM5α(rh)) targets the HIV-1 capsid and blocks infection at an early post-entry stage, prior to reverse transcription. Studies have shown that binding of TRIM5α to the assembled capsid is essential for restriction and requires the coiled-coil and B30.2/SPRY domains, but the molecular mechanism of restriction is not fully understood. In this study, we investigated, by cryoEM combined with mutagenesis and chemical cross-linking, the direct interactions between HIV-1 capsid protein (CA) assemblies and purified TRIM5α(rh) containing coiled-coil and SPRY domains (CC-SPRY(rh)). Concentration-dependent binding of CC-SPRY(rh) to CA assemblies was observed, while under equivalent conditions the human protein did not bind. Importantly, CC-SPRY(rh), but not its human counterpart, disrupted CA tubes in a non-random fashion, releasing fragments of protofilaments consisting of CA hexamers without dissociation into monomers. Furthermore, such structural destruction was prevented by inter-hexamer crosslinking using P207C/T216C mutant CA with disulfide bonds at the CTD-CTD trimer interface of capsid assemblies, but not by intra-hexamer crosslinking via A14C/E45C at the NTD-NTD interface. The same disruption effect by TRIM5α(rh) on the inter-hexamer interfaces also occurred with purified intact HIV-1 cores. These results provide insights concerning how TRIM5α disrupts the virion core and demonstrate that structural damage of the viral capsid by TRIM5α is likely one of the important components of the mechanism of TRIM5α-mediated HIV-1 restriction.

Size-controlled Peptide-directed Synthesis of Hollow Spherical Gold Nanoparticle Superstructures

Historical and Current Records of Aquarium Cetaceans in China

The number of cetaceans housed in aquariums in China is increasing. Detailed information on the historical and current population status has not been reported, despite its importance for successful breeding and population management. Questionnaires were conducted between December 2006 and May 2009, and the information was used to construct studbooks. Our survey showed that 10 species had been introduced to aquariums since 1978, including 26 (with 15 in the current population) finless porpoises (Neophocaena phocaenoides), 5 (5) false killer whales (Pseudorca crassidens), 94 (80) common bottlenose dolphins (Tursiops truncatus), 48 (30) Indo-Pacific bottlenose dolphins (Tursiops aduncus), 36 (32) beluga whales (Delphinapterus leucas), 10 (10) pantropical spotted dolphins (Stenella attenuata), 8 (8) Risso's dolphins (Grampus griseus), 2 (2) short-finned pilot whales (Globicephala macrorhynchus), 2 (2) Pacific white-sided dolphins (Lagenorhynchus obliquidens), and 5 (0) baiji dolphins (Lipotes vexillifer). The number of cetaceans has increased markedly in the past 32 years, especially since 1995. Currently, 184 individuals are under human care throughout China, a number larger than any other country with an International Species Information System membership. In addition, the Annual Survival Rates of bottlenose dolphins (0.959) and beluga whales (0.968) were found higher than those reported previously (0.93-0.951 and 0.94-0.954, respectively). Zoo Biol 30:1-14, 2011. © 2011 Wiley-Liss, Inc.

An Intramolecular Salt Bridge Drives the Soluble Domain of GTP-bound Atlastin into the Postfusion Conformation

Endoplasmic reticulum (ER) network branching requires homotypic tethering and fusion of tubules mediated by the atlastin (ATL) guanosine triphosphatase (GTPase). Recent structural studies on the ATL soluble domain reveal two dimeric conformers proposed to correspond to a tethered prefusion state and a postfusion state. How the prefusion conformer transitions to the postfusion conformer is unknown. In this paper, we identify an intramolecular salt bridge mediated by two residues outside the GTPase domain near the point of rotation that converts the prefusion dimer to the postfusion state. Charge reversal of either residue blocked ER network branching, whereas a compensatory charge reversal to reestablish electrostatic attraction restored function. In vitro assays using the soluble domain revealed that the salt bridge was dispensable for GTP binding and hydrolysis but was required for forming the postfusion dimer. Unexpectedly, the postfusion conformation of the soluble domain was achieved when bound to the nonhydrolyzable GTP analogue guanosine 5'-[β,γ-imido]triphosphate, suggesting that nucleotide hydrolysis might not be required for the prefusion to postfusion conformational change.

Direct Visualization of HIV-1 with Correlative Live-cell Microscopy and Cryo-electron Tomography

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.

3D Structure Determination of Native Mammalian Cells Using Cryo-FIB and Cryo-electron Tomography

Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB "lift-out".

MEDIATOR25 Acts As an Integrative Hub for the Regulation of Jasmonate-responsive Gene Expression in Arabidopsis

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression.

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.

Structural Insight into HIV-1 Capsid Recognition by Rhesus TRIM5α

Tripartite motif protein isoform 5 alpha (TRIM5α) is a potent antiviral protein that restricts infection by HIV-1 and other retroviruses. TRIM5α recognizes the lattice of the retrovirus capsid through its B30.2 (PRY/SPRY) domain in a species-specific manner. Upon binding, TRIM5α induces premature disassembly of the viral capsid and activates the downstream innate immune response. We have determined the crystal structure of the rhesus TRIM5α PRY/SPRY domain that reveals essential features for capsid binding. Combined cryo-electron microscopy and biochemical data show that the monomeric rhesus TRIM5α PRY/SPRY, but not the human TRIM5α PRY/SPRY, can bind to HIV-1 capsid protein assemblies without causing disruption of the capsid. This suggests that the PRY/SPRY domain alone constitutes an important pattern-sensing component of TRIM5α that is capable of interacting with viral capsids of different curvatures. Our results provide molecular insights into the mechanisms of TRIM5α-mediated retroviral restriction.

Sex Identification of Four Penguin Species Using Locus-specific PCR

Traditional methods for sex identification are not applicable to sexually monomorphic species, leading to difficulties in the management of their breeding programs. To identify sex in sexually monomorphic birds, molecular methods have been established. Two established primer pairs (2550F/2718R and p8/p2) amplify the CHD1 gene region from both the Z and W chromosomes. Here, we evaluated the use of these primers for sex identification in four sexually monomorphic penguin species: king penguins (Aptenodytes patagonicus), rockhopper penguins (Eudyptes chrysocome), gentoo penguins (Pygoscelis papua), and Magellanic penguins (Spheniscus magellanicus). For all species except rockhopper penguins, primer pair 2550F/2718R resulted in two distinct CHD1Z and CHD1W PCR bands, allowing for sex identification. For rockhopper penguins, only primer pair p8/p2 yielded different CHD1Z and CHD1W bands, which were faint and similar in size making them difficult to distinguish. As a result, we designed a new primer pair (PL/PR) that efficiently determined the gender of individuals from all four penguin species. Sequencing of the PCR products confirmed that they were from the CHD1 gene region. Primer pair PL/PR can be evaluated for use in sexing other penguin species, which will be crucial for the management of new penguin breeding programs. Zoo Biol 32:257-261, 2013. © 2012 Wiley Periodicals, Inc.

Tubular Crystals and Helical Arrays: Structural Determination of HIV-1 Capsid Assemblies Using Iterative Helical Real-space Reconstruction

Helical structures are important in many different life forms and are well-suited for structural studies by cryo-EM. A unique feature of helical objects is that a single projection image contains all the views needed to perform a three-dimensional (3D) crystallographic reconstruction. Here, we use HIV-1 capsid assemblies to illustrate the detailed approaches to obtain 3D density maps from helical objects. Mature HIV-1 particles contain a conical- or tubular-shaped capsid that encloses the viral RNA genome and performs essential functions in the virus life cycle. The capsid is composed of capsid protein (CA) oligomers which are helically arranged on the surface. The N-terminal domain (NTD) of CA is connected to its C-terminal domain (CTD) through a flexible hinge. Structural analysis of two- and three-dimensional crystals provided molecular models of the capsid protein (CA) and its oligomer forms. We determined the 3D density map of helically assembled HIV-1 CA hexamers at 16 Ã… resolution using an iterative helical real-space reconstruction method. Docking of atomic models of CA-NTD and CA-CTD dimer into the electron density map indicated that the CTD dimer interface is retained in the assembled CA. Furthermore, molecular docking revealed an additional, novel CTD trimer interface.

Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species

Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.

Nanoassembly of Surfactants with Interfacial Drug-interactive Motifs As Tailor-designed Drug Carriers

PEGylated lipopeptide surfactants carrying drug-interactive motifs specific for a peptide-nitroxide antioxidant, JP4-039, were designed and constructed to facilitate the solubilization of this drug candidate as micelles and emulsion nanoparticles. A simple screening process based on the ability that prevents the formation of crystals of JP4-039 in aqueous solution was used to identify agents that have potential drug-interactive activities. Several protected lysine derivatives possessing this activity were identified, of which α-Fmoc-ε-t-Boc lysine is the most potent, followed by α-Cbz- and α-iso-butyloxycarbonyl-ε-t-Boc-lysine. Using a polymer-supported liquid-phase synthesis approach, a series of synthetic lipopeptide surfactants with PEG headgroup, varied numbers and geometries of α-Fmoc or α-Cbz-lysyl groups located at interfacial region as the drug-interactive domains, and oleoyl chains as the hydrophobic tails were synthesized. All α-Fmoc-lysyl-containing lipopeptide surfactants were able to solubilize JP4-039 as micelles, with enhanced solubilizing activity for surfactants with increased numbers of α-Fmoc groups. The PEGylated lipopeptide surfactants with α-Fmoc-lysyl groups alone tend to form filamentous or wormlike micelles. The presence of JP4-039 transformed α-Fmoc-containing filamentous micelles into dots and barlike mixed micelles with substantially reduced sizes. Fluorescence quenching and NMR studies revealed that the drug and surfactant molecules were in close proximity in the complex. JP4-039-loaded emulsion carrying α-Cbz-containing surfactants demonstrated enhanced stability over drug-loaded emulsion without lipopeptide surfactants. JP4-039 emulsion showed a significant mitigation effect on mice exposed to a lethal dose of radiation. PEGylated lipopeptides with an interfacially located drug-interactive domain are therefore tailor-designed formulation materials potentially useful for drug development.

Safety and Efficacy Studies on Trivalent Inactivated Vaccines Against Infectious Coryza

The safety and efficacy of an inactivated oil-emulsion infectious coryza vaccine containing three Avibacterium paragallinarum isolates (one each of Page serovars A, B, and C) was evaluated. The safety of six batches of the vaccine was confirmed by testing with chickens vaccinated with a single large dose or vaccinated repeatedly with a normal dose. Efficacy tests were carried out on three batches of vaccine using both specific pathogen free (SPF) chickens and conventional chickens. In SPF chickens given a single vaccination at 42 days of age, the protection rate against all three serovars of Av. paragallinarum was at least 80% at 30 days post vaccination. The conventional chickens, which were immunized at 42 and 110 days of age, were challenged at 9 months post the second vaccination and the protection rate was at least 80% for all three serovars. The effect of storage on the vaccine was evaluated in SPF chickens using three batches of vaccine stored at 4-8°C for 1 year. The protection rate against challenge from all three serovars (single vaccination at 42 days of age and challenge at 30 days post-vaccination) was at least 80%.

A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis Leaves

A model is presented describing the gene regulatory network surrounding three similar NAC transcription factors that have roles in Arabidopsis leaf senescence and stress responses. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1-hybrid analysis and modelling using gene expression time course data has been applied to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modelling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions. Gene expression analysis in mutants of ANAC019 and ANAC055 at different times during leaf senescence has revealed a distinctly different role for each of these genes. Yeast 1-hybrid analysis is shown to be a valuable tool that can distinguish clades of binding proteins and be used to test and quantify protein binding to predicted promoter motifs.

Characterization of Spherulites As a Lipidic Carrier for Low and High Molecular Weight Agents

To develop spherulite formulations to achieve high entrapment efficiency for both small and macromolecules as well as cell-type specific delivery.

Draft Genome Sequence of Avibacterium Paragallinarum Strain 221

Avibacterium paragallinarum is the causative agent of infectious coryza. Here we report the draft genome sequence of reference strain 221 of A. paragallinarum serovar A. The genome is composed of 135 contigs for 2,685,568 bp with a 41% G+C content.

Mature HIV-1 Capsid Structure by Cryo-electron Microscopy and All-atom Molecular Dynamics

Retroviral capsid proteins are conserved structurally but assemble into different morphologies. The mature human immunodeficiency virus-1 (HIV-1) capsid is best described by a 'fullerene cone' model, in which hexamers of the capsid protein are linked to form a hexagonal surface lattice that is closed by incorporating 12 capsid-protein pentamers. HIV-1 capsid protein contains an amino-terminal domain (NTD) comprising seven α-helices and a β-hairpin, a carboxy-terminal domain (CTD) comprising four α-helices, and a flexible linker with a 310-helix connecting the two structural domains. Structures of the capsid-protein assembly units have been determined by X-ray crystallography; however, structural information regarding the assembled capsid and the contacts between the assembly units is incomplete. Here we report the cryo-electron microscopy structure of a tubular HIV-1 capsid-protein assembly at 8 Å resolution and the three-dimensional structure of a native HIV-1 core by cryo-electron tomography. The structure of the tubular assembly shows, at the three-fold interface, a three-helix bundle with critical hydrophobic interactions. Mutagenesis studies confirm that hydrophobic residues in the centre of the three-helix bundle are crucial for capsid assembly and stability, and for viral infectivity. The cryo-electron-microscopy structures enable modelling by large-scale molecular dynamics simulation, resulting in all-atom models for the hexamer-of-hexamer and pentamer-of-hexamer elements as well as for the entire capsid. Incorporation of pentamers results in closer trimer contacts and induces acute surface curvature. The complete atomic HIV-1 capsid model provides a platform for further studies of capsid function and for targeted pharmacological intervention.

Low-Dimensional Nanoparticle Clustering in Polymer Micelles and Their Transverse Relaxivity Rates

One- or two-dimensional arrays of iron oxide nanoparticles were formed in colloidal assemblies of amphiphilic polymers. Electron tomography imaging revealed that nanoparticles are arranged into one-dimensional strings in magneto-micelles or two-dimensional sheets in magneto-core/shell assemblies. The distinct directional assembly behavior was attributed to the interparticle interaction relative to the nanoparticle-polymer interaction, which was modulated by varying the cosolvent used for the solution phase self-assembly. Magneto-core/shell assemblies with varying structural parameters were formed with a range of different sized as-synthesized nanoparticles. The transverse magnetic relaxivity rates (r2) of a series of different assemblies were determined to examine the effect of nanoparticle arrangement on the magnetic relaxivity for their potential applications in MRI. The results indicated that the assembly structure of nanoparticles in polymer micelles significantly affects the r2 of surrounding water, providing a way to control magnetic relaxivity.

Tailorable Plasmonic Circular Dichroism Properties of Helical Nanoparticle Superstructures

We utilize a peptide-based methodology to prepare a diverse collection of double-helical gold nanoparticle superstructures having controllable handedness and structural metrics. These materials exhibit well-defined circular dichroism (CD) signatures at visible wavelengths owing to the collective dipole-dipole interactions between the nanoparticles. We couple theory and experiment to show how tuning the metrics and structure of the helices results in predictable and tailorable chirooptical properties. Finally, we experimentally and theoretically demonstrate that the intensity, position, and nature of the chirooptical activity can be carefully adjusted via silver overgrowth. These studies illustrate the utility of peptide-based nanoparticle assembly platforms for designing and preparing complex plasmonic materials with tailorable optical properties.

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